GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC GENES

Abstract

The present disclosure relates to genetically modified non-human animals that express a human or chimeric (e.g., humanized) IL4R and/or IL4, and methods of use thereof.

Description

CLAIM OF PRIORITY

[0001] This application is a divisional application of U.S. application Ser. No. 17/010,054, filed Sep. 2, 2020, which is a continuation, pursuant to 35 U.S.C. .sctn. 119(e), of International Application PCT/CN2019/110819, with an international filing date of Oct. 12, 2019 which claims the benefit of Chinese Patent Application App. No. 201811194052.1, filed on Oct. 12, 2018, and Chinese Patent Application App. No. 201811628008.7, filed on Dec. 28, 2018. The entire contents of the foregoing are incorporated herein by reference.

TECHNICAL FIELD

[0002] This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) genes, and methods of use thereof.

BACKGROUND

[0003] Interleukin-4 (IL4) is a cytokine produced by several different cell types, including e.g., activated T cells, mast cells and basophils. The immuno-regulatory role of IL4 in allergic diseases and activation of Th2 type responses has been well established. There is substantial evidence showing that targeting IL4/IL4R pathway can be a therapeutic strategy for treating immune-related disorders (e.g., allergy and autoimmune diseases) in humans.

[0004] The traditional drug research and development for therapeutic agents that target IL4/IL4R pathway typically use in vitro screening approaches. However, these screening approaches are still different from what happens in the in vivo environment (such as cell microenvironment, extracellular matrix components and immune cell interaction, etc.), resulting in a high rate of failure in drug development. There is a need for humanized animal models that are suitable for human antibody screening and efficacy evaluation.

SUMMARY

[0005] This disclosure is related to an animal model with human IL4R and/or IL4 or chimeric IL4R and/or IL4. The animal model can express human IL4R and/or IL4 or chimeric IL4R and/or IL4 (e.g., humanized IL4R and/or IL4) protein in its body. It can be used in the studies on the function of IL4R and/or IL4 gene, and can be used in the screening and evaluation of anti-human IL4R and anti-IL4 antibodies. In addition, the animal models prepared by the methods described herein can be used in drug screening, pharmacodynamics studies, treatments for immune-related diseases (e.g., autoimmune disease, allergies). They can also be used to facilitate the development and design of new drugs, and save time and cost. In summary, this disclosure provides a powerful tool for studying the function of IL4R and/or IL4 protein and a platform for screening treatments for immune-related diseases.

[0006] In one aspect, the disclosure provides a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric IL4R.

[0007] In some embodiments, the sequence encoding the human or chimeric IL4R is operably linked to an endogenous regulatory element at the endogenous IL4R gene locus in the at least one chromosome.

[0008] In some embodiments, the sequence encoding a human or chimeric IL4R comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human IL4R (NP 000409.1; SEQ ID NO: 42).

[0009] In some embodiments, the sequence encoding a human or chimeric IL4R comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 44.

[0010] In some embodiments, the sequence encoding a human or chimeric IL4R comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to amino acids 30-216 of SEQ ID NO: 42.

[0011] In some embodiments, the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 48, 49, 50, or 51.

[0012] In some embodiments, the animal is a mammal, e.g., a monkey, a rodent or a mouse. In some embodiments, the animal is a mouse.

[0013] In some embodiments, the animal does not express endogenous IL4R. In some embodiments, the animal has one or more cells expressing human or chimeric IL4R.

[0014] In some embodiments, the animal has one or more cells expressing human or chimeric IL4R, and the expressed human or chimeric IL4R can bind to endogenous IL4.

[0015] In some embodiments, the animal has one or more cells expressing human or chimeric IL4R, and the expressed human or chimeric IL4R cannot bind to endogenous IL4.

[0016] In another aspect, the disclosure is related to a genetically-modified, non-human animal, in some embodiments, the genome of the animal comprises a replacement of a sequence encoding a region of endogenous IL4R with a sequence encoding a corresponding region of human IL4R at an endogenous IL4R gene locus.

[0017] In some embodiments, the sequence encoding the corresponding region of human IL4R is operably linked to an endogenous regulatory element at the endogenous IL4R locus, and one or more cells of the animal express a chimeric IL4R.

[0018] In some embodiments, the animal does not express endogenous IL4R.

[0019] In some embodiments, the replaced locus is the extracellular region of IL4R.

[0020] In some embodiments, the animal has one or more cells expressing a chimeric IL4R having an extracellular region, in some embodiments, the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the extracellular region of human IL4R.

[0021] In some embodiments, the extracellular region of the chimeric IL4R has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 150 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human IL4R.

[0022] In some embodiments, the animal is a mouse, and the replaced endogenous IL4R region is exon 4, exon 5, exon 6, and/or exon 7 of the endogenous mouse IL4R gene.

[0023] In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous IL4R gene locus. In some embodiments, the animal is homozygous with respect to the replacement at the endogenous IL4R gene locus.

[0024] In one aspect, the disclosure relates to methods for making a genetically-modified, non-human animal. The methods involve replacing in at least one cell of the animal, at an endogenous IL4R gene locus, a sequence encoding a region of an endogenous IL4R with a sequence encoding a corresponding region of human IL4R.

[0025] In some embodiments, the sequence encoding the corresponding region of human IL4R comprises exon 4, exon 5, exon 6, and/or exon 7 of a human IL4R gene.

[0026] In some embodiments, the sequence encoding the corresponding region of IL4R comprises at least 100, 200, or 300 nucleotides of exon 4, exon 5, exon 6 and/or exon 7 of a human IL4R gene.

[0027] In some embodiments, the sequence encoding the corresponding region of human IL4R encodes a sequence that is at least 90% identical to amino acids 30-216 of SEQ ID NO: 42.

[0028] In some embodiments, the locus is located within the extracellular region of IL4R.

[0029] In some embodiments, the animal is a mouse, and the locus is exons 4, exon5, exon 6 and/or exon 7 of the mouse IL4R gene.

[0030] In one aspect, the disclosure relates to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL4R polypeptide, in some embodiments, the chimeric IL4R polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4R. In some embodiments, the animal expresses the chimeric IL4R.

[0031] In some embodiments, the chimeric IL4R polypeptide has at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4R extracellular region.

[0032] In some embodiments, the chimeric IL4R polypeptide comprises a sequence that is at least 90%, 95%, or 99% identical to amino acids 30-216 of SEQ ID NO: 42.

[0033] In some embodiments, the nucleotide sequence is operably linked to an endogenous IL4R regulatory element of the animal.

[0034] In some embodiments, the chimeric IL4R polypeptide comprises an endogenous IL4R transmembrane region and/or an endogenous cytoplasmic region.

[0035] In some embodiments, the nucleotide sequence is integrated to an endogenous IL4R gene locus of the animal.

[0036] In some embodiments, the chimeric IL4R has at least one mouse IL4R activity and/or at least one human IL4R activity.

[0037] In one aspect, the disclosure relates to methods of making a genetically-modified mouse cell that expresses a chimeric IL4R. The methods involve replacing, at an endogenous mouse IL4R gene locus, a nucleotide sequence encoding a region of mouse IL4R with a nucleotide sequence encoding a corresponding region of human IL4R, thereby generating a genetically-modified mouse cell that includes a nucleotide sequence that encodes the chimeric IL4R. In some embodiments, the mouse cell expresses the chimeric IL4R.

[0038] In some embodiments, the chimeric IL4R comprises: an extracellular region of human IL4R; a transmembrane region of mouse IL4R; and/or a cytoplasmic region of mouse IL4R.

[0039] In some embodiments, the nucleotide sequence encoding the chimeric IL4R is operably linked to an endogenous IL4R regulatory region, e.g., promoter.

[0040] In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4, IL33, IL13, programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte Activating 3 (LAG-3), B And T Lymphocyte Associated (BTLA), Programmed Cell Death 1 Ligand 1 (PD-L1), CD27, CD28, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT), T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), Glucocorticoid-Induced TNFR-Related Protein (GITR), CD137, TNF Receptor Superfamily Member 4 (OX40), CD47, or Signal regulatory protein .alpha. (SIRPa)). In some embodiments, the additional human or chimeric protein is IL4.

[0041] In one aspect, the disclosure provides a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric IL4.

[0042] In some embodiments, the sequence encoding the human or chimeric IL4 is operably linked to an endogenous regulatory element at the endogenous IL4 gene locus in the at least one chromosome. In some embodiments, the sequence encoding the human or chimeric IL4 is operably linked to a human regulatory element at the endogenous IL4 gene locus in the at least one chromosome.

[0043] In some embodiments, the sequence encoding a human or chimeric IL4 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human IL4 (NP 000580.1; SEQ ID NO: 4).

[0044] In some embodiments, the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8, 9, 10, 11, 23, 24, or 25.

[0045] In some embodiments, the animal is a mammal, e.g., a monkey, a rodent or a mouse. In some embodiments, the animal is a mouse.

[0046] In some embodiments, the animal does not express endogenous IL4. In some embodiments, the animal has one or more cells expressing human IL4. In some embodiments, the animal has one or more cells expressing human or chimeric IL4, and the expressed human or chimeric IL4 can bind to endogenous IL4R. In some embodiments, the animal has one or more cells expressing human or chimeric IL4, and the expressed human or chimeric IL4 cannot bind to endogenous IL4R.

[0047] In one aspect, the disclosure provides a genetically-modified, non-human animal. In some embodiments, the genome of the animal comprises a replacement of a sequence encoding a region of endogenous IL4 with a sequence encoding a corresponding region of human IL4 at an endogenous IL4 gene locus.

[0048] In some embodiments, the sequence encoding the corresponding region of human IL4 is operably linked to an endogenous regulatory element at the endogenous IL4 locus, and one or more cells of the animal expresses a human IL4.

[0049] In some embodiments, the sequence encoding the corresponding region of human IL4 is operably linked to a human regulatory element at the endogenous IL4 locus, and one or more cells of the animal expresses a human IL4.

[0050] In some embodiments, the animal does not express endogenous IL4.

[0051] In some embodiments, the replaced locus comprises a sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

[0052] In some embodiments, the animal is a mouse, and the replaced endogenous IL4 region is exon 1, exon 2, exon 3 and/or exon 4 of the endogenous mouse IL4 gene.

[0053] In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous IL4 gene locus. In some embodiments, the animal is homozygous with respect to the replacement at the endogenous IL4 gene locus.

[0054] In another aspect, the disclosure relates to methods for making a genetically-modified, non-human animal. The methods involve replacing in at least one cell of the animal, at an endogenous IL4 gene locus, a sequence encoding a region of an endogenous IL4 with a sequence encoding a corresponding region of human IL4.

[0055] In some embodiments, the sequence encoding the corresponding region of human IL4 comprises exon 1, exon 2, exon 3 and/or exon 4 of a human IL4 gene. In some embodiments, the sequence encoding the corresponding region of IL4 comprises at least 50, 100, 150, or 200 nucleotides of exon 1, exon 2, exon 3 and/or exon 4 of a human IL4 gene.

[0056] In some embodiments, the sequence encoding the corresponding region of human IL4 encodes a sequence that is at least 90% identical to SEQ ID NO: 4. In some embodiments, replaced locus comprises a sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

[0057] In some embodiments, the animal is a mouse, and the locus is exon 1, exon 2, exon 3 and/or exon 4 of the mouse IL4 gene.

[0058] In another aspect, the disclosure provides a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL4 polypeptide, in some embodiments, the chimeric IL4 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4. In some embodiments, the animal expresses the chimeric IL4.

[0059] In some embodiments, the chimeric IL4 polypeptide has at least 100 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4.

[0060] In some embodiments, the nucleotide sequence is operably linked to an endogenous IL4 regulatory element of the animal. In some embodiments, the nucleotide sequence is operably linked to a human IL4 regulatory element of the animal.

[0061] In some embodiments, the nucleotide sequence is integrated to an endogenous IL4 gene locus of the animal.

[0062] In some embodiments, the chimeric IL4 has at least one mouse IL4 activity and/or at least one human IL4 activity.

[0063] In one aspect, the disclosure relates to methods of making a genetically-modified mouse cell that expresses a chimeric IL4 or a human IL4. The methods involve replacing, at an endogenous mouse IL4 gene locus, a nucleotide sequence encoding a region of mouse IL4 with a nucleotide sequence encoding a corresponding region of human IL4, thereby generating a genetically-modified mouse cell that includes a nucleotide sequence that encodes the chimeric IL4. In some embodiments, the mouse cell expresses the chimeric IL4 or human IL4.

[0064] In some embodiments, the nucleotide sequence encoding the chimeric IL4 is operably linked to an endogenous IL4 regulatory region, e.g., promoter, 5'-UTR, or 3'-UTR.

[0065] In some embodiments, the nucleotide sequence encoding the chimeric IL4 is operably linked to a human IL4 regulatory region, e.g., promoter, 5'-UTR, or 3'-UTR. In some embodiments, the animal or mouse further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4R, IL33, IL13, PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, TIGIT, TIM-3, GITR, CD137, OX40, CD47, or SIRPa). In some embodiments, the additional human or chimeric protein is IL4R.

[0066] In one aspect, the disclosure relates to methods of determining effectiveness of an IL4-IL4R pathway inhibitor for treating an allergic disorder. The methods involve administering the IL4-IL4R pathway inhibitor to the animal as described herein, where the animal has an allergic disorder; and determining the inhibitory effects of the IL4-IL4R pathway inhibitor. In some embodiments, the allergic disorder is asthma. In some embodiments, the allergic disorder is atopic dermatitis. In some embodiments, the allergic disorder is chromic sinusitis.

[0067] In some embodiments, the IL4-IL4R pathway inhibitor is an anti-IL4 antibody (e.g., anti-human IL4 antibody). In some embodiments, the IL4-IL4R pathway inhibitor is an anti-IL4R antibody (e.g., anti-human IL4R antibody). In some embodiments, the IL4-IL4R pathway inhibitor is an anti-IL13 antibody (e.g., anti-human IL13 antibody).

[0068] In some embodiments, the inhibitory effects are evaluated by serum IgE levels; pathological lung histology features; number of leukocytes (CD45+ cells), eosinophils (Eos) or neutrophils in bronchoalveolar lavage fluid (BALF); or ratio of eosinophils or neutrophils cells in CD45+ cells in bronchoalveolar lavage fluid (BALF).

[0069] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4-IL4R pathway inhibitor for reducing inflammation. The methods involve administering the IL4-IL4R pathway inhibitor to the animal as described herein, where the animal has inflammation; and determining the inhibitory effects of the IL4-IL4R pathway inhibitor.

[0070] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4-IL4R pathway inhibitor for treating autoimmune disorder. The methods involve administering the IL4-IL4R pathway inhibitor to the animal as described herein, where the animal has autoimmune disorder; and determining the inhibitory effects of the IL4-IL4R pathway inhibitor.

[0071] In another aspect, the disclosure also provides methods of determining effectiveness of an IL4-IL4R pathway inhibitor for treating cancer. The methods involve administering the IL4-IL4R pathway inhibitor to the animal as described herein, where the animal has a tumor; and determining the inhibitory effects of the IL4-IL4R pathway inhibitor. In some embodiments, determining the inhibitory effects of the treatment involves measuring the tumor volume in the animal.

[0072] In one aspect, the disclosure also provides methods of determining toxicity of an anti-IL4R antibody or an anti-IL4 antibody, the methods involve administering the anti-IL4R antibody or the anti-IL4 antibody to the animal as described herein; and determining weight change of the animal. In some embodiments, the method further comprising performing a blood test (e.g., determining red blood cell count).

[0073] In one aspect, the disclosure relates to proteins comprising an amino acid sequence, in some embodiments, the amino acid sequence is one of the following:

[0074] (a) an amino acid sequence set forth in SEQ ID NO: 44;

[0075] (b) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44;

[0076] (c) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

[0077] (d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 44.

[0078] In one aspect, the disclosure relates to nucleic acids comprising a nucleotide sequence, in some embodiments, the nucleotide sequence is one of the following:

[0079] (a) a sequence that encodes the protein as described herein;

[0080] (b) SEQ ID NO: 8, 9, 10, 11, 23, 24, 25, 43, 48, 49, 50, or 51; or

[0081] (c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, 9, 10, 11, 23, 24, 25, 43, 48, 49, 50, or 51.

[0082] In some embodiments, the mice described in the present disclosure can be mated with the mice containing other human or chimeric genes (e.g., chimeric IL4, chimeric PD-1, chimeric PD-L1, chimeric CTLA-4, or other immunomodulatory factors), so as to obtain a mouse expressing two or more human or chimeric proteins. The mice can also, e.g., be used for screening antibodies in the case of a combined use of drugs, as well as evaluating the efficacy of the combination therapy.

[0083] In one aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal's endogenous IL4R gene, wherein the disruption of the endogenous IL4R gene comprises deletion of exon 4, exon 5, exon 6, and/or exon 7, or part thereof of the endogenous IL4R gene.

[0084] In some embodiments, the disruption of the endogenous IL4R gene further comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11 of the endogenous IL4R gene.

[0085] In some embodiments, the disruption of the endogenous IL4R gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9 and/or intron 10 of the endogenous IL4R gene.

[0086] In some embodiments, wherein the deletion can comprise deleting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, or more nucleotides.

[0087] In some embodiments, the disruption of the endogenous IL4R gene comprises the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11 (e.g., deletion of at least 100 nucleotides of exon 5).

[0088] In one aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal's endogenous IL4 gene, wherein the disruption of the endogenous IL4 gene comprises deletion of exon 1, exon 2, exon 3, and/or exon 4, or part thereof of the endogenous IL4 gene.

[0089] In some embodiments, the disruption of the endogenous IL4 gene further comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3, and/or exon 4 of the endogenous IL4 gene.

[0090] In some embodiments, the disruption of the endogenous IL4 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, and/or intron 3 of the endogenous IL4 gene.

[0091] In some embodiments, wherein the deletion can comprise deleting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, or more nucleotides.

[0092] In some embodiments, the disruption of the endogenous IL4 gene comprises the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides of exon 1, exon 2, exon 3, and/or exon 4 (e.g., deletion of at least 100 nucleotides of exon 3).

[0093] The disclosure also relates to a method for establishing a genetically-modified non-human animal expressing two human or chimeric (e.g., humanized) genes. The method includes the steps of (a) using the method for establishing a IL4R gene humanized animal model to obtain a IL4R gene genetically modified humanized mouse; (b) mating the IL4R gene genetically modified humanized mouse obtained in step (a) with another humanized mouse, and then screening to obtain a double humanized mouse model. In some embodiments, in step (b), the IL4R gene genetically modified humanized mouse obtained in step (a) is mated with an IL4 humanized mouse to obtain a IL4R and IL4 double humanized mouse model.

[0094] The disclosure also relates to a method for establishing a genetically-modified non-human animal expressing two human or chimeric (e.g., humanized) genes. The method includes the steps of (a) using the method for establishing a IL4 gene humanized animal model to obtain a IL4 gene genetically modified humanized mouse; (b) mating the IL4 gene genetically modified humanized mouse obtained in step (a) with another humanized mouse, and then screening to obtain a double humanized mouse model. In some embodiments, in step (b), the IL4 gene genetically modified humanized mouse obtained in step (a) is mated with an IL4R humanized mouse to obtain an IL4 and IL4R double humanized mouse model.

[0095] The disclosure also relates to non-human mammal generated through the methods as described herein. In some embodiments, the genome thereof contains human gene(s).

[0096] In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse. In some embodiments, the non-human mammal expresses a protein encoded by a humanized IL4R and/or IL4 gene.

[0097] The disclosure also relates to an offspring of the non-human mammal.

[0098] In one aspect, the disclosure relates to a non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein. In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0099] The disclosure also relates to a cell (e.g., stem cell or embryonic stem cell) or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal. The disclosure further relates to the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.

[0100] In one aspect, the disclosure relates to a tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.

[0101] The disclosure further relates to a IL4R and/or IL4 genomic DNA sequence of a humanized mouse, a DNA sequence obtained by a reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell comprising the construct thereof; a tissue comprising the cell thereof.

[0102] The disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the development of a product related to an immunization processes of human cells, the manufacture of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

[0103] The disclosure also relates to the use of the non-human mammal or an offspring thereof, or the non-human mammal, the animal model generated through the method as described herein in the production and utilization of an animal experimental disease model of immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.

[0104] The disclosure further relates to the use of the non-human mammal or an offspring thereof, or the non-human mammal, the animal model generated through the methods as described herein, in the screening, verifying, evaluating or studying the IL4R and/or IL4 gene function, human IL4R and/or IL4 antibodies, the drugs or efficacies for human IL4R and/or IL4 targeting sites, and the drugs for immune-related diseases.

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0106] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

[0107] FIG. 1 is a schematic diagram showing mouse and human IL4 gene locus.

[0108] FIG. 2 is a schematic diagram showing humanized IL4 gene locus (replacing coding sequence).

[0109] FIG. 3 is a schematic diagram showing humanized IL4 gene (replacing coding sequencing and 5'-UTR and 3'-UTR).

[0110] FIG. 4 is a schematic diagram showing an IL4 gene targeting strategy.

[0111] FIG. 5A is a graph showing recombinant vector digestion results, wherein No. 1, 2, 3 refer to 3 vector plasmids respectively, ck represents the undigested plasmid control, M is the Marker.

[0112] FIG. 5B is a graph showing DNA ladder for the Marker.

[0113] FIG. 6 is a graph showing PCR identification results of cells, wherein + is the positive control, WT is the wild-type control, M is the Marker, H.sub.2O is a blank control. The clones marked with the clone numbers were identified as positive clones.

[0114] FIG. 7 is a graph showing Southern blot results.

[0115] FIG. 8 is a schematic diagram showing an IL4 gene targeting strategy.

[0116] FIG. 9A is a graph showing recombinant vector digestion results, wherein ck represents the undigested plasmid control, M is the Marker. The plasmids were treated with BamHI, EcoRI or ScaI, respectively.

[0117] FIG. 9B is a graph showing DNA ladder for the Marker.

[0118] FIG. 10 is a graph showing PCR identification results, wherein M is the Marker, + is the positive control, WT is the wild-type control, and H.sub.2O is a blank control. The lane marked with clone number 1-G9 was identified as a positive clone.

[0119] FIG. 11 is a graph showing Southern blot results.

[0120] FIG. 12 is a schematic diagram showing the FRT recombination process.

[0121] FIG. 13A shows PCR identification results for F1 generation mice (short fragment replacement), wherein primer pairs WT-F and WT-R were used to amplify wild-type mouse IL4 gene exon 4. WT is wild-type, H.sub.2O is a blank control, and + is the positive control.

[0122] FIG. 13B shows PCR identification results for F1 generation mice (short fragment replacement), wherein primer pairs Mut-F and WT-R were used to amplify the engineered exon 4 of the mouse IL4 gene to verify the correct insertion of the recombinant vector into the genomic locus; wherein WT is wild-type and + is the positive control.

[0123] FIG. 13C shows PCR identification results for F1 generation mice (short fragment replacement), wherein primer pairs Frt-F and Frt-R are used to amplify the Neo fragment to determine whether the resistance gene NeoR was removed; wherein WT is wild-type, and + is the positive control.

[0124] FIG. 13D shows PCR identification results for F1 generation mice (short fragment replacement), wherein primer pairs Flp-F and Flp-R were used to confirm the presence of the Flp fragment; wherein WT is wild-type and + is the positive control.

[0125] FIG. 14A is a graph showing the ELISA detection results of the mouse IL4 protein in IL4 humanized mice. One wild-type C57BL/6 mouse and two IL4 humanized heterozygotes were selected, wherein +/+ represents wild-type C57BL/6 mice; B-hIL4 (V1) H/+ represents the IL4 humanized mouse heterozygote obtained by short fragment replacement (replacing coding sequences); and B-hIL4 (V2) H/+ represents the IL4 humanized mouse heterozygote obtained by long fragment replacement (replacing coding sequences and 5'- and 3'-UTR).

[0126] FIG. 14B is a graph showing the ELISA detection results of the human IL4 protein in IL4 humanized mice. One wild-type C57BL/6 mouse and two IL4 humanized heterozygotes were selected, wherein +/+ represents wild-type C57BL/6 mice; B-hIL4 (V1) H/+ represents the IL4 humanized mouse heterozygote obtained by short fragment replacement; and B-hIL4 (V2) H/+ represents the IL4 humanized mouse heterozygote obtained by long fragment replacement.

[0127] FIG. 15 is a schematic diagram showing mouse and human IL4R gene locus.

[0128] FIG. 16 is a schematic diagram showing humanized IL4R gene locus.

[0129] FIG. 17 is a schematic diagram showing the targeting strategy for IL4R gene locus.

[0130] FIG. 18 is a graph showing the recombinant vector restriction enzyme digestion results, wherein No. 1, 2, 3, 4 refer to 4 vector plasmids respectively, ck represents the undigested plasmid control, and M is the Marker.

[0131] FIG. 19A is a graph showing the PCR identification results using primer pairs F1/R1, wherein + is the positive control, WT is the wild-type control, and M is the Marker.

[0132] FIG. 19B is a graph showing the PCR identification results using primer pairs F2/R2, wherein + is the positive control, WT is the wild-type control, M is the Marker.

[0133] FIG. 20 is a graph showing Southern blot results. WT is the wild-type.

[0134] FIG. 21A shows PCR identification results of F1 generation mice, wherein primer pairs WT-F2 and WT-R2 were used to amplify wild-type mouse IL4Ra gene exon 7; wherein WT is wild-type, + is the positive control, H.sub.2O is a blank control, and M is the Marker.

[0135] FIG. 21B shows PCR identification results of F1 generation mice, wherein primer pairs Mut-F2 and WT-R2 were used to amplify modified exon 7 of the mouse IL4Ra gene to confirm the insertion of the recombinant vector into the genomic locus; wherein WT is wild-type, + is the positive control and, M is the Marker.

[0136] FIG. 22A shows PCR identification results of F1 generation mice, wherein primer pairs Flp-F2 and Flp-R2 were used to confirm the presence of the Flp fragment; wherein WT is wild-type, and + is the positive control.

[0137] FIG. 22B shows PCR identification results of F1 generation mice, wherein primer pairs Frt-F2 and Frt-R2 were used to amplify the Neo fragment to verify whether the resistance gene Neo was removed; wherein WT is wild-type, and + is the positive control.

[0138] FIG. 23A is a graph showing the flow cytometry analysis result of wild-type C57BL/6 mice, wherein cells were stained by anti-mouse IL4Ra antibody (mIL4RA PE) and anti-mouse CD19 antibody (mCD19 FITC).

[0139] FIG. 23B is a graph showing the flow cytometry analysis result of anti-mouse CD3 antibody-stimulated wild-type C57BL/6 mice, wherein cells were stained by anti-mouse IL4Ra antibody (mIL4RA PE) and anti-mouse CD19 antibody (mCD19 FITC).

[0140] FIG. 23C is a graph showing the flow cytometry analysis result of anti-mouse CD3 antibody-stimulated humanized IL4Ra gene heterozygous mice, wherein cells were stained by anti-mouse IL4Ra antibody (mIL4RA PE) and anti-mouse CD19 antibody (mCD19 FITC).

[0141] FIG. 23D is a graph showing the flow cytometry analysis result of wild-type C57BL/6 mice, wherein cells were stained by anti-human IL4Ra antibody (hIL4RA APC) and anti-mouse CD19 antibody (mCD19 FITC).

[0142] FIG. 23E is a graph showing the flow cytometry analysis result of anti-mouse CD3 antibody-stimulated wild-type C57BL/6 mice, wherein cells were stained by anti-human IL4Ra antibody (hIL4RA APC) and anti-mouse CD19 antibody (mCD19 FITC).

[0143] FIG. 23F is a graph showing the flow cytometry analysis result of anti-mouse CD3 antibody-stimulated humanized IL4Ra gene heterozygous mice, wherein cells were stained by anti-human IL4Ra antibody (hIL4RA APC) and anti-mouse CD19 antibody (mCD19 FITC).

[0144] FIG. 24A is a graph showing the results of sgRNA1-sgRNA7 activity assay, in which Con. is the negative control and PC is the positive control.

[0145] FIG. 24B is a graph showing the results of sgRNA8-sgRNA15 activity assay, in which Con. is the negative control and PC is the positive control.

[0146] FIG. 25A is the ELISA detection result showing mouse IL4 protein levels in unstimulated mouse serum, wherein three wild-type C57BL/6 mice and three double-humanized IL4/IL4Ra homozygous mice (B-hIL4/IL4R mice) were selected.

[0147] FIG. 25B is the ELISA detection result showing human IL4 protein levels in unstimulated mouse serum, wherein three wild-type C57BL/6 mice and three double-humanized IL4/IL4Ra homozygous mice (B-hIL4/IL4R mice) were selected.

[0148] FIG. 25C is the ELISA detection result showing mouse IL4 protein levels in stimulated mouse serum, wherein three wild-type C57BL/6 mice and three double-humanized IL4/IL4Ra homozygous mice (B-hIL4/IL4R mice) were selected.

[0149] FIG. 25D is the ELISA detection result showing human IL4 protein levels in stimulated mouse serum, wherein three wild-type C57BL/6 mice and three double-humanized IL4/IL4Ra homozygous mice (B-hIL4/IL4R mice) were selected.

[0150] FIG. 26A is a graph showing the flow cytometry analysis result of three unstimulated wild-type C57BL/6 mice, wherein cells were labeled by anti-mouse IL4Ra antibody (mIL4RA PE) and anti-mouse CD19 antibody (mCD19 APC-Cy7).

[0151] FIG. 26B is a graph showing the flow cytometry analysis result of three unstimulated double-humanized IL4/IL4Ra homozygous (B-HIL4/IL4R (H/H)) mice, wherein cells were labeled by anti-mouse IL4Ra antibody (mIL4RA PE) and anti-mouse CD19 antibody (mCD19 APC-Cy7).

[0152] FIG. 26C is a graph showing the flow cytometry analysis result of three anti-mouse CD3 antibody-stimulated wild-type C57BL/6 mice, wherein cells were labeled by anti-mouse IL4Ra antibody (mIL4RA PE) and anti-mouse CD19 antibody (mCD19 APC-Cy7).

[0153] FIG. 26D is a graph showing the flow cytometry analysis result of three anti-mouse CD3 antibody-stimulated double-humanized IL4/IL4Ra homozygous (B-HIL4/IL4R (H/H)) mice, wherein cells were labeled by anti-mouse IL4Ra antibody (mIL4RA PE) and anti-mouse CD19 antibody (mCD19 APC-Cy7).

[0154] FIG. 26E is a graph showing the flow cytometry analysis result of three unstimulated wild-type C57BL/6 mice, wherein cells were labeled by anti-human IL4Ra antibody (hIL4RA PE) and anti-mouse CD19 antibody (mCD19 APC-Cy7).

[0155] FIG. 26F is a graph showing the flow cytometry analysis result of three unstimulated double-humanized IL4/IL4Ra homozygous (B-HIL4/IL4R (H/H)) mice, wherein cells were labeled by anti-human IL4Ra antibody (hIL4RA PE) and anti-mouse CD19 antibody (mCD19 APC-Cy7).

[0156] FIG. 26G is a graph showing the flow cytometry analysis result of three anti-mouse CD3 antibody-stimulated wild-type C57BL/6 mice, wherein cells were labeled by anti-human IL4Ra antibody (hIL4RA PE) and anti-mouse CD19 antibody (mCD19 APC-Cy7).

[0157] FIG. 26H is a graph showing the flow cytometry analysis result of three anti-mouse CD3 antibody-stimulated double-humanized IL4/IL4Ra homozygous (B-HIL4/IL4R (H/H)) mice, wherein cells were labeled by anti-human IL4Ra antibody (hIL4RA PE) and anti-mouse CD19 antibody (mCD19 APC-Cy7).

[0158] FIG. 27A is a graph showing the flow cytometry analysis result of double-humanized IL4/IL4Ra homozygous mice, wherein the cells were labeled by anti-hIgG-AF647 and anti-mCD19 antibody (mCD19APC-Cy7).

[0159] FIG. 27B is a graph showing the flow cytometry analysis result of double-humanized IL4/IL4Ra homozygous mice, wherein the cells were labeled by anti-IgG4-kappa hIgG-APC/anti-hIgG-AF647 and anti-mouse CD19 antibody (mCD19APC-Cy7).

[0160] FIG. 27C is a graph showing the flow cytometry analysis result of double-humanized IL4/IL4Ra homozygous mice, wherein the cells were labeled by anti-human IL4Ra antibody (Dupilumab)/anti-hIgG-AF647 and anti-mouse CD19 antibody (mCD19APC-Cy7). The results show that Dupilumab binds well to IL4Ra expressed in the double-humanized homozygous mice.

[0161] FIG. 28 is a graph showing LPS-induced spleen lymphocyte proliferation result, wherein G1 was induced only by LPS, G2 was induced by LPS and mIL4, G3 was induced by LPS and hIL4. The result shows that the level of IgE in double-humanized IL4/IL4Ra mice was comparable to that of wild-type C57BL/6 mice, and the lymphocytes of wild-type mice can only be induced by mIL4 to produce IgE, while lymphocytes of double-humanized IL4/IL4Ra mice can only be induced by hIL4.

[0162] FIG. 29 is a graph showing IgE production induced by hIL4 in double-humanized IL4/IL4Ra mice was effectively blocked by anti-human IL4Ra antibody Dupilumab.

[0163] FIG. 30 is the experimental protocol of using double-humanized IL4/IL4Ra mice to make an inducible asthma model.

[0164] FIG. 31 is a graph showing serum IgE levels of control group (PBS) and the asthma model (OVA). The serum IgE levels in the asthma model mice are significantly higher than those in the PBS control group.

[0165] FIG. 32 is a graph showing lung histology results of double-humanized IL4/IL4Ra mice induced by ovalbumin combined with aluminum hydroxide (OVA) or PBS (control group). The OVA-induced group had more darkly stained area than the PBS control group, showing higher rate of inflammatory cell infiltration and obvious pathological features of asthma.

[0166] FIG. 33A is a graph showing total number of eosinophils (Eos) cells in bronchoalveolar lavage fluid (BALF) of double-humanized IL4/IL4Ra mice induced by ovalbumin combined with aluminum hydroxide or PBS (control group), wherein OVA-induced group had more Eos cells than the PBS control group.

[0167] FIG. 33B is a graph showing the proportion of eosinophils cells (Eos %) in bronchoalveolar lavage fluid (BALF) of double-humanized IL4/IL4Ra mice induced by ovalbumin combined with aluminum hydroxide (OVA) or PBS (control group). The result shows mice in the OVA-induced group had significantly more eosinophils compared than the PBS control group, indicating a sensitizing phenotype.

[0168] FIG. 33C is a graph showing the proportion of neutrophil cells (Neu %) in bronchoalveolar lavage fluid (BALF) of double-humanized IL4/IL4Ra mice induced by ovalbumin combined with aluminum hydroxide (OVA) or PBS (control group). The result shows mice in OVA-induced group had significantly more neutrophils compared with the PBS control group, indicating a sensitizing phenotype.

[0169] FIG. 34 is the experimental design to assess treatment efficacy of anti-human IL4Ra antibody Dupilumab in the double-humanized IL4/IL4Ra mice induced by ovalbumin combined with aluminum hydroxide.

[0170] FIG. 35A is a graph showing total number of leukocytes (CD45+ cells) in bronchoalveolar lavage fluid (BALF) of double-humanized IL4/IL4Ra mice induced by ovalbumin combined with aluminum hydroxide and control group, wherein OVA-induced group (G2) had slightly more leukocytes than the control group (G1) and the treatment groups (G3, G4).

[0171] FIG. 35B is a graph showing total number of eosinophils (Eos) cells in bronchoalveolar lavage fluid (BALF) of double-humanized IL4/IL4Ra mice induced by ovalbumin combined with aluminum hydroxide, wherein OVA-induced group (G2) had the most eosinophils cells, and the treatment group (G3, G4) had slightly more eosinophils cells than the control group (G1).

[0172] FIG. 35C is a graph showing the proportion of eosinophils (Eos %) cells in leukocytes (CD45+ cells) in bronchoalveolar lavage fluid (BALF) of double-humanized IL4/IL4Ra mice induced by ovalbumin combined with aluminum hydroxide, wherein OVA-induced group (G2) had the highest proportion, the treatment group (G3) had the lowest proportion, and the treatment group (G4) had slightly lower proportion than the control group (G1).

[0173] FIG. 36 is a graph showing serum IgE levels of double-humanized IL4/IL4Ra mice induced by ovalbumin combined with aluminum hydroxide, wherein high serum IgE levels were only detected in OVA-induced group (G2).

[0174] FIG. 37 is a graph showing airway tissue section H&E staining result of double-humanized IL4/IL4Ra mice induced by ovalbumin combined with aluminum hydroxide, wherein airway of the control group (G1) mice showed no inflammation while the OVA-induced group (G2) had peribronchial and perivascular inflammation and increased mucus secretion levels. The mice in the treatment group (G3, G4) had decreased inflammatory infiltration and mucus secretion as compared to the G2 group.

[0175] FIG. 38 is the experimental design to assess treatment efficacy of anti-human IL4Ra antibody Dupilumab in the double-humanized IL4/IL4Ra mouse asthma model.

[0176] FIG. 39A is a graph showing total number of leukocytes (CD45+ cells) in BALF in the double-humanized IL4/IL4Ra mouse asthma model, wherein OVA-induced groups (G2, G4) have significantly more leukocytes than the control group (G1) and the treatment group (G3).

[0177] FIG. 39B is a graph showing total number of eosinophils cells in BALF in the double-humanized IL4/IL4Ra mouse asthma model, wherein OVA-induced groups (G2, G4) had significantly more eosinophils cells than the control group (G1) and the treatment group (G3).

[0178] FIG. 39C is a graph showing total number of neutrophils cells in BALF in the double-humanized IL4/IL4Ra mouse asthma model, wherein OVA-induced groups (G2, G4) had significantly more neutrophils cells than the control group (G1) and the treatment group (G3).

[0179] FIG. 40 is a graph showing serum IgE levels in the double-humanized IL4/IL4Ra mouse asthma model, wherein OVA-induced groups (G2, G4) had significantly higher serum IgE levels than the control group (G1) and the treatment group (G3).

[0180] FIG. 41 is a graph showing airway tissue section H&E staining results in the double-humanized IL4/IL4Ra mouse asthma model, wherein airway of the control group (G1) mice had no inflammation while OVA-induced groups (G2, G4) had peribronchial and perivascular inflammation and increased mucus secretion levels. Decreased inflammatory infiltration and mucus secretion were observed in the treatment group (G3) as compared to the OVA-induced groups.

[0181] FIG. 42 shows amino acid sequence alignment result between human IL4 protein and mouse IL4 protein.

[0182] FIG. 43 shows amino acid sequence alignment result between human IL4Ra protein and mouse IL4Ra protein.

[0183] FIG. 44A is a graph showing total number of leukocytes (CD45+ cells) in BALF in the double-humanized IL4/IL4Ra mouse asthma model.

[0184] FIG. 44B is a graph showing total number of eosinophils cells in BALF in the double-humanized IL4/IL4Ra mouse asthma model.

[0185] FIG. 45A is a graph showing serum IgE levels in the double-humanized IL4/IL4Ra mouse asthma model.

[0186] FIG. 45B is a graph showing IgE levels in BALF of the double-humanized IL4/IL4Ra mouse asthma model.

DETAILED DESCRIPTION

[0187] This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) IL4R and/or IL4, and methods of use thereof.

[0188] IL4/IL4R signaling is implicated in many immune diseases including e.g., allergies, autoimmune diseases, asthma, and atopic dermatitis. In the immune system, IL4 controls the development, survival, and maturation of B cells and the proliferation and differentiation of Th2 T lymphocytes. IL4 supports this enhanced proliferation and survival in part by inducing glucose uptake and metabolism. IL4 can also polarize macrophages to the `M2` or alternatively activated phenotype.

[0189] In normal tissues, IL4 receptor .alpha. (IL4Ra) is often expressed on T and B lymphocytes, eosinophils, macrophages, endothelial cells, lung fibroblasts, bronchial epithelial cells, myeloid-derived suppressor cells (MDSCs), and smooth muscle cells. There are two types of IL4 receptors. Each type of receptors has two protein subunits that heterodimerize upon IL4 binding the IL4Ra subunit. The type I receptor is predominantly expressed by hematopoietic cells, and has the IL4Ra and common gamma C (.gamma.c) subunits. The type II receptor can be expressed by non-hematopoetic cells, and has the IL13R.alpha.1 and IL4Ra subunits. Interestingly, while the majority of normal epithelial tissues do not express IL4 receptors, the type II receptor is overexpressed on the surface of many solid tumors including, but not limited to, renal cell carcinoma, melanoma, breast cancer, ovarian cancer, colon cancer, AIDS-related kaposi's sarcoma, and head and neck squamous cell carcinoma, suggesting targeting the IL4/IL4R signaling axis can be a potential anti-tumor therapy (Bankaitis et al. "Targeting IL4/IL4R for the treatment of epithelial cancer metastasis." Clinical & experimental metastasis 32.8 (2015): 847-856).

[0190] Unlike type I receptor, the type II receptor can also bind to IL13. IL13 signaling through the type II receptor on airway epithelial cells can lead to the airway hyper-responsiveness and increased mucus secretion in asthma. It was hypothesized that continued IL13 signaling through the type II receptor may contribute to the limited clinical efficacy of IL4-targeted treatments in asthma. Therefore, blocking the receptor subunit, IL4Ra, to inhibit both IL4- and IL13-induced signaling was an enticing alternative. However, the use of monoclonal antibodies against IL4Ra has yielded mixed results in patients. The humanized anti-IL4Ra antibody, AMG317, showed no clinical efficacy in asthma patients, and development was abandoned after phase II clinical trials. A second monoclonal antibody directed against IL4Ra, Dupilumab, has shown efficacy in the treatment of atopic dermatitis, and is possibly more effective for the treatment of asthma.

[0191] Experimental animal models are an indispensable research tool for studying the effects of these antibodies before clinical trials. Common experimental animals include mice, rats, guinea pigs, hamsters, rabbits, dogs, monkeys, pigs, fish and so on. However, there are differences between human and animal genes and protein sequences, and many human proteins cannot bind to the animal's homologous proteins to produce biological activity, leading to that the results of many clinical trials do not match the results obtained from animal experiments. A large number of clinical studies are in urgent need of better animal models. With the continuous development and maturation of genetic engineering technologies, the use of human cells or genes to replace or substitute an animal's endogenous similar cells or genes to establish a biological system or disease model closer to human, and establish the humanized experimental animal models (humanized animal model) has provided an important tool for new clinical approaches or means. In this context, the genetically engineered animal model, that is, the use of genetic manipulation techniques, the use of human normal or mutant genes to replace animal homologous genes, can be used to establish the genetically modified animal models that are closer to human gene systems. The humanized animal models have various important applications. For example, due to the presence of human or humanized genes, the animals can express or express in part of the proteins with human functions, so as to greatly reduce the differences in clinical trials between humans and animals, and provide the possibility of drug screening at animal levels. Furthermore, because of interaction between human IL4R and human IL4, a desirable animal model for the investigation of anti-IL4R or anti-IL4 antibodies should faithfully mimic the interaction between human IL4R and human IL4, elicit robust responses from both the innate and adaptive immunity, and recapitulate side effects of IL4 blockade in human patients.

[0192] Unless otherwise specified, the practice of the methods described herein can take advantage of the techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology. These techniques are explained in detail in the following literature, for examples: Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gaited., 1984); Mullis et al U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames& S. J. Higginseds. 1984); Transcription And Translation (B. D. Hames& S. J. Higginseds. 1984); Culture Of Animal Cell (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984), the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Hand book Of Experimental Immunology, Volumes V (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986); each of which is incorporated herein by reference in its entirety.

Interleukin 4 (IL4)

[0193] The interleukin 4 (IL4) has a compact, globular fold (similar to other cytokines), stabilized by 3 disulfide bonds. One half of the structure is dominated by a 4 alpha-helix bundle with a left-handed twist. The helices are anti-parallel, with 2 overhand connections, which fall into a 2-stranded anti-parallel beta-sheet.

[0194] IL4 is a cytokine that induces differentiation of naive helper T cells (Th0 cells) to Th2 cells. Upon activation by IL4, Th2 cells subsequently produce additional IL4 in a positive feedback loop. The cell that initially produces IL4, thus inducing Th2 differentiation, has not been identified, but recent studies suggest that basophils may be the effector cell. It is closely related and has functions similar to interleukin 13.

[0195] Tissue macrophages play an important role in chronic inflammation and wound repair. The presence of IL4 in extravascular tissues promotes alternative activation of macrophages into M2 cells and inhibits classical activation of macrophages into M1 cells. An increase in repair macrophages (M2) is coupled with secretion of IL10 and TGF-.beta. that result in a diminution of pathological inflammation. Release of arginase, proline, polyaminase and TGF-.beta. by the activated M2 cell is tied with wound repair and fibrosis.

[0196] A detailed description of IL4 and its function can be found, e.g., in Pillai et al. "Evolution of IL4 and pathogen antagonism." Growth Factors 29.4 (2011): 153-160; Li-Weber et al. "Regulation of IL4 gene expression by T cells and therapeutic perspectives." Nature Reviews Immunology 3.7 (2003): 534; which are incorporated by reference herein in the entirety.

[0197] In human genomes, IL4 gene (Gene ID: 3565) locus has 4 exons, exon 1, exon 2, exon 3, and exon 4. The nucleotide sequence for human IL4 mRNA is NM 000589.3 (SEQ ID NO: 3), and the amino acid sequence for human IL4 is NP 000580.1 (SEQ ID NO: 4). The location for each exon and each region in human IL4 nucleotide sequence and amino acid sequence is listed below:

TABLE-US-00001 TABLE 1 Human IL4 NM_000589.3 NP_000580.1 (approximate 642 bp 153 aa location) (SEQ ID NO: 3) (SEQ ID NO: 4) Exon 1 1-200 1-45 Exon 2 201-248 46-61 Exon 3 249-425 62-120 Exon 4 426-618 121-153 Signal peptide 66-137 1-24 Donor region in 66-527 1-153 Example FIG. 4 Donor region in 1-642 1-153 Example FIG. 8

[0198] In mice, IL4 gene locus has 4 exons, exon 1, exon 2, exon 3, and exon 4. The nucleotide sequence for mouse IL4 mRNA is NM_021283.2 (SEQ ID NO: 1), the amino acid sequence for mouse IL4 is NP 067258.1 (SEQ ID NO: 2). The location for each exon and each region in the mouse IL4 nucleotide sequence and amino acid sequence is listed below:

TABLE-US-00002 TABLE 2 Mouse IL4 NM_021283.2 NP_067258.1 (approximate 605 bp 140 aa location) (SEQ ID NO: 1) (SEQ ID NO: 2) Exon 1 1-191 1-44 Exon 2 192-239 45-60 Exon 3 240-392 61-111 Exon 4 393-586 112-140 Signal peptide 60-119 1-20 Replaced region in 60-482 1-140 Example FIG. 4 Replaced region in 1-605 1-140 Example FIG. 8

[0199] The mouse IL4 gene (Gene ID: 16189) located in Chromosome 11 of the mouse genome, which is located from 53612460 to 53618665, of NC 000077.6 (GRCm38.p4, GCF_000001635.24). The 5'-UTR is from 53618669 to 53618607, exon 1 is from 53618606 to 53618475, the first intron is from 53618474 to 53618218, exon 2 is from 53618217 to 53618170, the second intron is from 53618169 to 53614057, exon 3 is from 53614056 to 53613904, the third intron is from 53613903 to 53612654, exon 4 is from 53612653 to 53612564, the 3'-UTR is from 53612563 to 53612460, based on transcript NM_021283.2. All relevant information for mouse IL4 locus can be found in the NCBI website with Gene ID: 16189, which is incorporated by reference herein in its entirety.

[0200] FIG. 42 shows the alignment between human IL4 amino acid sequence (NP_000580.1; SEQ ID NO: 4) and mouse IL4 amino acid sequence (NP_067258.1; SEQ ID NO: 2). Thus, the corresponding amino acid residue or region between human and mouse IL4 can also be found in FIG. 42.

[0201] IL4 genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for IL4 in Rattus norvegicus is 287287, the gene ID for IL4 in Macaca mulatta (Rhesus monkey) is 574281, the gene ID for IL4 in Canis lupus familiaris (dog) is 403785, and the gene ID for IL4 in Cavia porcellus (domestic guinea pig) is 100720403. The relevant information for these genes (e.g., intron sequences, exon sequences, amino acid residues of these proteins) can be found, e.g., in NCBI database, which are incorporated herein by reference in the entirety.

[0202] The present disclosure provides human or chimeric (e.g., humanized) IL4 nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse signal peptide, exon 1, exon 2, exon 3, and/or exon 4, are replaced by the corresponding human sequence.

[0203] In some embodiments, a "region" or "portion" of mouse signal peptide, exon 1, exon 2, exon 3, and/or exon 4 is replaced by the corresponding human sequence.

[0204] In some embodiments, the "region" or "portion" can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to signal peptide, exon 1, exon 2, exon 3, and/or exon 4. In some embodiments, a region, a portion, or the entire sequence of mouse signal peptide, exon 1, exon 2, exon 3 and/or exon 4 is replaced by a region, a portion, or the entire sequence of human signal peptide, exon 1, exon 2, exon 3, and/or exon 4.

[0205] In some embodiments, a "region" or "portion" of mouse signal peptide, exon 1, exon 2, exon 3, and/or exon 4 is deleted.

[0206] Thus, in some embodiments, the present disclosure also provides a chimeric (e.g., humanized) IL4 nucleotide sequence and/or amino acid sequences, wherein in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the sequence are identical to or derived from mouse IL4 mRNA sequence (e.g., SEQ ID NO: 1), mouse IL4 amino acid sequence (e.g., SEQ ID NO: 2), or a portion thereof (e.g., exon 1, exon 2, exon 3, and/or exon 4); and in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the sequence are identical to or derived from human IL4 mRNA sequence (e.g., SEQ ID NO: 3), human IL4 amino acid sequence (e.g., SEQ ID NO: 4), or a portion thereof (e.g., exon 1, exon 2, exon 3, and/or exon 4).

[0207] In some embodiments, the sequence encoding full-length amino acid sequence of mouse IL4 (SEQ ID NO: 2) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human IL4 (e.g., full-length amino acid sequence of human IL4 (SEQ ID NO: 4)).

[0208] In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse IL4 promotor, a human IL4 promotor, an inducible promoter, a human enhancer, a mouse enhancer, and/or mouse or human regulatory elements.

[0209] In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, or 60 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from a portion of or the entire mouse IL4 nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, or SEQ ID NO: 1).

[0210] In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, or 60 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire mouse IL4 nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, or SEQ ID NO: 1).

[0211] In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from a portion of or the entire human IL4 nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, or SEQ ID NO: 3).

[0212] In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire human IL4 nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, or SEQ ID NO: 3).

[0213] In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire mouse IL4 amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, SEQ ID NO: 2).

[0214] In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire mouse IL4 amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, SEQ ID NO: 2).

[0215] In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire human IL4 amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, or SEQ ID NO: 4).

[0216] In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire human IL4 amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, or SEQ ID NO: 4).

[0217] In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 2 or 4 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

[0218] Cells, tissues, and animals (e.g., mouse) are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express human or chimeric (e.g., humanized) IL4 from an endogenous non-human IL4 locus.

[0219] In one aspect, the disclosure provides a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric IL4.

[0220] In some embodiments, the sequence encoding the human or chimeric IL4 is operably linked to an endogenous regulatory element, or a human regulatory element at the endogenous IL4 gene locus in the at least one chromosome.

[0221] In some embodiments, the sequence encoding a human or chimeric IL4 comprises a sequence encoding an amino acid sequence that is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human IL4 (SEQ ID NO: 4).

[0222] In some embodiments, the animal is a mammal, e.g., a monkey, a rodent or a mouse. In some embodiments, the animal is a BALB/c mouse or a C57BL/6 mouse.

[0223] In some embodiments, the animal does not express endogenous IL4. In some embodiments, the animal has one or more cells expressing human or chimeric IL4.

[0224] In some embodiments, the animal has one or more cells expressing human or chimeric IL4, and the expressed human or chimeric IL4 can bind to endogenous IL4R. In some embodiments, the animal has one or more cells expressing human or chimeric IL4, and the expressed human or chimeric IL4 cannot bind to endogenous IL4R.

[0225] In another aspect, the disclosure is related to a genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous IL4 with a sequence encoding a corresponding region of human IL4 at an endogenous IL4 gene locus.

[0226] In some embodiments, the sequence encoding the corresponding region of human IL4 is operably linked to an endogenous regulatory element, or a human regulatory element at the endogenous IL4 locus, and one or more cells of the animal expresses a chimeric IL4.

[0227] In some embodiments, the animal is a mouse, and the replaced endogenous IL4 locus is exon 1, exon 2, exon 3, and/or exon 4 of the endogenous mouse IL4 gene.

[0228] In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous IL4 gene locus. In some embodiments, the animal is homozygous with respect to the replacement at the endogenous IL4 gene locus.

[0229] In another aspect, the disclosure is related to methods for making a genetically-modified, non-human animal. The methods involve replacing in at least one cell of the animal, at an endogenous IL4 gene locus, a sequence encoding a region of an endogenous IL4 with a sequence encoding a corresponding region of human IL4.

[0230] In some embodiments, the sequence encoding the corresponding region of human IL4 comprises exon 1, exon 2, exon 3 and/or exon 4 of a human IL4 gene.

[0231] In some embodiments, the sequence encoding the corresponding region of IL4 comprises at least 50, 75, 100, 125, 150, 175, or 200 nucleotides of exon 1, exon 2, exon 3 and/or exon 4 of a human IL4 gene.

[0232] In some embodiments, the sequence encoding the corresponding region of human IL4 encodes a sequence that is at least 90% identical to full-length amino acid sequence of SEQ ID NO: 4.

[0233] In some embodiments, the animal is a mouse, and the locus is exon 1, exon 2, exon 3, and/or exon 4 of the mouse IL4 gene.

[0234] In another aspect, the disclosure is also related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL4 polypeptide, wherein the chimeric IL4 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4, wherein the animal expresses the chimeric IL4.

[0235] In some embodiments, the chimeric IL4 polypeptide comprises a sequence that is at least 90%, 95%, or 99% identical to full-length amino acid sequence of SEQ ID NO: 4.

[0236] In some embodiments, the nucleotide sequence is operably linked to an endogenous IL4 regulatory element of the animal, a human IL4 regulatory element, a mouse 5'-UTR, a mouse 3'-UTR, a human 5'-UTR, or a human 3'-UTR.

[0237] In some embodiments, the nucleotide sequence is integrated to an endogenous IL4 gene locus of the animal.

[0238] In some embodiments, the chimeric IL4 has at least one mouse IL4 activity and/or at least one human IL4 activity.

[0239] In another aspect, the disclosure is also related to methods of making a genetically-modified mouse cell that expresses a chimeric IL4. The methods involve replacing, at an endogenous mouse IL4 gene locus, a nucleotide sequence encoding a region of mouse IL4 with a nucleotide sequence encoding a corresponding region of human IL4, thereby generating a genetically-modified mouse cell that includes a nucleotide sequence that encodes the chimeric IL4, wherein the mouse cell expresses the chimeric IL4.

[0240] In some embodiments, the nucleotide sequence encoding the chimeric IL4 is operably linked to an endogenous regulatory region, or a human IL4 regulatory region, e.g., promoter.

[0241] In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4R, Interleukin 33 (IL33), Interleukin 13 (IL13), programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte Activating 3 (LAG-3), B And T Lymphocyte Associated (BTLA), Programmed Cell Death 1 Ligand 1 (PD-L1), CD27, CD28, T-Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), Glucocorticoid-Induced TNFR-Related Protein (GITR), CD137, TNF Receptor Superfamily Member 4 (OX40), CD47, or Signal Regulatory Protein alpha (SIRPa)).

[0242] In some embodiments, the additional human or chimeric protein is IL4R.

[0243] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4 antagonist (e.g., an anti-IL4 antibody) for reducing inflammation. The methods involve administering the IL4 antagonist to the animal described herein, wherein the animal has an inflammation; and determining the inhibitory effects of the IL4 antagonist to the reduction of inflammation.

[0244] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4 antagonist (e.g., an anti-IL4 antibody) for treating autoimmune disorder or allergy. The methods involve administering the IL4 antagonist to the animal described herein, wherein the animal has an autoimmune disorder or allergy; and determining the inhibitory effects of the IL4 antagonist to the treatment of autoimmune disorder or allergy.

[0245] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4 antagonist (e.g., an anti-IL4 antibody) for treating cancer. The methods involve administering the IL4 antagonist to the animal as described herein, wherein the animal has a tumor; and determining the inhibitory effects of the IL4 antagonist to the tumor.

[0246] In some embodiments, the animal further comprises a sequence encoding a human or chimeric IL4R. In some embodiments, the additional therapeutic agent is an anti-IL4R antibody.

[0247] In some embodiments the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-CD20 antibody, an anti-EGFR antibody, or an anti-CD319 antibody.

[0248] In another aspect, the disclosure further provides methods of determining toxicity of an agent (e.g., an IL4 antagonist). The methods involve administering the agent to the animal as described herein; and determining weight change of the animal. In some embodiments, the method further involve performing a blood test (e.g., determining red blood cell count).

[0249] In one aspect, the disclosure relates to proteins comprising an amino acid sequence, wherein the amino acid sequence is one of the following:

[0250] (a) an amino acid sequence set forth in SEQ ID NO: 4;

[0251] (b) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4;

[0252] (c) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 4 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

[0253] (d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 4.

[0254] In some embodiments, provided herein are cells comprising the proteins disclosed herein. In some embodiments, provided herein are animals having the proteins disclosed herein.

[0255] In another aspect, the disclosure relates to nucleic acids comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following:

[0256] (a) a sequence that encodes the protein as described herein;

[0257] (b) SEQ ID NO: 3;

[0258] (c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, 8, 9, 10, 11, 23, 24, or 25;

[0259] In some embodiments, provided herein are cells comprising the nucleic acids disclosed herein. In some embodiments, provided herein are animals having the nucleic acids disclosed herein.

[0260] In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal's endogenous IL4 gene, wherein the disruption of the endogenous IL4 gene comprises deletion of exon1, exon2, exon 3 and/or exon 4 or part thereof of the endogenous IL4 gene.

[0261] In some embodiments, the disruption of the endogenous IL4 gene further comprises deletion of one or more exons or part of exons selected from the group consisting of exon 1, exon 2, exon 3 and/or exon 4 of the endogenous IL4 gene.

[0262] In some embodiments, the disruption of the endogenous IL4 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, and/or intron 3 of the endogenous IL4 gene.

[0263] In some embodiments, wherein the deletion can comprise deleting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500 or more nucleotides.

[0264] In some embodiments, the disruption of the endogenous IL4 gene comprises the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides of exon 1, exon 2, exon 3, and/or exon 4 (e.g., deletion of the entire exon 1, exon 2, exon 3, and exon 4).

Interleukin 4 Receptor (IL4R)

[0265] The interleukin 4 receptor (IL4R), also named as interleukin 4 receptor subunit alpha, or interleukin 4 receptor .alpha. (IL4Ra), is a type I cytokine receptor. It is encoded by IL4Ra gene. The N-terminal (extracellular) portion of interleukin-4 receptor is related in overall topology to fibronectin type III modules and folds into a sandwich comprising seven antiparallel beta sheets arranged in a three-strand and a four-strand beta-pleated sheet. They are required for binding of IL4 to the receptor alpha chain, which is a crucial event for the generation of a Th2-dominated early immune response.

[0266] The IL4Ra gene encodes the alpha chain of the IL4 receptor. The IL4 receptor is a type I transmembrane protein that can bind IL4 and IL13 to regulate IgE antibody production in B cells. Among T cells, the encoded protein also can bind IL4 to promote differentiation of Th2 cells. A soluble form of the encoded protein can be produced by an alternate splice variant or by proteolysis of the membrane-bound protein, and this soluble form can inhibit IL4-mediated cell proliferation and IL5 upregulation by T-cells. Allelic variations in this gene have been associated with atopy, a condition that can manifest itself as allergic rhinitis, sinusitis, asthma, or eczema. Two transcript variants encoding different isoforms, a membrane-bound and a soluble form, have been found for this gene. Interactions of IL4 with TNF.alpha. promote structural changes to vascular endothelial cells, thus playing an important role in tissue inflammation.

[0267] The binding of IL4 or IL13 to the IL4 receptor on the surface of macrophages results in the alternative activation of those macrophages. Alternatively activated macrophages (AAM.PHI.) downregulate inflammatory mediators such as IFN.gamma. during immune responses, particularly with regards to helminth infections.

[0268] A detailed description of IL4R and its function can be found, e.g., in Bankaitis et al. "Targeting IL4/IL4R for the treatment of epithelial cancer metastasis." Clinical & experimental metastasis 32.8 (2015): 847-856; Zhang et al. "Association of IL4 and IL4R polymorphisms with multiple sclerosis susceptibility in Caucasian population: a meta-analysis." Journal of the neurological sciences 363 (2016): 107-113; which are incorporated by reference herein in the entirety.

[0269] In human genomes, IL4Ra gene (Gene ID: 3566) locus has 11 exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and exon 11. The IL4R protein has an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for human IL4Ra mRNA is NM_000418.3 (SEQ ID NO: 41), and the amino acid sequence for human IL4R is NP_000409.1 (SEQ ID NO: 42). The location for each exon and each region in human IL4Ra nucleotide sequence and amino acid sequence is listed below:

TABLE-US-00003 TABLE 3 Human IL4R NM_000418.3 NP_000409.1 (approximate 3710 bp 825 aa location) (SEQ ID NO: 41) (SEQ ID NO: 42) Exon 1 1-112 Non-coding sequence Exon 2 48-245 Non-coding sequence Exon 3 224-333 1-23 Exon 4 312-472 24-70 Exon 5 454-624 71-121 Exon 6 606-776 122-171 Exon 7 758-933 172-223 Exon 8 915-1033 224-257 Exon 9 1015-1112 258-283 Exon 10 1094-1162 284-300 Exon 11 1144-3689 301-825 Signal peptide 264-338 1-25 Extracellular 339-959 26-232 Transmembrane 960-1031 233-256 Cytoplasmic 1032-2738 257-825 Donor Range 351-911 30-216

[0270] In mice, IL4Ra gene locus has 11 exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and exon 11 (FIG. 15). The mouse IL4R protein also has an extracellular region, a transmembrane region, and a cytoplasmic region. The nucleotide sequence for mouse IL4Ra mRNA is NM_001008700.3 (SEQ ID NO: 39), the amino acid sequence for mouse IL4R is NP_001008700.1 (SEQ ID NO: 40). The location for each exon and each region in the mouse IL4Ra nucleotide sequence and amino acid sequence is listed below:

TABLE-US-00004 TABLE 4 Mouse IL4Ra NM_001008700.3 NP_001008700.1 (approximate 5122 bp 810 aa location) (SEQ ID NO: 39) (SEQ ID NO: 40) Exon 1 1-47 Non-coding sequence Exon 2 48-223 Non-coding sequence Exon 3 224-311 1-23 Exon 4 312-453 24-71 Exon 5 454-605 72-121 Exon 6 606-757 122-172 Exon 7 758-914 173-224 Exon 8 915-1014 225-258 Exon 9 1015-1093 259-284 Exon 10 1094-1143 285-301 Exon 11 1144-5094 302-810 Signal peptide 242-316 1-25 Extracellular 317-940 26-233 Transmembrane 941-1012 234-257 Cytoplasmic 1013-2671 258-810 Replaced region 329-892 30-217 in Example

[0271] The mouse IL4Ra gene (Gene ID: 16190) located in Chromosome 7 of the mouse genome, which is located from 125552282 to 125579474, of NC 000073.6 (GRCm38.p4, GCF_000001635.24). The 5'-UTR is from 125,552,120 to 125,552,328, 125,564,552 to 125,564,727 and, 125,565,637 to 125,565,654, exon 1 is from 125,552,120 to 125,552,328, the first intron is from 125,552,329 to 125,564,551, exon 2 is from 125,564,552 to 125,564,727, the second intron is from 125,564,728 to 125,565,636, exon 3 is from 125,565,637 to 125,565,724, the third intron is from 125,565,725 to 125,567,155, exon 4 is from 125,567,156 to 125,567,297, the fourth intron is from 125,567,298 to 125,569,022, exon 5 is from 125,569,023 to 125,569,174, the fifth intron is from 125,569,175 to 125,569,941, exon 6 is from 125,569,942 to 125,570,093, the sixth intron is from 125,570,094 to 125,571,433, exon 7 is from 125,571,434 to 125,571,590, the seventh intron is from 125,571,591 to 125,572,850, exon 8 is from 125,572,851 to 125,572,950, the eighth intron is from 125,572,951 to 125,574,637, exon 9 is from 125,574,638 to 125,574,716, the ninth intron is from 125,574,717 to 125,575,139, exon 10 is from 125,575,140 to 125,575,189, the ten intron is from 125,575,190 to 125,575,523, exon 11 is from 125,575,524 to 125,579,474, the 3'-UTR is from 125577055 to 125,579,474, based on transcript NM_001008700.3. All relevant information for mouse IL4Ra locus can be found in the NCBI website with Gene ID: 16190, which is incorporated by reference herein in its entirety.

[0272] FIG. 43 shows the alignment between human IL4R amino acid sequence (NP_000409.1; SEQ ID NO: 42) and mouse IL4R amino acid sequence (NP_001008700.1; SEQ ID NO: 40). Thus, the corresponding amino acid residue or region between human and mouse IL4R can also be found in FIG. 43.

[0273] IL4Ra genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for IL4Ra in Rattus norvegicus is 25084, the gene ID for IL4Ra in Macaca mulatta (Rhesus monkey) is 705404, the gene ID for IL4Ra in Canis lupus familiaris (dog) is 489957, and the gene ID for IL4Ra in Sus scrofa (pig) is 397614. The relevant information for these genes (e.g., intron sequences, exon sequences, amino acid residues of these proteins) can be found, e.g., in NCBI database.

[0274] The present disclosure provides human or chimeric (e.g., humanized) IL4Ra nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, the signal peptide, the extracellular region, the transmembrane region, and/or the cytoplasmic region are replaced by the corresponding human sequence.

[0275] In some embodiments, a "region" or "portion" of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, signal peptide, the extracellular region, the transmembrane region, and/or the cytoplasmic region is replaced by the corresponding human sequence. The term "region" or "portion" can refer to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, or 400 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 150 amino acid residues.

[0276] In some embodiments, the "region" or "portion" can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, signal peptide, the extracellular region, the transmembrane region, and/or the cytoplasmic region. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and/or exon 11 (e.g., exon 4, exon 5, exon 6, and exon 7) is replaced by a region, a portion, or the entire sequence of human exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and/or exon 11 (e.g., exon 4, exon 5, exon 6, and exon 7).

[0277] In some embodiments, a "region" or "portion" of mouse exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, signal peptide, the extracellular region, the transmembrane region, and/or the cytoplasmic region is deleted. For example, a region or a portion of exon 4, exon 5, exon 6, and exon 7 is deleted.

[0278] Thus, in some embodiments, the present disclosure also provides a chimeric (e.g., humanized) IL4Ra nucleotide sequence and/or amino acid sequences, wherein in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the sequence are identical to or derived from mouse IL4Ra mRNA sequence (e.g., SEQ ID NO: 39), mouse IL4R amino acid sequence (e.g., SEQ ID NO: 40), or a portion thereof (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and/or exon 11). In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the sequence are identical to or derived from human IL4Ra mRNA sequence (e.g., SEQ ID NO: 41), human IL4R amino acid sequence (e.g., SEQ ID NO: 42), or a portion thereof (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, and/or exon 11).

[0279] In some embodiments, the sequence encoding amino acids 30-217 of mouse IL4R (SEQ ID NO: 40) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human IL4R (e.g., amino acids 30-216 of human IL4R (SEQ ID NO: 42).

[0280] In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse IL4R promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

[0281] In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from a portion of or the entire mouse IL4Ra nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, or SEQ ID NO: 39).

[0282] In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire mouse IL4Ra nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, or SEQ ID NO: 39).

[0283] In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from a portion of or the entire human IL4Ra nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, or SEQ ID NO: 41).

[0284] In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire human IL4Ra nucleotide sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, or SEQ ID NO: 41).

[0285] In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire mouse IL4R amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, or NP_001008700.1 (SEQ ID NO: 40)).

[0286] In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire mouse IL4R amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, or NP_001008700.1 (SEQ ID NO: 40)).

[0287] In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire human IL4R amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, or SEQ ID NO: 42).

[0288] In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire human IL4R amino acid sequence (e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, or SEQ ID NO: 42).

[0289] The present disclosure also provides a humanized IL4R mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

[0290] a) an amino acid sequence shown in SEQ ID NO: 44;

[0291] b) an amino acid sequence having a homology of at least 90% with or at least 90% identical to the amino acid sequence shown in SEQ ID NO: 44;

[0292] c) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 44 under a low stringency condition or a strict stringency condition;

[0293] d) an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 44;

[0294] e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

[0295] f) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 44.

[0296] The present disclosure also relates to a nucleic acid (e.g., DNA or RNA) sequence, wherein the nucleic acid sequence can be selected from the group consisting of:

[0297] a) a nucleic acid sequence as shown in SEQ ID NO: 39, 41, or 43, or a nucleic acid sequence encoding a homologous IL4R amino acid sequence of a humanized mouse;

[0298] b) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 39, 41, or 43 under a low stringency condition or a strict stringency condition;

[0299] c) a nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence as shown in SEQ ID NO: 39, 41, 43, 48, 49, 50, or 51;

[0300] d) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90% with or at least 90% identical to the amino acid sequence shown in SEQ ID NO: 40, 42, or 44;

[0301] e) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 40, 42, or 44;

[0302] f) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 40, 42, or 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

[0303] g) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 40, 42, or 44.

[0304] The present disclosure further relates to an IL4R genomic DNA sequence of a humanized mouse. The DNA sequence is obtained by a reverse transcription of the mRNA obtained by transcription thereof is consistent with or complementary to the DNA sequence homologous to the sequence shown in SEQ ID NO: 39, 41, or 43.

[0305] The disclosure also provides an amino acid sequence that has a homology of at least 90% with, or at least 90% identical to the sequence shown in SEQ ID NO: 40, 42, or 44, and has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 40, 42, or 44 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

[0306] In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 40, 42, or 44 is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

[0307] The disclosure also provides a nucleotide sequence that has a homology of at least 90%, or at least 90% identical to the sequence shown in SEQ ID NO: 39, 41, or 43, and encodes a polypeptide that has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 39, 41, or 43 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

[0308] Cells, tissues, and animals (e.g., mouse) are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express human or chimeric (e.g., humanized) IL4R from an endogenous non-human IL4R locus.

[0309] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4R antagonist (e.g., an anti-IL4R antibody) for reducing inflammation. The methods involve administering the IL4R antagonist to the animal described herein, wherein the animal has an inflammation; and determining the inhibitory effects of the IL4R antagonist to the reduction of inflammation.

[0310] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4R antagonist (e.g., an anti-IL4R antibody) for treating autoimmune disorder or allergy. The methods involve administering the IL4R antagonist to the animal described herein, wherein the animal has an autoimmune disorder or allergy; and determining the inhibitory effects of the IL4R antagonist to the treatment of autoimmune disorder or allergy.

[0311] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4R antagonist (e.g., an anti-IL4R antibody) for treating cancer. The methods involve administering the IL4R antagonist to the animal described herein, wherein the animal has a tumor; and determining the inhibitory effects of the IL4R antagonist to the tumor. In some embodiments, the tumor comprises one or more cancer cells that are injected into the animal. In some embodiments, determining the inhibitory effects of the IL4R antagonist (e.g., an anti-IL4R antibody) to the tumor involves measuring the tumor volume in the animal.

[0312] In another aspect, the disclosure also provides a genetically-modified, non-human animal whose genome comprise a disruption in the animal's endogenous IL4R gene, wherein the disruption of the endogenous IL4R gene comprises deletion of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11 (e.g., exon 4, exon 5, exon 6 and/or exon 7) or part thereof of the endogenous IL4R gene.

[0313] In some embodiments, the disruption of the endogenous IL4R gene comprises deletion of one or more exons or part of exons selected from the group consisting of exon 4, exon 5, exon 6 and/or exon 7 of the endogenous IL4R gene.

[0314] In some embodiments, the disruption of the endogenous IL4R gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 4, intron 5, and/or intron 6 of the endogenous IL4R gene.

[0315] In some embodiments, wherein the deletion can comprise deleting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 10, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500 or more nucleotides.

[0316] In some embodiments, the disruption of the endogenous IL4R gene comprises the deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11 (e.g., exon 4, exon 5, exon 6 and/or exon 7)

Genetically Modified Animals

[0317] As used herein, the term "genetically-modified non-human animal" refers to a non-human animal having genetic modification (e.g., exogenous DNA) in at least one chromosome of the animal's genome. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50% of cells of the genetically-modified non-human animal have the genetic modification in its genome. The cell having exogenous DNA can be various kinds of cells, e.g., an endogenous cell, a somatic cell, an immune cell, a T cell, a B cell, a germ cell, a blastocyst, or an endogenous tumor cell. In some embodiments, genetically-modified non-human animals are provided that comprise a modified endogenous IL4R or IL4 locus that comprises an exogenous sequence (e.g., a human sequence), e.g., a replacement of one or more non-human sequences with one or more human sequences. The animals are generally able to pass the modification to progeny, i.e., through germline transmission.

[0318] As used herein, the term "chimeric gene" or "chimeric nucleic acid" refers to a gene or a nucleic acid, wherein two or more portions of the gene or the nucleic acid are from different species, or at least one of the sequences of the gene or the nucleic acid does not correspond to the wild-type nucleic acid in the animal. In some embodiments, the chimeric gene or chimeric nucleic acid has at least one portion of the sequence that is derived from two or more different sources, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) protein of two or more different species. In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized gene or humanized nucleic acid.

[0319] As used herein, the term "chimeric protein" or "chimeric polypeptide" refers to a protein or a polypeptide, wherein two or more portions of the protein or the polypeptide are from different species, or at least one portion of the sequences of the protein or the polypeptide does not correspond to wild-type amino acid sequence in the animal. In some embodiments, the chimeric protein or the chimeric polypeptide has at least one portion of the sequence that is derived from two or more different sources, e.g., same (or homologous) proteins of different species. In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized protein or a humanized polypeptide.

[0320] In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized IL4R gene or a humanized IL4R nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human IL4R gene, at least one or more portions of the gene or the nucleic acid is from a non-human IL4R gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an IL4R protein. The encoded IL4R protein is functional or has at least one activity of the human IL4R protein or the non-human IL4R protein, e.g., binding to human or non-human IL4, and/or upregulating immune response.

[0321] In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized IL4R protein or a humanized IL4R polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a human IL4R protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human IL4R protein. The humanized IL4R protein or the humanized IL4R polypeptide is functional or has at least one activity of the human IL4R protein or the non-human IL4R protein.

[0322] In some embodiments, the humanized IL4R protein or the humanized IL4R polypeptide can bind to mouse IL4, and/or upregulate immune response. In some embodiments, the humanized IL4R protein or the humanized IL4R polypeptide cannot bind to mouse IL4, thus cannot upregulate immune response.

[0323] In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized IL4 gene or a humanized IL4 nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human IL4 gene, at least one or more portions of the gene or the nucleic acid is from a non-human IL4 gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes an IL4 protein. The encoded IL4 protein is functional or has at least one activity of the human IL4 protein or the non-human IL4 protein, e.g., binding to human or non-human IL4R, and/or upregulating immune response.

[0324] In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized IL4 protein or a humanized IL4 polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a human IL4 protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human IL4 protein. The humanized IL4 protein or the humanized IL4 polypeptide is functional or has at least one activity of the human IL4 protein or the non-human IL4 protein.

[0325] In some embodiments, the humanized IL4 protein or the humanized IL4 polypeptide can bind to mouse IL4R, and/or upregulate immune response. In some embodiments, the humanized IL4 protein or the humanized IL4 polypeptide cannot bind to mouse IL4R, thus cannot upregulate immune response.

[0326] The genetically modified non-human animal can be various animals, e.g., a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). For the non-human animals where suitable genetically modifiable embryonic stem (ES) cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. These methods are known in the art, and are described, e.g., in A. Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)," Cold Spring Harbor Laboratory Press, 2003, which is incorporated by reference herein in its entirety.

[0327] In one aspect, the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats, malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In some embodiments, the genetically modified rodent is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a crested rat. In some embodiments, the non-human animal is a mouse.

[0328] In some embodiments, the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/01a. In some embodiments, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 12951/SV, 12951/SvIm), 129S2, 129S4, 129S5, 12959/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000), both of which are incorporated herein by reference in the entirety. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains. In some embodiments, the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50% BALB/c-50% 12954/Sv; or 50% C57BL/6-50% 129).

[0329] In some embodiments, the animal is a rat. The rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti. The animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the humanized IL4R or IL4 animal is made. For example, suitable mice for maintaining a xenograft, can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part. Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin), physical means (e.g., irradiating the animal), and/or genetic modification (e.g., knocking out one or more genes). Non-limiting examples of such mice include, e.g., NOD mice, SCID mice, NOD/SCID mice, IL2Ry knockout mice, NOD/SCID/.gamma.cnull mice (Ito, M. et al., NOD/SCID/.gamma.cnull mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100(9): 3175-3182, 2002), nude mice, and Rag1 and/or Rag2 knockout mice. These mice can optionally be irradiated, or otherwise treated to destroy one or more immune cell type. Thus, in various embodiments, a genetically modified mouse is provided that can include a humanization of at least a portion of an endogenous non-human IL4R or IL4 locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD mice, SCID mice, NOD/SCID mice, IL-2Ry knockout mice, NOD/SOD/ye null mice, nude mice, Rag1 and/or Rag2 knockout mice, and a combination thereof. These genetically modified animals are described, e.g., in US20150106961, which is incorporated herein by reference in its entirety. In some embodiments, the mouse can include a replacement of all or part of mature IL4R or IL4 coding sequence with human mature IL4R or IL4 coding sequence.

[0330] Genetically modified non-human animals can comprise a modification of an endogenous non-human IL4 or IL4R locus. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a mature IL4 or IL4R protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the mature IL4 or IL4R protein sequence). Although genetically modified cells are also provided that can comprise the modifications described herein (e.g., ES cells, somatic cells), in many embodiments, the genetically modified non-human animals comprise the modification of the endogenous IL4 or IL4R locus in the germline of the animal.

[0331] Genetically modified animals can express a human IL4 or IL4R (or a chimeric IL4 or IL4R) from endogenous mouse loci, wherein the endogenous mouse gene has been replaced with a human gene and/or a nucleotide sequence that encodes a region of human IL4 or IL4R sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human IL4 or IL4R sequence. In various embodiments, an endogenous non-human locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a mature protein.

[0332] In some embodiments, the genetically modified mice express the human IL4 or IL4R (or chimeric IL4 or IL4R) from endogenous loci that are under control of mouse promoters and/or mouse regulatory elements. The replacement(s) at the endogenous mouse loci provide non-human animals that express human protein or chimeric protein in appropriate cell types and in a manner that does not result in the potential pathologies observed in some other transgenic mice known in the art. The human protein or the chimeric protein expressed in animal can maintain one or more functions of the wild-type mouse or human protein in the animal. For example, IL4R can bind to human or non-human IL4, and upregulate immune response, e.g., upregulate immune response by at least 10%, 20%, 30%, 40%, or 50%. As used herein, the term "endogenous IL4R" refers to IL4R protein that is expressed from an endogenous IL4R nucleotide sequence of the non-human animal (e.g., mouse) before any genetic modification. Similarly, the term "endogenous IL4" refers to IL4 protein that is expressed from an endogenous IL4 nucleotide sequence of the non-human animal (e.g., mouse) before any genetic modification.

[0333] The genome of the animal can comprise a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 40, 42, or 44, and/or a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 4.

[0334] The genome of the genetically modified animal can comprise a replacement at an endogenous IL4R gene locus of a sequence encoding a region of endogenous IL4R with a sequence encoding a corresponding region of human IL4R. In some embodiments, the sequence that is replaced is any sequence within the endogenous IL4R gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11 5'-UTR, 3'UTR, the first intron, the second intron, and the third intron, the fourth intron, the fifth intron, the sixth intron, the seventh intron, the eighth intron, the ninth intron, the tenth intron or the eleventh intron, etc. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous IL4R gene. In some embodiments, the sequence that is replaced is exon 4, exon 5, exon 6 and/or exon 7 or part thereof, of an endogenous mouse IL4R gene locus.

[0335] The genetically modified animal can have one or more cells expressing a human or chimeric IL4R (e.g., humanized IL4R) having an extracellular region and a cytoplasmic region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% identical to the extracellular region of human IL4R. In some embodiments, the extracellular region of the humanized IL4R has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 amino acids (e.g., contiguously or non-contiguously) that are identical to human IL4R.

[0336] The genome of the genetically modified animal can comprise a replacement at an endogenous IL4 gene locus of a sequence encoding a region of endogenous IL4 with a sequence encoding a corresponding region of human IL4. In some embodiments, the sequence that is replaced is any sequence within the endogenous IL4 gene locus, e.g., exon 1, exon 2, exon 3, exon 4, 5'-UTR, 3'UTR, the first intron, the second intron, or the third intron, etc. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous IL4 gene. In some embodiments, the sequence that is replaced is within the regulatory region of the human IL4 gene.

[0337] Because human protein and non-human protein sequences, in many cases, are different, antibodies that bind to human protein will not necessarily have the same binding affinity with non-human protein or have the same effects to non-human protein. Therefore, the genetically modified animal expressing human IL4 and the genetically modified animal having a human or a humanized extracellular region of IL4R can be used to better evaluate the effects of anti-IL4 or IL4R antibodies in an animal model. In some embodiments, the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to part or the entire sequence of exon 4, exon 5, exon 6 and/or exon 7 of human IL4R, part or the entire sequence of the extracellular region of human IL4R (with or without signal peptide), or part or the entire sequence of amino acids 30-216 of SEQ ID NO: 42.

[0338] In some embodiments, the non-human animal can have, at an endogenous IL4R gene locus, a nucleotide sequence encoding a chimeric human/non-human IL4R polypeptide, wherein a human portion of the chimeric human/non-human IL4R polypeptide comprises a portion of human IL4R extracellular region, and wherein the animal expresses a functional IL4R on a surface of a cell of the animal. The human portion of the chimeric human/non-human IL4R polypeptide can comprise a portion of exon 4, exon 5, exon 6 and/or exon 7 of human IL4R. In some embodiments, the human portion of the chimeric human/non-human IL4R polypeptide can comprise a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to amino acids 30-216 of SEQ ID NO: 42.

[0339] In some embodiments, the non-human portion of the chimeric human/non-human IL4R polypeptide comprises the transmembrane region, and/or the cytoplasmic region of an endogenous non-human IL4R polypeptide. There may be several advantages that are associated with the transmembrane and/or cytoplasmic regions of an endogenous non-human IL4R polypeptide. For example, once IL4 binds to IL4R, they can properly transmit extracellular signals into the cells and regulate the downstream pathway. A human or humanized transmembrane and/or cytoplasmic regions may not function properly in non-human animal cells. In some embodiments, a few extracellular amino acids that are close to the transmembrane region of IL4R are also derived from endogenous sequence.

[0340] In some embodiments, the humanized IL4R locus lacks a human IL4R 5'-UTR. In some embodiment, the humanized IL4R locus comprises a rodent (e.g., mouse) 5'-UTR. In some embodiments, the humanization comprises a human 3'-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human IL4R genes appear to be similarly regulated based on the similarity of their 5'-flanking sequence. As shown in the present disclosure, humanized IL4R mice that comprise a replacement at an endogenous mouse IL4R locus, which retain mouse regulatory elements but comprise a humanization of IL4R encoding sequence, do not exhibit obvious pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized IL4R are grossly normal.

[0341] In some embodiments, the humanized IL4 locus has a human IL4 5'-UTR or an endogenous IL4 5'-UTR. In some embodiment, the humanized IL4 locus comprises a rodent (e.g., mouse) 5'-UTR. In some embodiments, the humanization comprises a human 3'-UTR or an endogenous 3'-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human IL4 genes appear to be similarly regulated based on the similarity of their 5'-flanking sequence. As shown in the present disclosure, humanized IL4 mice that comprise a replacement at an endogenous mouse IL4 locus, which has mouse or human regulatory elements, do not exhibit obvious pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized IL4 are grossly normal.

[0342] The present disclosure further relates to a non-human mammal generated through the method mentioned above. In some embodiments, the genome thereof contains human gene(s). In some embodiments, the non-human mammal is a rodent, and preferably, the non-human mammal is a mouse.

[0343] In some embodiments, the non-human mammal expresses a protein encoded by a humanized IL4R or IL4 gene.

[0344] In addition, the present disclosure also relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein. In some embodiments, the non-human mammal is a rodent (e.g., a mouse).

[0345] The present disclosure further relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; and the tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.

[0346] The present disclosure also provides non-human mammals produced by any of the methods described herein. In some embodiments, a non-human mammal is provided; and the genetically modified animal contains the DNA encoding human or humanized IL4R or IL4 in the genome of the animal.

[0347] In some embodiments, the non-human mammal comprises the genetic construct as described herein. In some embodiments, a non-human mammal expressing human or humanized IL4R or IL4 is provided. In some embodiments, the tissue-specific expression of human or humanized IL4R or IL4 protein is provided.

[0348] In some embodiments, the expression of human or humanized IL4R or IL4 in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance.

[0349] Non-human mammals can be any non-human animal known in the art and which can be used in the methods as described herein. Preferred non-human mammals are mammals, (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

[0350] Genetic, molecular and behavioral analyses for the non-human mammals described above can performed. The present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes.

[0351] The present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof. A model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cell can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques. The integration of genetic constructs containing DNA sequences encoding human IL4R or IL4 protein can be detected by a variety of methods.

[0352] There are many analytical methods that can be used to detect exogenous DNA, including methods at the level of nucleic acid (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies). In addition, the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art. Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human or humanized IL4R or IL4 protein.

[0353] The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence as described herein. In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

[0354] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

[0355] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

[0356] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0357] The percentage of residues conserved with similar physicochemical properties (percent homology), e.g. leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The homology percentage, in many cases, is higher than the identity percentage.

[0358] Cells, tissues, and animals (e.g., mouse) are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express human or chimeric (e.g., humanized) amino acid sequence from an endogenous non-human IL4R or IL4 locus.

Vectors

[0359] The present disclosure relates to a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of a region to be altered (5' arm), which is selected from the IL4Ra or IL4 gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3' end of the region to be altered (3' arm), which is selected from the IL4Ra or IL4 gene genomic DNAs in the length of 100 to 10,000 nucleotides.

[0360] In some embodiments, a) the DNA fragment homologous to the 5' end of a conversion region to be altered (5' arm) is selected from the nucleotide sequences that have at least 90% homology to the NCBI accession number NC_000077.6; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotide sequences that have at least 90% homology to the NCBI accession number NC_000077.6.

[0361] In some embodiments, a) the DNA fragment homologous to the 5' end of a region to be altered (5' arm) is selected from the nucleotides from the position 53622826 to the position 53618607 of the NCBI accession number NC_000077.6; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotides from the position 53612065 to the position 53607981 of the NCBI accession number NC_000077.6.

[0362] In some embodiments, a) the DNA fragment homologous to the 5' end of a region to be altered (5' arm) is selected from the nucleotides from the position 53625623 to the position 53620071 of the NCBI accession number NC_000077.6; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotides from the position 53612200 to the position 53607005 of the NCBI accession number NC_000077.6.

[0363] In some embodiments, a) the DNA fragment homologous to the 5' end of a conversion region to be altered (5' arm) is selected from the nucleotide sequences that have at least 90% homology to the NCBI accession number NC_000073.6; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotide sequences that have at least 90% homology to the NCBI accession number NC_000073.6.

[0364] In some embodiments, a) the DNA fragment homologous to the 5' end of a region to be altered (5' arm) is selected from the nucleotides from the position 125562909 to the position 125567172 of the NCBI accession number NC_000073.6; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotides from the position 125572100 to the position 125576624 of the NCBI accession number NC_000073.6.

[0365] In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be about or at least 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb or 10 kb.

[0366] In some embodiments, the region to be altered is exon 1, exon 2, exon 3, and/or exon 4 of IL4 gene (e.g., exon 1, exon 2, exon 3 and/or exon 4 of mouse IL4 gene). In some embodiments, the region to be altered is exon 4, exon 5, exon 6, and/or exon 7 of IL4Ra gene (e.g., exon 4, exon 5, exon 6 and/or exon 7 of mouse IL4Ra gene).

[0367] The targeting vector can further include a selected gene marker.

[0368] In some embodiments, the sequence of the 5' arm is shown in SEQ ID NO: 5; and the sequence of the 3' arm is shown in SEQ ID NO: 6.

[0369] In some embodiments, the sequence of the 5' arm is shown in SEQ ID NO: 20; and the sequence of the 3' arm is shown in SEQ ID NO: 21.

[0370] In some embodiments, the sequence of the 5' arm is shown in SEQ ID NO: 45; and the sequence of the 3' arm is shown in SEQ ID NO: 46.

[0371] In some embodiments, the sequence is derived from human (e.g., 132674051-132682587 of NC_000005.10, or 132672342-132682914 of NC_000005.10). For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human IL4, preferably exon 1, exon 2, exon 3 and/or exon 4 of the human IL4. In some embodiments, the nucleotide sequence of the humanized IL4 encodes the entire or the part of human IL4 protein (e.g., SEQ ID NO: 4).

[0372] In some embodiments, the sequence is derived from human (e.g., 27342138-27352674 of NC_000016.10). For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human IL4Ra, preferably exon 4, exon 5, exon 6 and/or exon 7 of the human IL4Ra. In some embodiments, the nucleotide sequence of the humanized IL4Ra encodes the entire or the part of human IL4Ra protein (e.g., SEQ ID NO: 42).

[0373] In some embodiments, the target region is derived from human. In some embodiments, the target region is a part or entirety of the nucleotide sequence of a humanized IL4R. In some embodiments, the nucleotide sequence is shown as one or more of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11 of the human IL4R. In some embodiments, the target region is a part or entirety of the nucleotide sequence of a humanized IL4. In some embodiments, the nucleotide sequence is shown as one or more of exon 1, exon 2, exon 3, and/or exon 4 of the human IL4.

[0374] In some embodiments, the nucleotide sequence of the human IL4R encodes the human IL4R protein with the NCBI accession number NP_000409.1 (SEQ ID NO: 42). In some emboldens, the nucleotide sequence of the human IL4R is selected from the nucleotides from the position 27342138 to the position 27352674 of NC_000016.10 (SEQ ID NO: 47).

[0375] In some embodiments, the nucleotide sequence of the human IL4 encodes the human IL4 protein with the NCBI accession number NP_000580.1 (SEQ ID NO: 4). In some emboldens, the nucleotide sequence of the human IL4R is selected from the nucleotides from the position 132674051 to the position 132682587 of NC_000005.10 (SEQ ID NO: 7), or position 132672342 to the position 132682914 of NC_000005.10 (SEQ ID NO: 22).

[0376] The disclosure also relates to a cell comprising the targeting vectors as described herein.

[0377] In addition, the present disclosure further relates to a non-human mammalian cell, having any one of the foregoing targeting vectors, and one or more in vitro transcripts of the construct as described herein. In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.

[0378] In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.

[0379] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.

Methods of Making Genetically Modified Animals

[0380] Genetically modified animals can be made by several techniques that are known in the art, including, e.g., nonhomologous end-joining (NHEJ), homologous recombination (HR), zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are known in the art, and is described, e.g., in Yin et al., "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated by reference in its entirety. Many other methods are also provided and can be used in genome editing, e.g., micro-injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.

[0381] Thus, in some embodiments, the disclosure provides replacing in at least one cell of the animal, at an endogenous IL4Ra or IL4 gene locus, a sequence encoding a region of an endogenous IL4R or IL4 with a sequence encoding a corresponding region of human or chimeric IL4R or IL4. In some embodiments, the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.

[0382] FIG. 17 shows a humanization strategy for a mouse IL4R locus. In FIG. 17, the targeting strategy involves a vector comprising the 5' end homologous arm, human IL4Ra gene fragment, 3' homologous arm. The process can involve replacing endogenous IL4Ra sequence with human sequence by homologous recombination. FIG. 4 and FIG. 8 show a humanization strategy for a mouse IL4 locus. In FIG. 4 and FIG. 8, the targeting strategy involves a vector comprising the 5' end homologous arm, human IL4 gene fragment, 3' homologous arm. The process can involve replacing endogenous IL4 sequence with human sequence by homologous recombination. In some embodiments, the cleavage at the upstream and the downstream of the target site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strand break, and the homologous recombination is used to replace endogenous IL4Ra or IL4 sequence with human IL4Ra or IL4 sequence.

[0383] Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous IL4Ra locus (or site), a nucleic acid encoding a sequence encoding a region of endogenous IL4R with a sequence encoding a corresponding region of human IL4R. The sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10 and/or exon 11 of a human IL4Ra gene. In some embodiments, the sequence includes a region of exon 4, exon 5, exon 6 and/or exon 7 of a human IL4Ra gene (e.g., amino acids 30-216 of SEQ ID NO: 42). In some embodiments, the region is located within the extracellular region of IL4R. In some embodiments, the endogenous IL4Ra locus is exon 4, exon 5, exon 6 and/or exon 7 of mouse IL4Ra.

[0384] Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous IL4 locus (or site), a nucleic acid encoding a sequence encoding a region of endogenous IL4 with a sequence encoding a corresponding region of human IL4. The sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, and/or exon 4 of a human IL4 gene. In some embodiments, the sequence includes a region of exon 1, exon 2, exon 3 and/or exon 4 of a human IL4 gene (e.g., full-length sequence of SEQ ID NO: 4). In some embodiments, the endogenous IL4 locus is exon 1, exon 2, exon 3 and/or exon 4 of mouse IL4.

[0385] In some embodiments, the methods of modifying an IL4Ra or IL4 locus of a mouse to express a chimeric human/mouse IL4R or IL4 peptide can include the steps of replacing at the endogenous mouse IL4Ra or IL4 locus a nucleotide sequence encoding a mouse IL4R or IL4 with a nucleotide sequence encoding a human IL4R or IL4, thereby generating a sequence encoding a chimeric human/mouse IL4R or IL4.

[0386] In some embodiments, the nucleotide sequence encoding the chimeric human/mouse IL4R can include a first nucleotide sequence encoding a region of the extracellular region of mouse IL4R (with or without the mouse or human signal peptide sequence); a second nucleotide sequence encoding a region of the extracellular region of human IL4R; a third nucleotide sequence encoding the transmembrane region, and/or the cytoplasmic region of a mouse IL4R.

[0387] In some embodiments, the nucleotide sequences as described herein do not overlap with each other (e.g., the first nucleotide sequence, the second nucleotide sequence, and/or the third nucleotide sequence do not overlap). In some embodiments, the amino acid sequences as described herein do not overlap with each other.

[0388] The present disclosure further provides a method for establishing an IL4Ra or IL4 gene humanized animal model, involving the following steps:

[0389] (a) providing the cell (e.g. a fertilized egg cell) based on the methods described herein;

[0390] (b) culturing the cell in a liquid culture medium;

[0391] (c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;

[0392] (d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c).

[0393] In some embodiments, the non-human mammal in the foregoing method is a mouse (e.g., a C57BL/6 or BALB/c mouse).

[0394] In some embodiments, the non-human mammal in step (c) is a female with pseudo pregnancy (or false pregnancy).

[0395] In some embodiments, the fertilized eggs for the methods described above are C57BL/6 or BALB/c fertilized eggs. Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, FVB/N fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.

[0396] Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein. In some embodiments, the fertilized egg cells are derived from rodents. The genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the method described above.

Methods of Using Genetically Modified Animals

[0397] Replacement of non-human genes in a non-human animal with homologous or orthologous human genes or human sequences, at the endogenous non-human locus and under control of endogenous promoters and/or regulatory elements, can result in a non-human animal with qualities and characteristics that may be substantially different from a typical knockout-plus-transgene animal. In the typical knockout-plus-transgene animal, an endogenous locus is removed or damaged and a fully human transgene is inserted into the animal's genome and presumably integrates at random into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of the human gene and/or protein assay and/or functional assay. Inclusion in the human transgene of upstream and/or downstream human sequences are apparently presumed to be sufficient to provide suitable support for expression and/or regulation of the transgene.

[0398] In some cases, the transgene with human regulatory elements expresses in a manner that is unphysiological or otherwise unsatisfactory, and can be actually detrimental to the animal. The disclosure demonstrates that a replacement with human sequence at an endogenous locus under control of endogenous regulatory elements provides a physiologically appropriate expression pattern and level that results in a useful humanized animal whose physiology with respect to the replaced gene are meaningful and appropriate in the context of the humanized animal's physiology.

[0399] Genetically modified animals that express human or humanized IL4R and/or IL4 protein, e.g., in a physiologically appropriate manner, provide a variety of uses that include, but are not limited to, developing therapeutics for human diseases and disorders, and assessing the toxicity and/or efficacy of these human therapeutics in the animal models.

[0400] In various aspects, genetically modified animals are provided that express human or humanized IL4R and/or IL4, which are useful for testing agents that can decrease or block the interaction between IL4R and IL4 or the interaction between IL4R and other IL4R ligands, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an IL4R or IL4 agonist or antagonist. The genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (a knock-in or knockout). In various embodiments, the genetically modified non-human animals further comprise an impaired immune system, e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor or a blood cell tumor (e.g., a lymphocyte tumor, e.g., a B or T cell tumor).

[0401] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4R or IL4 antagonist (e.g., an anti-IL4R or an anti-IL4 antibody) for reducing inflammation. The methods involve administering the IL4R or IL4 antagonist to the animal described herein, wherein the animal has an inflammation; and determining the inhibitory effects of the IL4R or IL4 antagonist to the reduction of inflammation.

[0402] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4R or IL4 antagonist (e.g., an anti-IL4R or anti-IL4 antibody) for treating an immune disorder (e.g., an autoimmune disorder or allergy). The methods involve administering the IL4R or IL4 antagonist to the animal described herein, wherein the animal has an immune disorder; and determining the inhibitory effects of the IL4R or IL4 antagonist.

[0403] In one aspect, the disclosure also provides methods of determining effectiveness of an IL4R or IL4 antagonist (e.g., an anti-IL4R or anti-IL4 antibody) for treating cancer. The methods involve administering the IL4R or IL4 antagonist to the animal described herein, wherein the animal has a tumor; and determining the inhibitory effects of the IL4R or IL4 antagonist to the tumor. In some embodiments, the tumor comprises one or more cancer cells that are injected into the animal. The inhibitory effects that can be determined include, e.g., a decrease of tumor size or tumor volume, a decrease of tumor growth, a reduction of the increase rate of tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or in another subject without such treatment), a decrease in the risk of developing a metastasis or the risk of developing one or more additional metastasis, an increase of survival rate, and an increase of life expectancy, etc. The tumor volume in a subject can be determined by various methods, e.g., as determined by direct measurement, Mill or CT.

[0404] In some embodiments, the anti-IL4R antibody or anti-IL4 antibody prevents IL4 from binding to IL4R. In some embodiments, the anti-IL4R antibody or anti-IL4 antibody cannot prevent IL4 from binding to IL4R (e.g., endogenous IL4R).

[0405] In some embodiments, the genetically modified animals can be used for determining whether an anti-IL4R antibody is an IL4R agonist or antagonist. In some embodiments, the genetically modified animals can be used for determining whether an anti-IL4 antibody is an IL4 agonist or antagonist. In some embodiments, the methods as described herein are also designed to determine the effects of the agent (e.g., anti-IL4R or anti-IL4 antibodies) on IL4R and/or IL4, e.g., whether the agent can stimulate macrophages, and/or whether the agent can upregulate the immune response or downregulate immune response. In some embodiments, the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., an immune disorder, an allergy, or autoimmune diseases.

[0406] In some embodiments, the inhibitory effects of treating inflammation are evaluated by serum IgE levels; pathological lung histology features; number of leukocytes (CD45+ cells), eosinophils (Eos) or neutrophils in bronchoalveolar lavage fluid (BALF); or ratio of eosinophils or neutrophils cells in CD45+ cells in bronchoalveolar lavage fluid (BALF).

[0407] The inhibitory effects on tumors can also be determined by methods known in the art, e.g., measuring the tumor volume in the animal, and/or determining tumor (volume) inhibition rate (TGITv). The tumor growth inhibition rate can be calculated using the formula TGITv (%)=(1-TVt/TVc).times.100, where TVt and TVc are the mean tumor volume (or weight) of treated and control groups.

[0408] In some embodiments, the anti-IL4R or anti-IL4 antibody is designed for treating various cancers. As used herein, the term "cancer" refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term "tumor" as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. As IL-4 blockade improves the response to anti-OX40 antibody or CpG oligodeoxynucleotide immunotherapies, in some embodiments, the anti-IL4R antibody or anti-IL4 antibody are used in connection with anti-OX40 antibody or CpG oligodeoxynucleotide immunotherapies.

[0409] In some embodiments, the antibody is designed for treating various autoimmune diseases or allergy (e.g., allergic rhinitis, sinusitis, asthma, or eczema). Thus, the methods as described herein can be used to determine the effectiveness of an antibody in inhibiting immune response.

[0410] The present disclosure also provides methods of determining toxicity of an antibody (e.g., anti-IL4R antibody or anti-IL4 antibody). The methods involve administering the antibody to the animal as described herein. The animal is then evaluated for its weight change, red blood cell count, hematocrit, and/or hemoglobin. In some embodiments, the antibody can decrease the red blood cells (RBC), hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%.

[0411] The present disclosure also relates to the use of the animal model generated through the methods as described herein in the development of a product related to an immunization processes of human cells, the manufacturing of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

[0412] In some embodiments, the disclosure provides the use of the animal model generated through the methods as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.

[0413] The disclosure also relates to the use of the animal model generated through the methods as described herein in the screening, verifying, evaluating or studying the IL4R or IL4 gene function, human IL4R or IL4 antibodies, drugs for human IL4R or IL4 targeting sites, the drugs or efficacies for human IL4R or IL4 targeting sites, the drugs for immune-related diseases and antitumor drugs.

Genetically Modified Animal Model with Two or More Human or Chimeric Genes

[0414] The present disclosure further relates to methods for generating genetically modified animal model with two or more human or chimeric genes. The animal can comprise a human or chimeric IL4Ra gene and a sequence encoding one or more additional human or chimeric protein (e.g., IL4). Alternatively, the animal can comprise a human or chimeric IL4 gene and a sequence encoding one or more additional human or chimeric protein (e.g., IL4R).

[0415] In some embodiments, the additional human or chimeric protein can be IL4, IL4R, Interleukin 33 (IL33), Interleukin 13 (IL13), programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte Activating 3 (LAG-3), B And T Lymphocyte Associated (BTLA), Programmed Cell Death 1 Ligand 1 (PD-L1), CD27, CD28, T-Cell Immunoreceptor with Ig and ITIM Domains (TIGIT), T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), Glucocorticoid-Induced TNFR-Related Protein (GITR), CD137, TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40), CD47 or SIRPa.

[0416] The methods of generating genetically modified animal model with two or more human or chimeric genes (e.g., humanized genes) can include the following steps:

[0417] (a) using the methods of introducing human IL4R gene or chimeric IL4Ra gene as described herein to obtain a genetically modified non-human animal;

[0418] (b) mating the genetically modified non-human animal with another genetically modified non-human animal, and then screening the progeny to obtain a genetically modified non-human animal with two or more human or chimeric genes.

[0419] In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal with human or chimeric IL4, IL33, IL13, PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, TIGIT, TIM-3, GITR, OX40, CD137, CD47, CD40, CD3e or SIRPa. Some of these genetically modified non-human animal are described, e.g., in PCT/CN2017/090320, PCT/CN2017/099577, PCT/CN2017/099575, PCT/CN2017/099576, PCT/CN2017/099574, PCT/CN2017/106024, PCT/CN2017/110494, PCT/CN2017/110435, PCT/CN2017/117984, PCT/CN2017/120388, PCT/CN2018/091846, PCT/CN2018/091845, PCT/CN2018/120713, PCT/CN2018/110069; each of which is incorporated herein by reference in its entirety.

[0420] Similarly, the methods of generating genetically modified animal model can include the following steps:

[0421] (a) using the methods of introducing human IL4 gene or chimeric IL4 gene as described herein to obtain a genetically modified non-human animal;

[0422] (b) mating the genetically modified non-human animal with another genetically modified non-human animal, and then screening the progeny to obtain a genetically modified non-human animal with two or more human or chimeric genes.

[0423] In some embodiments, the humanization is directly performed on a genetically modified animal having a human or chimeric IL4, IL4R, IL33, IL13, PD-1, CTLA-4, BTLA, PD-L1, CD27, CD28, TIGIT, TIM-3, GITR, CD137, OX40, CD47, CD40, CD3e or SIRPa gene.

[0424] In some embodiments, the IL4R humanization is directly performed on a genetically modified animal having a human or chimeric IL4. In some embodiments, the IL4 humanization is directly performed on a genetically modified animal having a human or chimeric IL4R.

[0425] As these proteins may involve different mechanisms, a combination therapy that targets two or more of these proteins thereof may be a more effective treatment. In fact, many related clinical trials are in progress and have shown a good effect. The genetically modified animal model with two or more human or humanized genes can be used for determining effectiveness of a combination therapy that targets two or more of these proteins, e.g., an anti-IL4R antibody and an additional therapeutic agent for the treatment. The methods include administering the anti-IL4R antibody and/or the anti-IL4 antibody, and the additional therapeutic agent to the animal, wherein the animal has a tumor; and determining the inhibitory effects of the combined treatment to the tumor. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to IL4, IL4R, IL33, IL13, PD-1, CTLA-4, BTLA, PD-L1, CD27, CD28, TIGIT, TIM-3, GITR, CD137, OX40, CD47, CD40, CD3e or SIRPa. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-CD20 antibody (e.g., rituximab), an anti-EGFR antibody (e.g., cetuximab), and an anti-CD319 antibody (e.g., elotuzumab), or anti-PD-1 antibody (e.g., nivolumab).

EXAMPLES

[0426] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

[0427] The following materials were used in the following examples.

[0428] C57BL/6 mice were purchased from the China Food and Drugs Research Institute National Rodent Experimental Animal Center.

[0429] AIO kit was obtained from Beijing Biocytogen Co., Ltd. (Catalog number: BCG-DX-004).

[0430] Mouse IL-4 ELISA kit was obtained from RayBiotech, Inc. (Catalog number ELM-IL4-1).

[0431] Human IL-4 ELISA kit was obtained from RayBiotech, Inc. (Catalog number ELH-IL4-5).

[0432] BamHI, HindIII, XhoI, EcoRI, EcoRV, NotI, NdeI, SacI, BglII, AseI, MfeI and SmaI restriction enzymes were purchased from NEB with Catalog numbers: R3136M, R3104M, R0146S, R3101M, R0195S, R3189M, R3193M, R3156M, R0144M, R0526M, R3589S and R0141S, respectively.

[0433] Bacterial Artificial Chromosome (BAC) bacteria containing mouse IL4 and IL4R genes, and BAC bacteria containing human IL4 and IL4R genes were ordered from Invitrogen (Catalog number RPCI23.0 and RPCI11.C).

[0434] Purified NA/LE Hamster anti-mouse CD3e (mCD3) antibody was purchased from Becton, Dickinson and Company (BD Biosciences, Catalog number 553057).

[0435] FITC anti-mouse CD19 antibody (mCD19 FITC) was purchased from Biolegend (Catalog number 115506).

[0436] APC anti-human CD124 (IL4Ra) antibody (hIL4RA APC) was purchased from Biolegend (Catalog number: 355006).

[0437] PE anti-mouse CD124 (IL4Ra) antibody (mIL4RA PE) was purchased from Biolegend (Catalog number 144804).

[0438] APC/Cy7 anti-mouse CD19 antibody (mCD19 APC-Cy7) was purchased from Biolegend (Catalog number: 115530).

[0439] PE anti-human CD124 (IL-4Ra) antibody (hIL4RA PE) was purchased from Biolegend (Catalog number 355003).

[0440] Alexa Fluor.RTM. 647 AffiniPure F(ab').sub.2 Fragment Goat Anti-Human IgG, Fc.gamma. fragment specific (anti-hIgG-AF647) was purchased from Jackson ImmunoResearch Inc. (Catalog number: 109-606-170).

[0441] In vivo Grade Human IgG4 kappa Isotype Control--CrownVivo.TM. Antibody (anti-IgG4-kappa hIgG-APC) was purchased from Crown Bio Inc. (Catalog number: C0004).

Example 1: Mice with Humanized IL4 Gene

[0442] The transcript of mouse IL4 gene (NCBI Gene ID: 16189, Primary source: MGI: 96556, UniProt ID: P07750) is NM_021283.2 (SEQ ID NO: 1) with the corresponding protein NP_067258.1 (SEQ ID NO: 2). The transcript of human IL4 gene (NCBI Gene ID: 3565, Primary source: HGNC: 6014, UniProt ID: P05112) is NM_000589.3 (SEQ ID NO: 3) with the corresponding protein NP_000580.1 (SEQ ID NO: 4). A schematic diagram that compares the mouse IL4 gene and the human IL4 gene was shown in FIG. 1.

[0443] Two humanization strategies were used to introduce a gene sequence encoding a human IL4 protein into the endogenous mouse IL4 locus. In one method, the mouse IL4 gene sequence was replaced with human IL4 gene sequences at the endogenous IL4 locus. About .about.8.5 kb sequence starting from ATG (start codon) to TGA (stop codon) was replaced with the corresponding human DNA sequence to obtain a humanized IL4 locus (FIG. 2; version 1, the mRNA sequence of the engineered mouse IL4 after humanization was shown in SEQ ID NO: 80). The sequence is still under the control of mouse IL4 regulatory elements, and has mouse 5'-UTR and 3'-UTR. Another humanization strategy is shown in FIG. 3 (version 2). A longer sequence at the IL4 locus was replaced. The sequence includes human 5'-UTR and human 3'-UTR, and is under the control of human IL4 regulatory elements.

[0444] Mouse and human IL4 DNA were obtained using Bacterial Artificial Chromosome (BAC) RP23-464K4 and RP11-17K19, respectively. As shown in the schematic diagram of the targeting strategy in FIG. 4, the recombinant vector contained the homology arm sequence upstream and downstream of mouse IL4 (4220 bp upstream of ATG of endogenous IL4 gene and 4085 bp downstream of TGA), and 8537 bp human IL4 Sequence (extending from start codon ATG in exon 1 to stop codon TGA in exon 4). The upstream homology arm sequence (5' homology arm, SEQ ID NO: 5) was identical to the nucleotide sequence of 53622826-53618607 with NCBI accession number NC_000077.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 6) was identical to the nucleotide sequence of 53612065-53607981 with NCBI accession number NC_000077.6. The DNA fragment sequence of human IL4 (SEQ ID NO: 7) was identical to the nucleotide sequence of 132674051-132682587 with NCBI accession number NC_000005.10. The sequence containing the human IL4 gene and the upstream connection site at the mouse locus was designed as 5'-ACTTTAACTCTATATATAGAGAGACCTCTGCCAGCATTGCATTGTTAGCAT CTCTTGATAAACTTAATTGTCTCTCGTCACTGACGGCACAGAGCTATTGATGG GTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCGGCA ACTTTGTCCACGGACACAAGTGCGATATCACCTTACAGGAGA-3' (SEQ ID NO: 8), wherein the "G" in the sequence "TATTG" was the last nucleotide of the mouse sequence, and the "A" in the sequence "ATGGG" was the first nucleotide of the human sequence.

[0445] The sequence containing the human IL4 gene and the downstream connection site at the mouse locus was designed as 5'-TGGAAAACTTCTTGGAAAGGCTAAAGACGATCATGAGAGAGAAATATTCA AAGTGTTCGAGCTGATACTGAGCCACCATGCTTTAACTTATGAATTTTTAATG GTTTTATTTTTAATATTTATATATTTATAATTCATAAAATAAAATA-3' (SEQ ID NO: 9), wherein the "A" in the sequence "GCTGA" was the last nucleotide of the human sequence, and the "T" in the sequence "TACTG" was the first nucleotide of the mouse sequence.

[0446] The targeting vector also included an antibiotic resistance gene for positive clone screening (neomycin phosphotransferase Neo), and two Frt recombination sites on both sides of the antibiotic resistance gene. The locus formed a Neo cassette. The connection between the 5' end of the Neo cassette and the mouse IL4 locus was designed as 5'-ACATGCCTGTAGGCAAGACACCCACACACATAAAAACAAAATAAAATAA GGATAGAAAGGCCAGGGGGATGAATCCTCGAGGTCGACGGTATCGATAAGC TTGATATCGAATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGGT CT-3' (SEQ ID NO: 10), wherein the "C" of the sequence "GAATC" was the last nucleotide of the mouse sequence, and the "C" of the sequence "CTCGA" was the first nucleotide of the Neo cassette. The connection between the 3' end of the Neo cassette with the mouse IL4 locus was designed as 5'-AGAAAGTATAGGAACTTCATCAGTCAGGTACATAATGG TGGATCCATTAATCAGAGGTAGAAGAAAACTTATTCC-3' (SEQ ID NO: 11), wherein the last "T" of the sequence "TTAAT" was the last nucleotide of the Neo cassette, and the "C" of the sequence "CAGAG" was the first nucleotide of the mouse sequence. In addition, a coding gene with a negative selectable marker (a gene encoding diphtheria toxin A subunit (DTA)) was also inserted downstream of the 3' homology arm of the recombinant vector.

[0447] Vector construction can be carried out by restriction enzyme digestion and ligation. The constructed recombinant vector sequence can be initially verified by restriction enzyme digestion. The verification results were shown in FIGS. 5A-5B. The restriction enzyme digestion results were verified by using three groups of enzymes. Among them, EcoRI+NotI should generate 634 bp+1678 bp+5953 bp+16413 bp fragments, EcoRV+NdeI should generate 587 bp+2637 bp+8928 bp+12526 bp fragments, BglII+XhoI should generate 1183 bp+5178 bp+6292 bp+12025 bp fragments. The results of restriction enzyme digestion were in line with expectations and the sequences of plasmids 1 and 2 were verified by sequencing.

[0448] The correct recombinant vector was electroporated and transfected into embryonic stem cells of C57BL/6 mice. The positive selectable marker gene was used to screen the cells, and the integration of exogenous genes was confirmed by PCR and Southern Blot. PCR and Southern Blot results (digested with EcoRI or AseI, respectively, and hybridized with 3 probes) for some of the clones were shown in FIG. 6 and FIG. 7. As shown in FIG. 7, among the 12 PCR-positive clones, 11 clones were selected and identified as positive heterozygous clones and no random insertions were detected.

[0449] The following primers were used in PCR:

TABLE-US-00005 F: (SEQ ID NO: 12) 5'-GACAACTTTCAGGGAGGGAGTCAG-3', R: (SEQ ID NO: 13) 5'-ATCGCACTTGTGTCCGTGGAC-3'.

[0450] The following probes were used in Southern Blot assays:

TABLE-US-00006 5' Probe: F: (SEQ ID NO: 14) 5'-GCCTCTCTCAACCCAGTATAAC-3', R: (SEQ ID NO: 15) 5'-CTTGGAGTCAACCACCTCTG-3'; 3' Probe: F: (SEQ ID NO: 16) 5'-GATCTTTCACTGAAACTTGACTG-3', R: (SEQ ID NO: 17) 5'-CTCCAACTACATCTACTTTCTG-3'; Neo Probe: F: (SEQ ID NO: 18) 5'-GGATCGGCCATTGAACAAGATGG - 3', R: (SEQ ID NO: 19) 5'-CAGAAGAACTCGTCAAGAAGGCG-3'.

[0451] Another IL4 mouse humanization strategy was to perform a sequence substitution for a longer sequence near the IL4 locus. The humanized mouse IL4 locus was shown in FIG. 3, and the targeting strategy was shown in FIG. 8. The recombinant vector contained an upstream homology arm sequence of 5553 bp and a downstream homology arm sequence of 5196 bp of the mouse IL4 locus, and a 10573 bp sequence containing human IL4 DNA fragments. The upstream homology arm sequence (5' homology arm, SEQ ID NO: 20) was identical to the nucleotide sequence of 53625623-53620071 of NCBI accession number NC_000077.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 21) was identical to the nucleotide sequence at position 53612200-53607005 of NCBI Accession No. NC_000077.6; the sequence of human IL4 DNA fragment (SEQ ID NO: 22) was identical to the nucleotide sequence of 132672342-132682914 with NCBI accession number NC_000005.10.

[0452] The sequence containing the human IL4 gene and the upstream connection site of the mouse locus was designed as 5'-TGGGGTATGGTGGCTTATATCTGTAACTTCAACACTTGAGAGGTGGAG GCAGGAGAGTGACCATGAATCTGAGGGCTTCCAGAATAAATTCATAGGGAG GCCCAGGCACAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGG-3' (SEQ ID NO: 23), wherein the last "G" of the sequence "GAGGG" was the last nucleotide of the mouse sequence, and the first "C" of the sequence "CTTCC" was the first nucleotide of the human sequence.

[0453] Downstream of the sequence containing the human IL4 gene was linked to the Neo cassette, and the connection site was designed as 5'-GGGGTTCCCTCTCGAGTTAGGGACATAACACACAAGATAATTAAAGAACA CAAGGCCATACAAGATGTAAATAAGACACCTTGGGTCCAAGAGTGCGTCGA CGGTATCGATAAGCTTGATATCGAATTCCGAAGTTCCTATTCTCTAGAAAGTA TAGGAACTTCAGGTCTGAAGAGGAGTTTACGTCCAGCCAAGC-3' (SEQ ID NO: 24), wherein the "C" of the sequence "GAGTGC" was the last nucleotide of the human sequence, and the first "G" of the sequence "GTCGA" was the first nucleotide of the Neo cassette.

[0454] The Neo cassette downstream was connected to the mouse sequence, and the connection sequence was designed as 5'-TGCGGAACCCTTCGAAGTTCCTATTCTCTAGAAAGTATA GGAACTTCATCAGTCAGGTACATAATGGTGGATCCTAACTCAAGTTCTGGGG GAGCTGATGCTCTCCTCTGGCCTCCTGTGGAGGTACACAGACCACATGCCTGT AGGCAA-3' (SEQ ID NO: 25), wherein the last "C" of the sequence "GATCC" was the last nucleotide of the Neo cassette, and the first "T" of the sequence "TAACT" was the first nucleotide of the mouse sequence.

[0455] In addition, a coding gene with a negative selectable marker (a gene encoding the diphtheria toxin A subunit (DTA)) was also inserted downstream of the 3' homology arm of the recombinant vector. The verified results of the constructed recombinant vector digestion were shown in FIGS. 9A-9B, wherein BamHI should generate 9938 bp+6990 bp+5266 bp+3577 bp+2930 bp fragments, EcoRI should generate 9606 bp+6558 bp+5432 bp+4470 bp+2178 bp+457 bp fragments, ScaI should generate 10389 bp+7026 bp+5739 bp+2818 bp+2074 bp+655 bp fragments. The results of restriction enzyme digestion were in line with expectations and the plasmid was verified by sequencing.

[0456] The embryonic stem cells from C57BL/6 mice were transfected with the correct recombinant vector, screened, then confirmed by PCR and Southern Blot. Some of the PCR and Southern Blot (digested with MfeI, BamHI or EcoRV, respectively, and hybridized with 3 probes) results were shown in FIG. 10 and FIG. 11. As shown in FIG. 11, the results showed that among the 12 PCR-positive clones, except for 1-Ell and 2-F2, the remaining 10 clones were confirmed as positive heterozygous clones and no random insertions were detected.

[0457] The following primers were used in PCR:

TABLE-US-00007 F: (SEQ ID NO: 26) 5'-CTGTGATCATGGTTCCTTATCTGG-3', R: (SEQ ID NO: 27) 5'-CCTCCCCGAGTAGCTGGGACTAC-3'.

[0458] The following probe were used in Southern Blot assays:

TABLE-US-00008 5' Probe: F: (SEQ ID NO: 28) 5'-CCACTAGGGGTCCACAGCTAGTCAT-3', R: (SEQ ID NO: 29) 5'-CTTCAGTGAAACCTCCTGAGCCTGG-3'; 3' Probe: F: (SEQ ID NO: 30) 5'- AGTCAGAGCTACAGAAGTGGAGGGT-3', R: (SEQ ID NO: 31) 5'- CTGCTCTGCAGGAAGTAAGGGTTCC-3'; SEQ ID NO: 18; Neo Probe F: SEQ ID NO: 19. Neo Probe R:

[0459] The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice), and the obtained chimeric blastocysts were transferred to the culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white). The F2 generation homozygous mice can be obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then mating the F1 generation heterozygous mice with each other. The positive mice can also be mated with the Flp tool mice to remove the positive selectable marker gene (the schematic diagram of the process was shown in FIG. 12), and then the humanized IL4 homozygous mice expressing human IL4 protein can be obtained by mating with each other. The genotype of the progeny mice can be identified by PCR, and the results for the F1 generation mice (Neo-removed) were shown in FIGS. 13A-13D, wherein the mice numbered 1 and 2 were positive heterozygous mice. The following primers were used in PCR:

TABLE-US-00009 WT-F: (SEQ ID NO: 32) 5'-ccacatcactgaaagacttcctgg-3'; WT-R: (SEQ ID NO: 33) 5'-gatcaagtagacaggcaggcaagac-3'; Mut-F: (SEQ ID NO: 34) 5'-ctgtgatcatggttccttatctgg-3'; (SEQ ID NO: 33); WT-R: Frt-F: (SEQ ID NO: 35) 5'-agggacagatgcaggctggg-3'; Frt-R: (SEQ ID NO: 36) 5'-gagatgcgtgttagaggttttggga-3'; Flp-F: (SEQ ID NO: 37) 5'-tcagcgatattaagaacgttgatccg-3'; Flp-R: (SEQ ID NO: 38) 5'-tgaagaattgccggtcctatttactcg-3'.

[0460] The expression of humanized IL4 protein in positive mice can be confirmed by ELISA. 7.5 .mu.g of anti-mouse CD3 antibody (mCD3) was intraperitoneally injected into the mice. After 1.5 hours, serum was taken and diluted 5.times. to measure the levels of mouse IL4 and human IL4 protein. The measurement results were shown in FIGS. 14A-14B. FIG. 14A showed that the expression of mouse IL4 protein was detected in wild-type C57BL/6 mice and the two versions of IL4 humanized heterozygotes. FIG. 14B showed that the expression of human IL4 protein was not detected in wild-type C57BL/6 mice, and human IL4 protein expression was detected in both versions of IL4 humanized mice.

Example 2: Mice with Humanized IL4Ra Genes

[0461] The transcript of mouse IL4Ra gene (NCBI Gene ID: 16190, Primary source: MGI: 105367, UniProt ID: P16382) is NM_001008700.3 (SEQ ID NO: 39) with the corresponding protein NP_001008700.1 (SEQ ID NO: 40). The transcript of human IL4Ra gene (NCBI Gene ID: 3566, Primary source: HGNC: 6015, UniProt ID: P24394) is NM_000418.3 (SEQ ID NO: 41) with the corresponding protein NP_000409.1 (SEQ ID NO: 42). A schematic diagram that compared the mouse IL4Ra gene locus and the human IL4Ra gene locus was shown in FIG. 15.

[0462] The extracellular region of the endogenous mouse IL4Ra gene was replaced with the human IL4Ra gene sequence. Two humanization strategies were used. One was to replace a 4.4 kb sequence encoding the extracellular region of the mouse IL4Ra from exon 4 to exon 7 with a 10.5 kb sequence spanning from exon 4 to exon 7 of the human IL4Ra locus. This humanized strategy resulted in a humanized IL4Ra gene as shown in FIG. 16. The mRNA sequence of the engineered mouse IL4Ra after humanization and its encoded protein sequence were shown in SEQ ID NO: 43 and SEQ ID NO: 44, respectively.

[0463] Mouse and human IL4Ra DNA were obtained using Bacterial Artificial Chromosome (BAC) RP23-261H16 and RP11-16E24, respectively. As shown in the schematic diagram of the targeting strategy in FIG. 17, the recombinant vector contained the homology arm sequence upstream and downstream of mouse IL4Ra sequence (4264 bp upstream of exon 4 of endogenous IL4Ra gene and 4525 bp downstream of exon 7), and 10537 bp human IL4Ra sequence (extending from part of exon 4 to exon 7). The upstream homology arm sequence (5' homology arm, SEQ ID NO: 45) was identical to the nucleotide sequence of 125562909-125567172 with NCBI accession number NC_000073.6, and the downstream homology arm sequence (3' homology arm, SEQ ID NO: 46) was identical to the nucleotide sequence of 125572100-125576624 with NCBI accession number NC_000073.6; the DNA fragment sequence of human IL4Ra (SEQ ID NO: 47) was identical to the nucleotide sequence of 27342138-27352674 with NCBI accession number NC_000016.10. The sequence containing the human IL4Ra gene and the upstream of the connecting site at the mouse locus was designed as 5'-CCTCCCTCTGACCTTAGTGGTGGGAGCCCCTGACCATGCCACCACTGATCT GGCCGTTCTGTCTCTGCAGGGAGCATCAAGGTCCTGCAGGAGCCCACCTGCG TCTCCGACTACATGAGCATCTCTACTTGCGAGTGGAAGATGAATGGTCCCACC AATTGCAGCACCGAGCTCCGCCTGTTG-3' (SEQ ID NO: 48), wherein the "G" of the sequence "TCCTG" was the last nucleotide of the mouse sequence, and the "C" of the sequence "CAGGA" was the first nucleotide of the human sequence. The sequence containing the human IL4Ra gene and the downstream of the connection site at the mouse locus was designed as 5'-CAGCACCCTGAAGTCTGGGATTTCCTACAGGGCA CGGGTGAGGGCCT GGGCTCAGTGCTATAACACCACCTGGAGTGAGTGGAGCCCTAGCATCACGTG GTACAACCGTGAGTATCAGGGTCGTAGGCTGTGAGGATCTCTACAGCCGTGT ATATTCTCTGTTCAGAAATTCCCTCTGGCTGA-3' (SEQ ID NO: 49), wherein the "C" of the sequence "GGAGC" was the last nucleotide of the human sequence, and the first "C" of the sequence "CCTAG" was the first nucleotide of the mouse sequence.

[0464] The connection sequence between the 5' end of the Neo cassette and the mouse IL4Ra locus was designed as 5'-AGGCCCGGAGTTTAAATCCCCAGAGCCCACGTAAAAGCCTGATATCGAA TTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3' (SEQ ID NO: 50), wherein the "T" of the sequence "AGCCT" was the last nucleotide of the mouse sequence, and the first "G" of the sequence "GATAT" was the first nucleotide of the Neo cassette.

[0465] The connection sequence of the 3' end of the Neo cassette with the mouse IL4Ra locus was designed as 5'-GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCATCAGTCAGGTACATAATG GTGGATCCAAGCTTTGCGCAGTAGCACGCATGCGTAATCCTGATGGAGCAAT TAGGAGAAGCCGGTGGCCGGCTAGCCTGTGCAGACTGTGAAAACAGAGCATC TGAAGCTGTGTGAAAGGCTAGCTCGC-3' (SEQ ID NO: 51), wherein the last "T" of the sequence "AGCTT" was the last nucleotide of the Neo cassette, and the "T" of the sequence "TGCGC" was the first nucleotide of the mouse sequence. In addition, a coding gene with a negative selectable marker (a gene encoding the diphtheria toxin A subunit (DTA)) was also added downstream of the 3' homology arm of the recombinant vector.

[0466] The constructed recombinant vector sequences were verified by restriction enzyme digestion as shown in FIG. 18. The restriction enzyme digestion results were verified by using three groups of enzymes. Among them, HindIII+EcoRV should generate 1866 bp+2513 bp+10385 bp+12455 bp fragments, XhoI+SmaI should generate 481 bp+1375 bp+2818 bp+3815 bp+5670 bp+13060 bp fragments, BglII should generate 754 bp+1610 bp+1856 bp+3705 bp+19294 bp fragments, and the results of restriction enzyme digestion were in line with expectations. Plasmid 3 was selected and the sequence was further verified by sequencing.

[0467] The recombinant vector was electroporated and transfected into embryonic stem cells of C57BL/6 mice. The positive selectable marker gene was used to screen the cells, and the integration of exogenous genes was confirmed by PCR and Southern Blot. Positive clones identified by PCR (see FIGS. 19A-19B for part of the cloning result) were then confirmed by Southern Blot (digested with SacI, HindIII or BglII, respectively, and hybridized with 3 probes). As shown in FIG. 20, the results indicated that four clones (1-A9, 1-D10, 1-F4, A-G5) were positive heterozygous clones and no random insertions were detected.

[0468] The following primers were used in PCR assays:

TABLE-US-00010 F1: (SEQ ID NO: 81) 5'-GGGAGGGTGAGTGGAGTCCCA-3'; R1: (SEQ ID NO: 82) 5'-CAGCGCAGGCTTACTCGGAGAG-3'; F2: (SEQ ID NO: 52) 5'-CGCATTGTCTGAGTAGGTGTC-3'; R2: (SEQ ID NO: 53) 5'-GCTGTTCAATGAATGGCCTCTGTG-3';

[0469] The following probes were used in Southern Blot assays:

TABLE-US-00011 5' Probe: F: (SEQ ID NO: 54) 5'-GTGGCCACCGTTTCTGGGAAC-3'; R: (SEQ ID NO: 55) 5'- TCAACAATCTAAGCACGGACCT-3'; 3' Probe: F: (SEQ ID NO: 56) 5'- GTGCTGAGGCCAGGGTTCCTC-3'; R: (SEQ ID NO: 57) 5'-GCTGTTCAATGAATGGCCTCTGTG-3'; Neo Probe: F: SEQ ID NO: 18, R: SEQ ID NO: 19.

[0470] The positive clones (black mice) were introduced into isolated blastocysts (white mice), according to the techniques known in the art (for example, the method as described in A. Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)," Cold Spring Harbor Laboratory Press, 2003). The obtained chimeric blastocysts were transferred to a culture medium for a short-term culture and then transplanted into the fallopian tubes of the recipient mother mice (white mice) to produce the F0 generation chimeric mice (black and white). The F1 generation mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice. F2 generation homozygous mice were then obtained by mating the F1 generation heterozygous mice with each other. The positive mice were also mated with the Flp tool mice to remove the positive selectable marker gene, and then the humanized IL4Ra gene homozygous mice expressing human IL4Ra protein were obtained by mating with each other. The genotype of the progeny mouse can be identified by PCR and the identification results of the F1 generation mice (Neo-removed) were shown in FIG. 21 and FIG. 22, wherein the mice number F1-1 to F1-7 were positive heterozygous mice in FIG. 20.

[0471] The following primers were used in PCR assays:

TABLE-US-00012 WT-F2: (SEQ ID NO: 58) 5'-gacacacgtgtctgccagctctgt-3'; WT-R2: (SEQ ID NO: 59) 5'-cacggtcagccagagggaatttctg-3'; Mut-F2: (SEQ ID NO: 60) 5'-gtggggtcagctaacgacagcaac-3'; Frt-F2: (SEQ ID NO: 61) 5'-cgtgtcaaaagcagaaacgcaggag-3'; Frt-R2: (SEQ ID NO: 62) 5'-gcgtatgtcaggatctgaggagcac-3'; Flp-F2: (SEQ ID NO: 63) 5'- gacaagcgttagtaggcacatatac-3'; Flp-R2: (SEQ ID NO: 64) 5'-gctccaatttcccacaacattagt-3'.

[0472] The expression of humanized IL4Ra protein in mice can be confirmed by routine detection methods. For example, IL4Ra protein expression can be detected by staining the mouse spleen cells with anti-mouse IL4Ra antibody (mIL4RA PE) combined with anti-mouse CD19 antibody (mCD19 FITC), or anti-human IL4Ra antibody (hIL4RA APC) combined with anti-mouse CD19 antibody (mCD19 FITC), followed by flow cytometry analysis. The results of the flow cytometry analysis (see FIGS. 23A-23F) showed that the mouse IL4Ra protein (FIG. 23C) and the humanized IL4Ra protein (FIG. 23F) were detected in the spleen of the humanized IL4Ra gene heterozygous mice upon stimulation by anti-mCD3 antibody. In the spleen of wild-type C57BL/6 mice, the mouse IL4Ra protein was detected regardless whether the mouse was stimulated by anti-mCD3 antibody (FIGS. 23A-23B), and no expression of human or humanized IL4Ra protein were detected in the wildtype mice (FIGS. 23D-23E).

[0473] A CRISPR/Cas system was also used in gene editing to obtain humanized IL4Ra mice. The target sequence in this system determines sgRNA targeting specificity and the efficiency of Cas9-induced cleavage at the target gene. Therefore, selection and design of an efficient and specific target sequence are prerequisites for the construction of sgRNA expression vector. A group of sgRNA sequences recognizing the 5'-end target site (sgRNA1-sgRNA7) and the 3'-end target site (sgRNA8-sgRNA15) were designed and synthesized. The 5'-end target site and the 3'-end target site were located in exon 4 and exon 7 of the IL4Ra gene, respectively. The sequence of the target site of each sgRNA on IL4Ra was as follows:

TABLE-US-00013 sgRNA1 target site sequence (SEQ ID NO: 65): 5'-AACATAGACTGGCGTTCACCTGG-3' sgRNA2 target site sequence (SEQ ID NO: 66): 5'-TCCGCACTTCCACGTGTGAGTGG-3' sgRNA3 target site sequence (SEQ ID NO: 67): 5'-GTGGTTCCTGGATAGCGCTGTGG-3' sgRNA4 target site sequence (SEQ ID NO: 68): 5'-ATCCAGGAACCACTCACACGTGG-3' sgRNA5 target site sequence (SEQ ID NO: 69): 5'-TATGTTGTGCTGTATGCTTGTGG-3' sgRNA6 target site sequence (SEQ ID NO: 70): 5'-CTCAGCTCTGCCTACACTACAGG-3' sgRNA7 target site sequence (SEQ ID NO: 71): 5'-AGAACATCAGCCTGTAGTGTAGG-3' sgRNA8 target site sequence (SEQ ID NO: 72): 5'-CTACTATACGGCGCGTGTGAGGG-3' sgRNA9 target site sequence (SEQ ID NO: 73): 5'-GATGTCAGGGGTCTACTATACGG 3' sgRNA10 target site sequence (SEQ ID NO: 74): 5'-TATAGTAGACCCCTGACATCAGG-3' sgRNA11 target site sequence (SEQ ID NO: 75): 5'-ACGTGTGTCGGTTCCCAGCCTGG-3' sgRNA12 target site sequence (SEQ ID NO: 76): 5'-CTGACATCAGGATGTTGATCGGG-3' sgRNA13 target site sequence (SEQ ID NO: 77): 5'-GTTGATCGGGAAGCTCAGCCTGG-3' sgRNA14 target site sequence (SEQ ID NO: 78): 5'-ATGTGACCTACAAGGAACCCAGG -3' sgRNA15 target site sequence (SEQ ID NO: 79): 5'- AGTCTATAATGTGACCTACAAGG -3'

[0474] The activity of multiple sgRNAs were detected using the UCA kit. The results showed that sgRNAs had different activities, and the detection results were shown in FIGS. 24A-24B and the table below. Two of the sgRNAs (sgRNA1 and sgRNA14 respectively) were selected for subsequent experiments. The positive clones can be obtained by transfecting the embryonic stem cells of C57BL/6 mice with the sgRNA1, sgRNA14 and Cas9 mRNAs together with the recombinant vector. In addition, due to the double-strand breakage of genomic DNA caused by Cas9-induced cleavage, the homologous recombination repair mechanism can randomly introduce insertions/deletions. Therefore, this method can also generate IL4Ra knockout mice.

TABLE-US-00014 TABLE 5 sgRNA activity test results 5'-end target site detection result 3'-end target site detection result Con. 1.00 Con. 1.00 PC 335.74 PC 403.91 sgRNA-1 181.76 sgRNA-8 57.47 sgRNA-2 13.08 sgRNA-9 90.55 sgRNA-3 63.34 sgRNA-10 141.49 sgRNA-4 100.09 sgRNA-11 41.83 sgRNA-5 189.77 sgRNA-12 88.48 sgRNA-6 10.68 sgRNA-13 53.77 sgRNA-7 62.37 sgRNA-14 196.53 / / sgRNA-15 89.55

Example 3: Mice with Both Humanized IL4/IL4Ra Genes

[0475] Mice containing the humanized IL4 or IL4Ra gene prepared using the methods as described in the present disclosure can also be used to further prepare an animal model with double-humanized IL4/IL4Ra genes or additional humanized genes. For example, the fertilized egg cells used in the microinjection and embryo transfer process can be selected from the embryos of genetically modified mice (e.g., the fertilized egg cells of IL4 mice and/or IL4Ra mice) and can be genetically edited to obtain IL4 and IL4Ra double humanized mice, or mice with additional gene modifications. Mice with humanized IL4 mice and mice with humanized IL4Ra (homozygous or heterozygous) can also mate each other or mate with other genetically modified homozygous or heterozygous mice. Their offspring can be screened. According to Mendel's laws, there is a chance to obtain the IL4 and IL4Ra double humanized mice, or multiple-gene modified heterozygous mice. The obtained heterozygotes can mate each other to finally obtain homozygotes with double- or multiple modified genes.

[0476] The mouse IL4 and IL4Ra genes are located on chromosomes 11 and 7, respectively. IL4 humanized mice (V1) and IL4Ra humanized mice were selected for mating. Positive cloned progeny mice were screened to obtain double-humanized IL4/IL4Ra mice (B-hIL4/hIL4Ra mouse). The protein expression of double-humanized IL4/IL4Ra mice was examined. Three double-humanized IL4/IL4Ra homozygous mice and three wild-type C57BL/6 mice were selected. Retro-orbital blood from these mice was collected. The mice were then stimulated by intraperitoneal injection of 7.5 .mu.g anti-mouse CD3 (mCD3) antibody. Serum and spleen samples were taken after 1.5 hours of stimulation, and the protein levels of IL4 and IL4Ra were detected by ELISA and flow cytometry. ELISA results showed that expression of mouse or human IL4 protein cannot be detected in either unstimulated double-humanized IL4/IL4Ra mouse homozygotes or wild-type C57BL/6 mice (FIG. 25A and FIG. 25B); however, human IL4 but not mouse IL4 protein expression was detected in anti-mCD3 antibody stimulated double-humanized IL4/IL4Ra homozygous mice. In contrast, in anti-mCD3 antibody stimulated wild-type C57BL/6 mice, only mouse IL4 protein expression was detected but no human IL4 protein was detected (FIGS. 25C-25D).

[0477] The anti-mouse IL4Ra antibody (mIL4RA PE) combined with anti-mouse CD19 antibody (mCD19 APC-Cy7), or anti-human IL4Ra antibody (hIL4RA PE) combined with anti-mouse CD19 antibody (mCD19 APC-Cy7) were used to detect the expression of IL4Ra protein in mouse spleen cells by flow cytometry. The results showed that the expression of humanized IL4Ra protein was detected in double-humanized IL4/IL4Ra mice, but the expression of mouse IL4Ra protein was not detected. No human or humanized IL4Ra protein was detected in wild type C57BL/6 mice. The detection results were shown in FIGS. 26A-26H.

[0478] The binding activity of humanized IL4Ra expressed in homozygous double-humanized IL4/IL4Ra mice to anti-human IL4Ra antibody was analyzed. Spleen cells from double-humanized IL4/IL4Ra homozygous mice stimulated by anti-mCD3 antibody were selected and divided into 3 groups. One randomly selected group was added with anti-human IL4Ra antibody (Dupilumab)/anti-hIgG-AF647 and anti-mouse CD19 antibody (mCD19 APC-Cy7) for staining. The control group was added with anti-IgG4-kappa (hIgG-APC)/anti-hIgG-AF647 or only anti-hIgG-AF647, and anti-mouse CD19 antibody (mCD19 APC-Cy7) for staining. The stained cells were washed with PBS, and then protein expression was detected by flow cytometry. The results showed that the anti-human IL4Ra antibody (Dupilumab) binds well to IL4Ra expressed in the double-humanized homozygotes (FIG. 27C) compared to the control groups (FIG. 27A or 27B).

[0479] Spleen cells from unstimulated double-humanized IL4/IL4Ra mice were further analyzed, and lymphocytes were sorted and compared to cells from wild-type C57BL/6 mice. Blood tests and other biochemical tests were performed on double-humanized IL4/IL4Ra mice and no obvious differences were observed as compared to wildtype mice.

[0480] In addition, spleen lymphocytes from double-humanized IL4/IL4Ra mice and wild-type C57BL/6 mice were divided into 3 groups. Lipopolysaccharide (LPS) was added to group G1, mIL4 was added to group G2, and hIL4 (50 ng/mL) was added to group G3. IgE levels of each group were determined on day 6, and the results were shown in FIG. 28, indicating that the in vitro induced IgE levels of spleen lymphocytes from double-humanized IL4/IL4Ra mice were comparable to the IgE levels from wild-type C57BL/6 mice. It further indicated that spleen cells isolated from double-humanized IL4/IL4Ra mice had functional IL4/IL4Ra signaling pathway.

[0481] In another experiment, double-humanized IL4/IL4Ra mouse spleen lymphocytes were first treated with different doses of anti-human IL4Ra antibody Dupilumab (0.01 ng/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL). After incubation for 0.5 hours, LPS and hIL4 (50 ng/mL) were added, and IgE levels of each group were determined on day 6. The results were shown in FIG. 29, indicating that at each test concentration, in vitro IgE production by exposing B cells to hIL4 was effectively blocked by anti-human IL4Ra antibody Dupilumab.

Example 4: Ovalbumin (OVA) Combined with Aluminum Hydroxide-Induced Asthma Model

[0482] Single-gene (IL4Ra) or double-humanized IL4/IL4Ra mice (5-8 weeks) were selected and exposed 3 times to ovalbumin (OVA) combined with aluminum hydroxide by intraperitoneal injection. After 3 weeks of the first injection, nebulization with 2% OVA was performed continuously for 5 days to make an inducible asthma model (modeling protocol was shown in FIG. 30). In the control group, OVA was replaced by PBS. All samples were obtained for analysis on day 26. When modeled with double-humanized IL4/IL4Ra mice, compared to the control group (PBS), the mice had typical symptoms such as elevated serum IgE levels and pathological lung histology features (FIGS. 31-32). Infiltrating cell analysis in bronchoalveolar lavage fluid (BALF) suggested an increase in total number and proportion of eosinophils (Eos) cells among CD45+ cells (FIGS. 33A-33C), indicating that double-humanized IL4/IL4Ra mice have IL4/IL4Ra signaling pathway in vivo. Treatment results with anti-human IL4 antibody or anti-human IL4Ra antibody can be evaluated by routine methods such as airway reactivity test, hematoxylin and eosin (HE) staining, immunohistochemistry (IHC) pathology detection, inflammatory cells and IgE detection to assess the efficacy of antibodies.

[0483] A number of double-humanized IL4/IL4Ra mice were randomly divided into 4 groups. The asthma model was induced according to the method above, in which the G3 and G4 groups were administered with Dupilumab. Different dosing schedules were followed after sensitization by intraperitoneal injection with anti-human IL4RA antibody Dupilumab (25 mg/kg) (see FIG. 34 for the dosing schedule). The results showed that the number of leukocytes (CD45+ cells) and eosinophils cells (Eos) in bronchoalveolar lavage fluid (BALF) was slightly higher in the OVA-inducing group (G2) than in the control group (G1) and the treatment group (G3, G4). The ratio of eosinophils cells (Eos) in CD45+ cells was the highest in the OVA-induced group (G2), the lowest in the G3 treatment group, and slightly lower in the G4 treatment group than in the control group (G1) (FIGS. 35A-35C). High serum IgE levels were only detected in the OVA-inducing group (G2) (FIG. 36). H&E staining showed that the airway of the control (G1) (PBS) mice had no inflammation, whereas peribronchial and perivascular inflammation was significantly increased in the OVA-induced group (G2) mice, with increased mucus secretion levels. In both of the treatment groups (G3, G4), inflammatory infiltration and mucus secretion were observed (compared to the G2 group) at reduced levels (FIG. 37).

TABLE-US-00015 TABLE 6 Alum/ovalbumin Group Mice sensitization Challenge Drug G1 B-hIL4/hIL4Ra PBS PBS NA G2 B-hIL4/hIL4Ra Al(OH).sub.3 + OVA 2% OVA NA G3 B-hIL4/hIL4Ra Al(OH).sub.3 + OVA 2% OVA Dupilumab (25 mg/kg) G4 B-hIL4/hIL4Ra Al(OH).sub.3 + OVA 2% OVA Dupilumab (25 mg/kg)

[0484] In another experiment, C57BL/6 wild-type mice and humanized IL4/IL4Ra mice were selected according to a similar scheme. The specific groupings were shown in the table below. The treatment group (G3) dosage schedule was shown in FIG. 38. All samples were obtained for analysis on day 26, and the results were consistent with the previous ones. The number of leukocytes (CD45+ cells), eosinophils (Eos) and neutrophils in bronchoalveolar lavage fluid (BALF) was higher in the OVA induction group (G2, G4), and was lower in the control group (G1) and the treatment group (G3) (see FIG. 39). Higher serum IgE levels were detected in the OVA-induced group (G2, G4) (FIG. 40). H&E staining results showed that the airway of the control (G1) (PBS) mice showed no inflammation, while the peribronchial and perivascular inflammation of the G2 and G4 mice in the OVA-induced group increased significantly with increased mucus secretion levels. Decreased inflammatory infiltration and mucus secretion were observed in the treatment group (G3) mice compared to the OVA-induced groups (see FIG. 41).

TABLE-US-00016 TABLE 7 Alum/ovalbumin Group Mice sensitization Challenge Drug G1 B-hIL4/hIL4Ra PBS PBS NA G2 B-hIL4/hIL4Ra Al(OH).sub.3 + OVA 2% OVA NA G3 B-hIL4/hIL4Ra Al(OH).sub.3 + OVA 2% OVA Dupilumab (25 mg/kg) G4 C57BL/6 WT Al(OH).sub.3 + OVA 2% OVA NA

Example 5: Mouse Asthma Models with Both Humanized IL4 (Version 2) and IL4Ra Genes

[0485] IL4 humanized mice (Version 2) and IL4Ra humanized mice were selected for mating to obtain double-humanized IL4/IL4Ra mice (B-hIL4 (V2)/hIL4Ra mouse). Similar protein expression and phenotypic analysis were performed using B-hIL4 (V2)/hIL4Ra mouse. The mice appeared normal. A few B-hIL4 (V2)/hIL4Ra mice were selected and randomly divided into 3 groups. The asthma model mice were induced and treated according to the G3 dosing schedule in FIG. 34. Different groups were shown in Table 8. All samples were collected for analysis on day 26, and the results were consistent with the results in Example 4. The number of leukocytes (CD45+ cells), eosinophils (Eos) and neutrophils in bronchoalveolar lavage fluid (BALF) in isotype control group (G2) was higher than treatment group (G3). The control group (G1) had the lowest level of leukocytes (CD45+ cells), eosinophils (Eos) and neutrophils in BALF (FIGS. 44A-44B). Higher serum IgE levels were detected in the isotype control group (G2) (FIGS. 45A-45B). H&E staining results were consistent with the B-hIL4/hIL4Ra mouse results in Example 4.

TABLE-US-00017 TABLE 8 Alum/ovalbumin Group Mice sensitization Challenge Drug G1 B-hIL4(V2)/IL4Ra PBS PBS NA G2 B-hIL4(V2)/IL4Ra Al(OH).sub.3 + OVA 2% OVA hIgG4(25 mg/kg) G3 B-hIL4(V2)/IL4Ra Al(OH).sub.3 + OVA 2% OVA DUPIXENT(25 mg/kg)

[0486] The experiments above (both Examples 4 and 5) showed that the anti-human IL4Ra antibody Dupilumab can block the IL4/IL4Ra signaling pathway in humanized IL4/IL4Ra mice, thereby reducing the number of eosinophils (Eos) in bronchoalveolar lavage fluid (BALF) and reducing IgE levels in serum to reduce inflammatory symptom. The results indicated that the humanized IL4/IL4Ra mice can be used as asthma models, can be used in the preclinical studies to screen and evaluate the in vivo efficacy of anti-human IL4/IL4Ra antibodies, and can be used to characterize anti-human IL4 and/or IL4Ra antibody characteristics.

Other Embodiments

[0487] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence CWU 1

1

821605DNAMus musculus 1ttgttagcat ctcttgataa acttaattgt ctctcgtcac tgacggcaca gagctattga 60tgggtctcaa cccccagcta gttgtcatcc tgctcttctt tctcgaatgt accaggagcc 120atatccacgg atgcgacaaa aatcacttga gagagatcat cggcattttg aacgaggtca 180caggagaagg gacgccatgc acggagatgg atgtgccaaa cgtcctcaca gcaacgaaga 240acaccacaga gagtgagctc gtctgtaggg cttccaaggt gcttcgcata ttttatttaa 300aacatgggaa aactccatgc ttgaagaaga actctagtgt tctcatggag ctgcagagac 360tctttcgggc ttttcgatgc ctggattcat cgataagctg caccatgaat gagtccaagt 420ccacatcact gaaagacttc ctggaaagcc taaagagcat catgcaaatg gattactcgt 480agtactgagc caccatgctt taacttatga atttttaatg gttttatttt taatatttat 540atatttataa ttcataaaat aaaatatttg tataatgtaa cagaaaaaaa aaaaaaaaaa 600aaaaa 6052140PRTMus musculus 2Met Gly Leu Asn Pro Gln Leu Val Val Ile Leu Leu Phe Phe Leu Glu1 5 10 15Cys Thr Arg Ser His Ile His Gly Cys Asp Lys Asn His Leu Arg Glu 20 25 30Ile Ile Gly Ile Leu Asn Glu Val Thr Gly Glu Gly Thr Pro Cys Thr 35 40 45Glu Met Asp Val Pro Asn Val Leu Thr Ala Thr Lys Asn Thr Thr Glu 50 55 60Ser Glu Leu Val Cys Arg Ala Ser Lys Val Leu Arg Ile Phe Tyr Leu65 70 75 80Lys His Gly Lys Thr Pro Cys Leu Lys Lys Asn Ser Ser Val Leu Met 85 90 95Glu Leu Gln Arg Leu Phe Arg Ala Phe Arg Cys Leu Asp Ser Ser Ile 100 105 110Ser Cys Thr Met Asn Glu Ser Lys Ser Thr Ser Leu Lys Asp Phe Leu 115 120 125Glu Ser Leu Lys Ser Ile Met Gln Met Asp Tyr Ser 130 135 1403642DNAHomo sapiens 3tgcatcgtta gcttctcctg ataaactaat tgcctcacat tgtcactgca aatcgacacc 60tattaatggg tctcacctcc caactgcttc cccctctgtt cttcctgcta gcatgtgccg 120gcaactttgt ccacggacac aagtgcgata tcaccttaca ggagatcatc aaaactttga 180acagcctcac agagcagaag actctgtgca ccgagttgac cgtaacagac atctttgctg 240cctccaagaa cacaactgag aaggaaacct tctgcagggc tgcgactgtg ctccggcagt 300tctacagcca ccatgagaag gacactcgct gcctgggtgc gactgcacag cagttccaca 360ggcacaagca gctgatccga ttcctgaaac ggctcgacag gaacctctgg ggcctggcgg 420gcttgaattc ctgtcctgtg aaggaagcca accagagtac gttggaaaac ttcttggaaa 480ggctaaagac gatcatgaga gagaaatatt caaagtgttc gagctgaata ttttaattta 540tgagtttttg atagctttat tttttaagta tttatatatt tataactcat cataaaataa 600agtatatata gaatctaaaa aaaaaaaaaa aaaaaaaaaa aa 6424153PRTHomo sapiens 4Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu Ala1 5 10 15Cys Ala Gly Asn Phe Val His Gly His Lys Cys Asp Ile Thr Leu Gln 20 25 30Glu Ile Ile Lys Thr Leu Asn Ser Leu Thr Glu Gln Lys Thr Leu Cys 35 40 45Thr Glu Leu Thr Val Thr Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr 50 55 60Glu Lys Glu Thr Phe Cys Arg Ala Ala Thr Val Leu Arg Gln Phe Tyr65 70 75 80Ser His His Glu Lys Asp Thr Arg Cys Leu Gly Ala Thr Ala Gln Gln 85 90 95Phe His Arg His Lys Gln Leu Ile Arg Phe Leu Lys Arg Leu Asp Arg 100 105 110Asn Leu Trp Gly Leu Ala Gly Leu Asn Ser Cys Pro Val Lys Glu Ala 115 120 125Asn Gln Ser Thr Leu Glu Asn Phe Leu Glu Arg Leu Lys Thr Ile Met 130 135 140Arg Glu Lys Tyr Ser Lys Cys Ser Ser145 15054220DNAArtificial SequenceSynthetic primer 5gtcatcagat tttgatctga gtcacctcac tggagtcctg actccaggtc ttaactctac 60catgatactg 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1800ccccaccctg gctctcgcgc aaggtcaacc ccagccaagc tcaactgagg cgggatagtt 1860gcttctccta gaggcgaccg acctttcaaa tgcttctaga gaagcccacc ctcggttaag 1920agcactggct gctcttccag aggacgctaa ttcagttctc agtacctaca agaaggctca 1980caaccacctg taacttcagt gtcaggggtc tcaacactct cttccggacc ccagaggcat 2040cgggtatatg tttggtacac acatgaaagc aaaataatca gatgtataaa attaataatt 2100ctaaaaaaaa aaaaaagcca aattgaaaca aagaatactc ctctacctgt acagcgaagg 2160atcagcttca gagccagaga gatgctcatc ggggagacgc tcgctcacca tgtggctgag 2220tggatctgta ggacatcaca gaagtggaag agggaaacca agcccaagcc ataagtaaat 2280aactcattta tctactaatt tattttaggt tttcgagaca gggtttctct gtactctgtg 2340tagccctggc tgtcctgaaa ctcactctgt agaccaggct gctctgaact caagagatcc 2400ccctgcctct gcctcccagt gctgggatta aaggtgtgcg ccaccactgc tcagcttaat 2460cttttttttt tttcttgtaa tgaacccctt tcacccaaag agagctcagg cctttaccct 2520ggctcgtcgc ccaggggaac agacttgaaa tttgcctcgt tgcttatcat cagtggctgg 2580aaggtaacag tttcccaaga attgactgtg tccctaggcc tacagaacat gactgtgtcc 2640aactttacaa cagtcacagt taatatatat atatttgtaa gtggggtatg gtggcttata 2700tctgtaactt caacacttga gaggtggagg caggagagtg accatgaatc tgagggcagc 2760cttacctata taagttccta ggtagctttg acttcagagt gagacccttc aaattttttt 2820attttaaaag ttaaaaagaa agaaagaaaa aaagaaagaa agaaagaaag aaagaaagaa 2880agaaagaaag aaagaaagaa agaaaggagg aaggaaggaa agaagaaggg aaggaaggct 2940tatatattat aaacatcttc agtttttatt aattaaataa cagtaatgga aaaataattt 3000aacacacaat gacaacctta gcaacaacta tttctagatg gtcttccaga aatgagaagt 3060cagaattgga ggcagggact tgggggatat gaagaaccct ttctactctg taaaacagag 3120cttcgcttcc aagcagatgg caggggtgcg gtgctcactg ctggtggggg gcaatgagta 3180cctcgacagg gcctgcattc gttagattaa ggcagtgggc tgggggcagg gtgctgactg 3240agagggtgag agagggctgt ttcctggtta gtgaaggata tgttttaagt tcattgaaaa 3300ggcagccagc cattcttggg ccaatgagat ggctcaggag ggaaggtgct tgccgccaag 3360cttgtgagtc tgagttcaag gatccacacg gtgcaaagag agacccggtc tcctgacctc 3420cacactgatg ctgtagtgca catagataca cacatgctca catgaagtaa tttaaaaaaa 3480ttttttttta aatcagccat ttctcaggct tctgtctaag gtaggaaaaa tcttcaacct 3540agcccagaac ctccatatag gtaaagcctc attccatggt cctgcctgcc ccactccatg 3600tcacctctct gtctccaaag accacaaact tgtaagatca gctggtctag gatgcgagaa 3660ggtctgcctc catcatcctt ctatgaggta agaccccaga gtcagctttc ccaagatatc 3720agagtttcca aggggccccc atagcaggaa gcagctaggc ccaggtgtgc tcaaggcaga 3780ctttcttgat attactctgt ctttccccag ggcgacacca gcaccctcgg acacctgtga 3840cctcttcctt ctctgcagga ggagagccag tggcaaccct acgctgataa gattagtctg 3900aaaggccgat tatggtgtaa tttcctatgc tgaaactttg tagatttaaa aaaaaagggg 3960ggggaggggt gtttcatttt ccaattggtc tgatttcaca ggaaaattta cctgtttctc 4020ttttttctcc tggaagagag gtgctgattg gcccagaata actgacaatc tggtgtaata 4080aaattttcca atgtaaactc attttccctt ggtttcagca actttaactc tatatataga 4140gagacctctg ccagcattgc attgttagca tctcttgata aacttaattg tctctcgtca 4200ctgacggcac agagctattg 422064085DNAArtificial SequenceSynthetic primer 6cagaggtaga agaaaactta ttccctggaa ttgtcctctg actcccctcc caaaacctct 60aacacgcatc tctctctctc tctctctctc tctctctctc tctctctctc acacacacat 120acacacacac atacacacac acacacacaa atgaggggta tgcatatata cacatgtata 180cacatacata cacacaaatg agatggtatg catactcaca catgtgcaca cacacagatg 240aggagtatgt acacacacac acatgcacac acactaagta aataaaacag agaagaacag 300aggcatggca ctacacccta ccttccatgg ggagggatta ctgagacaac agagcctgcg 360gcttgtgaaa caagaaccag aaatcaaaaa ataatttttt gaaaggacaa ggaatcggta 420gcacttacct acaatccagt ttgaagcagg ccagcatgca tccccagaat ccacataaaa 480gtggaaagag aaaatcaact ctacaaagat gtgttctgac ctccacatac gcatcataca 540tacacacagt catcatatat atatatatat atatatatat atatatatat atatatatat 600atatattaga gaaaatcaag agatttaagt gtttaaaact gctaacacag tagaactaca 660aaacttgact acaaaacatg tcatagggaa gccacacatg tgggtgtggc ctgagacctg 720tgggggactg tggtggattg aacgaaaatg gccgccatag acccatatgc ttgaatactt 780ggtccaccgt tggtggaact gtttgggaag gattaggagg tgtggccccg ttggaggagg 840agtgtcactg gggcaggctt tgaggtttca aaagcccagg ctattcccag tgtgttctct 900ctgcccccag ctttcagatc aagacatgag ctctgacaca gtgcctctgt tctgccatca 960tgtactctaa acccagaaac ctatagtccc aaattaaaca cttcctttta taagttgctt 1020tggtcatggt gtcttgtcca ttaagagcag taactaagac agggcaggga gactgtttaa 1080agaacaggag taattggtta ggtgtggtgg agtacacttt taattccagc acttgggaag 1140cacaggcagg ggtagctctg tgagtctgaa gccagcctgg tcggtatatt gagctccagg 1200ccagccagag ctacataatg agaacctgtt tctaaagtgg gggggtgggg ggtgtgagtt 1260gctgggcagg aatgatggct cagtggctaa gaccacctgc tgctcttggt tgtttccaag 1320caaccacata ctggctcaca attatctgta gctccagggg atttgacacc tctggcttct 1380gtgggcactg aactcacatg cacatatcca tgtgcagaca cacacatcta cacataactt 1440acagaaaaaa aaattacata tatatatata tatatatata catatacata tatatatata 1500tacacatata catatatata catatatata tatatatata tatatgtatg taaagttgat 1560aattaaaaat atagctaagg atagtcacga attggggctg gagagacagc tcagtggtta 1620tgagcaccaa ctgctcttcc agaggaccct ggttcaagtc ccagcaacca catggtggat 1680cacaaccatc tgtaactcca gttcttgggc atctgacacc atttcctgac ctctgtgagt 1740acttcgtgca gaacacagac atgcatgcag tcaatgccct cacacacaca aaagcaattt 1800gaacaaatct caatcttaat taacgctgtt attaaaaggg aaggaatcga gaggagagaa 1860cccaacggag ctccacactg tcaagcttgc ccgtcagaag atgctctatg cagtctcttc 1920tactagaggg atctagtaac agtgacttgg cactcttctg accctcaatc acatgcatgt 1980gacaaaaatt aacaccaaca ctgacatcac cccaaaaata cataccaact ttttaacccc 2040ttcaaaataa ggggaaaaaa atcaaaagct taaaatctca ccctgaccac aggcagtttc 2100accctgccca catgaaatac cagcatgcca agggcaatag cggcttctaa ggccaatcag 2160ggaaggtatg cagtgaaaaa tttaatttcc tctcttttga ttgaaactca tttgcatgtc 2220ctggctaatg tgagccctgt ctgccacagg atatgagttg tgggtttgca cttggtatat 2280gaggctgaga tgataaagga acggctgtca aaaagaaaga cctaaaatag accacgggac 2340ccaggcaggt tctaggtcag tcagtcacac ctacacttgg atggtcagaa cacactaact 2400cttttgtttg cctccacata gggagcccta gactctggac ttaaacatcc aatgctcagc 2460ctctgattcg atcaagggaa gaagaggtaa ggccagccat aaaacacgcg tggattaagg 2520gaacttgagg attaagtaag tcgtgaggct tgtgactgga gcagttactg aggccaccag 2580ccacagtcag tgctcagtgg agtgtgttcg ccacagatgg tgaaggtgga actgttaact 2640ctctctccca tgatgcacat gggctctgct tttgccctca gccagtggat gggttatctc 2700taactggcat ctggcaatgg gcaataagta cctcatggat aaatgcctaa caaaaccata 2760tatagctgtg tcttgcattc ttggggagcc gggaagaaat gctggcctgg gctggagaga 2820tggctcagtg gttaagagca ctgattgctc ttccagaggt cctgagttca attcccagca 2880accacatggc tcacaacctt ctgtaatggg gtctggtgtg tctgaagaca gctacagtgc 2940actcatacac ataaaataga gacaggtgga tctctgtaag tttgaggcca gcctggtcta 3000cagagtgagt tctaggacat ccagggctat acagagaaac cctgtctcaa aaaaccaaaa 3060gcaagtttaa agtagatttc cccctcctgc tgtactaggg cttgaaccca tggccttgtg 3120cacagcaggg aagcactgta ctatgaggta tacctctaga cctcctggtc tcccatccta 3180aagctatgat gttttcacct tagctctcta gactgccttt gtgtgtgtgc acaagcatgt 3240atgtacatgt gccttgacac taccatgtga agcccagaga tctgggtctt tcttaatcac 3300tatcttactt tttttttttt taataaatgt tagtgtgtgt gcatgtgtgc acataatgtg 3360tgcacctgtg tggcatggtg cgcacgttga agtatgtgag tatctgagat ggaactcagg 3420ccttgaggtt gtgcagcaag tgtcttttac ccgctgagcc atctccccgg catgcggtcc 3480cctcccttct gattagctct tcctgccagt ttcttcccag tgcaagctgc ggaagggacc 3540tgttccttct tggggagaac ctcctgtgta ctgtgaggaa gcatccgtca cctgtcaata 3600aagctggcgg ttggtctatt aactgaggca ggaaatgggg tggaagttcc agtagggaga 3660gaggaactct gggataggca ggtttaggaa gacattctcc cgggattctg agactcaaga 3720aactgaggca aggtaactag ccgtgtagca ctcacagaat agaataaatg gttaattaag 3780gtactgggta gtcaaggagt gggccaaagc ttgtggtcta ggtgtttatt cataaacagt 3840aagtctcaga gtcactgttt tgggcactag ggtgtcagtg gaaaagcctg aggttaaacc 3900ttcttgttct gtattggcca gtgttaaagc agacaaagtc tgctttcatg ctgtgggccc 3960agtgcatgcc cagtgtccat ttgacactac atttgaaaga catgtttgtc cccttggtct 4020aagagtggaa gctggcatat ctacctattg tctctccagc ccacgactgg cgtgtctgtt 4080ctcag 408578537DNAArtificial SequenceSynthetic primer 7atgggtctca cctcccaact gcttccccct ctgttcttcc tgctagcatg tgccggcaac 60tttgtccacg gacacaagtg cgatatcacc ttacaggaga tcatcaaaac tttgaacagc 120ctcacagagc agaaggtgag tacctatctg gcaccatctc tccagatgtt ctggtgatgc 180tctcagtatt tctaggcatg aaaacgttaa cagctgctag agaagttgga actggtggtt 240ggtggcagtc cagggcacac agcgaggctt ctcccctgcc actctttttt ctgagggttt 300gtaggaagtt tcctcagttg gagggagtga gagctgctca tcaaggactt ctctgtccgg 360ttggaggtta actctgtctc ttgctctctc atttctgcct ggaccaagac tctgtgcacc 420gagttgaccg taacagacat ctttgctgcc tccaaggtaa gaagccgtcc cacggtctgt 480tttagcaaat ggggagatcc atccccaaat gtctgaacaa gaaacttgtc taatggaaaa 540cgagcgggcc caaattaact ctaaggtgtt agatgttttc aaagaacgag aagtctgatc 600tttactctta agcatgtttt ggtctttctg gtttcacttg atttagaaga catgtaatag 660aaagcttaca tgctgtagtc ctgactcaga tcctggtcaa agaaaagccc tcttgggttt 720tacttagctt tggcatagtg cctggaacgt aggaggcact caataaatgc ctgttgaatg 780agagaatttt tctggcccat acatttctga aaaaccaaat actctcacag aaacagatat 840tgagatgaca ggttgaggga gctttcattt tgtctaagag acttcctatg gcaacagaaa 900aggtatcgcc agagcccctc ctcttccaca gcctggccac ctaacagccc tctgggttcc 960ggggctggcc gtccagagct cctcagcttg ctctggccgg ccgaactccc ctccagctcg 1020gtctggaacc atcctgctgg gcagcgtcca gcacatccct gcttcgggct gcctgggcac 1080ctcgcctctc tgcctcctgt gctgcctcac ccccacccct ctatctgtag tgggaggaga 1140tagatttgac agctgatagt gcattttctc tgacaaacac atgactacag ccgtatcaat 1200agttttgtgc atttcagttc ctgttttcat ggaaacacac ggctgagaat gaaagcccca 1260aagcctcaat ttcacagtgg tctcctaact acctgctttc catgcaaact agggagatga 1320tatggccagg agtgaagccc tgtgtgttgg gcagggtcac actccagcac ccagaccata 1380gaacagggcc catcctgctt catgagggaa actgctcttc gggcctttag ctggactatc 1440tcatttcatt agttatcccg ggagtccgat acaggatgag attctgaagg gcaaatacac 1500actttttttt ttttttgaga tagggtcttg ttctgtcacc caggctggag tgcagtggtg 1560cgatttcagc tcatagcagc ctccacctcc caggctcaag ctatcttcct acctcagcct 1620cccaagtagc cgggacgaca ggtgtgcacc accacgcctg gctaattttt gtattttttt 1680gtagagatgg agtcttgcca ttttgcccag gcttgtctcg aacttctggg ctcaagcaat 1740ccgtccacct cggcctctca aagtgctggg attagccact gcacctgggc aacagtttat 1800gtgtgtgtgt gtgtgtgtgt gtgtgtgtat atatgtgtgt gtgtatatat atgtgtgtat 1860gtatatatgt gtatgtatat gtgtgtgtgt gtgtgtgtgt gtgtgtataa aatctccaag 1920tccatccaac cgagatggct cctactagaa gccaagagtc caccgggttg agcactgggt 1980ctctggaggc ctgtggcact gctgagaagg ctctaacaaa gccaagggaa gggccacctc 2040actagaagcc aggcctggag gaagggtgag ggctgagggc ctggaggtaa gactgcctgt 2100ggttttagac ccagctctgc cactgactag ctgtgtggct ggccttcagc acatcttcac 2160acctctctgc acctcagttt ccacatgtga agatatgaaa gtgattctga aggtgattgc 2220aaggttgatt ggaatccagc tcttgagtta gtgcaaagtg ttattgtgag atgatataac 2280cacgattaaa agcaagaaca ggtgcagaga agcgatgatt ctaagaagga ggggaccggg 2340ttggaaagga tcaaaccatc caggatgccg agtctggggc aatccatctg ggctgtttct 2400ggaagacccc cgggtgcagg ccaggacact gctgccctcc cgtccttaac tcccctcttc 2460actcagtcct cactcacctc cctctcacac acacaaacat ctcctagaat aatccccact 2520gcctgccttc actcttaccc gtctcatttg cctcccctga acttcatcct cctggagttc 2580acgatctcac tcttcactct tttcttcccc tcgaagattc agcactgctt acttacatgt 2640taagatattt cagaacagtg aaatgttgct attttcaaaa acctacaaag gtggtatgca 2700gaggaaaagg tacttctttg tgttcccaaa gaaaacatct ttccaaaatc cagcctattg 2760attttatttc ttcgggggaa caagaatttt agtatctcta agttgggtag cattctactc 2820ttggcagttg ctggaaagaa ggcactggtc taggtcctgg gcttcacagg taacacctgt 2880cagggtgtct atgaagtcaa ggctgtctga ggaacagcaa agtgggaaga agcaagctgg 2940ctggctgatg aagggtttct tgggtggaca agtagttgga gctatttcct atttaccaaa 3000gagagctaaa gttcataatt ctacagagag ttccataatg aacctcaaat acctctgttt 3060tttgaaggag tttctcatat acagcactag ctgactatcc tgggcaggat gggagataat 3120gaatgcagtg ccaatcgggc tggatttata tggtcctcag tgaggctggt caagaaccga 3180gttagaactc tcacagagtc actgccacag aagaaatctc ccaagtggct gtttcctgac 3240attcccggga gggacaggcc tccttctgag tcactcccta agcagttctg aactgtgagg 3300tcagccaggc tgtccaagtg cactccctga gccactggca gacacactca gcagccagag

3360ctagacaggc aggtggtagg agtccagggc cacggcaggg atggagtgtc gccccctcgc 3420tgcgatacca gagcaactaa aacgttaagg ccttgcacta aagctgccct taggatgcat 3480tcttttaaag tttttccatt taatgcagac tcttttcaat tcttatttta tccttgtttc 3540ctttagaaag tcctttcaaa aatatcttta gagggttttt tcctatacta tgtggccata 3600tacgggtcaa aattaagttt aatttccagg ctccaagcca gcgtttcaga aaaatctcac 3660caaggtttgt ggtaaaagaa gcaaagggct gactttttgg ttttcttgaa tctcactgtt 3720ccctctgcag cagcatgcat gtctgcccac ctccagacac acaggcacca tctgccgccc 3780cccatcagcc cgtgtccctt ccacctcgac tcgcctacaa agcccagaga ggtctgtttc 3840ttggccccca gagcccaaag atactgacac actcttacat ttccaactag aatcaggaac 3900gaggagtgac tctcagtcag ttcattaagt aaatgtcttt ctaaccgctc tgcccatggg 3960acatcacgcc ccacagggga aaggggaagc ttctgtagcc tgggattctg gtgcctcagt 4020ctgggtctag actttcctga aaaaacgtta aaatatgaac tgcattccta gaatttagcc 4080tacataaata agagatgaac acaaagattt ctatagttta ctcactgccg cttatttaca 4140gaagcaaaaa tctgccacga taggggcctg acaaatgaca gtaccactgt gcaatgcgtt 4200tctacgcagc tctcaatccc atgttctcta ataccaccga agggcttagg aaatgcttat 4260ggtatatgta aagagtaaag aagttacaaa acagtatcaa cagttgaccc ctattttaaa 4320aagtattttt aaaagtgtga cgatatttac caaaatatta acagcaatag ttacctctgg 4380ctggtgggat gagtgaatgt atttttgttg aatatatgtt acctttatag taaatatatg 4440ttatcttgat catcagaaaa aaaaatatgt aagaacttga aagctgcttg gacagcgctg 4500ctgatagaaa cccctgagca tcttgtcact gttcttctga ttcagagggt ctgggtgggg 4560caggggtggt ctgagattct gtatttctaa gaagctccca gtgatgtcca tgctgctggt 4620ccatggacca cactttgagt atcaagggac cagagcatgt cgggggagag gctggggata 4680gctttcttta tctgaactgg ataaaggaac tgggctcaag ctaagaaccc tctccaggtt 4740ctgcatcttt gttcttcagt gaaaaatgag aggacacacc aggccaggtt cagactgaga 4800cacaatccct ctcctgggtt cccaatgact tgtctcttgt ccattccctt ctctaaggct 4860aagggccccc aggaagagcc atgtggccag accctcacag ttgctggcat tccaaggaga 4920ttctcactcc gcatcatttg gggccaaaag gccccttaca gaagctctgc ccaaggctca 4980gatcaatggc acctgctccc agagcctcct ctgatctccc aggacacctt tccctgatct 5040gtgcacttat ctcttgctgc ctggcaaaat gtcttagctc ctcacttggg ccatgtgctg 5100ctctcctctc ccatggggag agccacacgg agagtgctgg ccaaagcagc agagttcagg 5160ccaaaggatg tgcactcatt tattcaacag gcatgcagga tttccaggga aagctggatt 5220ttaaaacctc tgggaacaag agcagaacct gactgagagc tcatgtgggc acttttcata 5280gcagaatagc tcatgaggta tagagacacg gacgcagaac gtgggctgta gcgacagatg 5340gtcctgcatt ctagtcccca ctgtgccttt tcctcatggg atgactttat tcaggtaccc 5400tttcggcaaa atcctccaag agaaaggaaa ctgggaggtt ctggggagaa ggctgctgcg 5460tttgcaattg ggagaggttg ttgacagagg tttatgtctg tggcaagcag ccttccttca 5520gtggaatact tgaagacagg tctgtagttg agcaaactca cctccatttg tcctcctgga 5580aagaagaaat caagaggaaa aatctctctc ccatcctcca aatggagctg gcacattgct 5640atctgtggca tttgtctttc cagaacacaa ctgagaagga aaccttctgc agggctgcga 5700ctgtgctccg gcagttctac agccaccatg agaaggacac tcgctgcctg ggtgcgactg 5760cacagcagtt ccacaggcac aagcagctga tccgattcct gaaacggctc gacaggaacc 5820tctggggcct ggcgggcttg gtaagctgca ctgtattcct ggcaagccgg ccgcgtggct 5880cctggtggac agcagcctca cttctaaaca ctccttagga gctgcagcac ccttggtcaa 5940cccattcatt cattcactca ttcaataagt atttgctgaa gttccacaag tgctgggtgt 6000ggttctaggt gctgaggacg tgtcactaaa gacacgcagg ccgagtccct gttctcatgg 6060aatgttctaa tgggagagtt agaaaaacaa acatgtaaaa tgatggccag cagtgatacg 6120tgctacaaag aaaaacatag aaataaagaa cataagagtc atgggggagg gggctgactt 6180aggagctggt gacattatct gagcagatat ttgaattgag ggagcaggcc acatgactaa 6240ctagggagac cattccaggc agaaggagga ggtatgcaaa ggccttagga tggaaatgaa 6300ctaacttcct gtatttaaag accagtagga aggccagtgt ggctggatca gagtgagtga 6360ggggtagttt ccaggacagc agatcacaca aggcctttag attccaccac gagtatggca 6420gggaacacct gcagagcttt gggcaggaca aagactgtac aatctgattt acgtgattta 6480aaagggtcag tctggctact gtgtggtaaa taggctgaaa gggggaaagc atagaagcaa 6540gatggcctgt tgggaggcta ccacagtaaa ccaggctaga gatgatggtg gcgtggacag 6600aatgaagcaa gatggcctgt tgggaggcta ccacagtaaa ccaggctaga gacgatggtg 6660gcgtggacag aatgaagcaa gatggcctgt tgggaggcta ccacagtaaa ccaggctaga 6720gatgatggtg gcgtggacag aatgggagca gttgaggtga acagatttgg gatatgacta 6780aaaataaaac cagaaggatt tgctgacaga tcggttgtag ggggtaagat acaggggagg 6840aaaagatgac ctctttgttc ctgcccaaac ccctctggcg atggtcagta ctgtttacag 6900agagatgaaa gactggcggc aaggcagggc tggaggttca gcagaagatc aagagttcaa 6960ttttgtacat cgtacatgta aggtggctct tggatagcca agtgaaggtg ttgagaagat 7020ggttagaaaa gtctggaact taggggagag gtcagaactt gcaatacaaa aaggagagtc 7080cttagataga tactgctgaa aatctgaatg acagaaaggg agagatcaaa ggactgagcc 7140tgagatcaac acatggaggt caggagagga ggatccagcc aaggggcctg aggaggagtg 7200accagtgagg caggagaaca ctggagagtg ggcggtaccc caggaagccg ttgaggacac 7260tcaaggaggg agggttgact gtgtcaaatg tactgaaagg acaggtcagg tgaggaccaa 7320gaaaggcccc tgggtttggc tgatggaggc catgggtgag gctgatgtaa atggagaggc 7380aggaaggaaa gcccagctgg agtgggctca ccgaggatag ggtggcgaga ggagacaaag 7440aaggaacagt gagggcagaa cactctttga agatgtttag ctataaggct gcagagaaac 7500tgacccacag ctgcagggtg gttatggagt gagggaagct cttttaaggt tgggggtata 7560cccagcatgt taatgcacct gggggaatgg tccagtggag caggaagaac tgaagagagc 7620agaaagagga agaatcatta gggggcagaa gtccttgtag cccagagtgg atgttatcta 7680atatcgagtg gaggaattaa ttggctttag aggagaacaa ggacatgtat cccctctctg 7740ggcctatcac cttgtagaca atgggatagg tcatgggata ggaacttggc acaacacatg 7800ttctctcttt taattctctc cattatctta tgaagcaggc aagtaggcaa acaattgtcc 7860caactttaca aaagaaactg aagcttttat aaattaagta gtacatccta agcaatacaa 7920ttaataaatg gtagagctga gattcaaact gaagcagtgg cctgggggta gcatctggaa 7980tccttcccac ctttagggct gctgtgctgc ggtgctgctg tttaatggca caggagggcc 8040acatgactga atctctctca gcagtccagg cagtcatgca gaaggcccag tagagcaccg 8100ggcaggtctg agccagcatc ttcaagttcc accctctgag caagcaccta gctgtgacac 8160acctctccag agactgcact cccccccgcg ccacccaccc caaaagcaga taggtaatgg 8220tatacagtaa ccatttctag aagtgtaagt agtatgcacc caaaataggc aaaacctgct 8280ggcctagtga tagagacaac tcccagtcag gctagactgg aggccttggt tttataagtg 8340ttcaggtgac aagtgccaca gtaggcttga tcaagtagac aggcaggcaa gacaaatgct 8400taccaatgca agctaatgaa atgtttcttt tgcagaattc ctgtcctgtg aaggaagcca 8460accagagtac gttggaaaac ttcttggaaa ggctaaagac gatcatgaga gagaaatatt 8520caaagtgttc gagctga 85378200DNAArtificial SequenceSynthetic primer 8actttaactc tatatataga gagacctctg ccagcattgc attgttagca tctcttgata 60aacttaattg tctctcgtca ctgacggcac agagctattg atgggtctca cctcccaact 120gcttccccct ctgttcttcc tgctagcatg tgccggcaac tttgtccacg gacacaagtg 180cgatatcacc ttacaggaga 2009149DNAArtificial SequenceSynthetic primer 9tggaaaactt cttggaaagg ctaaagacga tcatgagaga gaaatattca aagtgttcga 60gctgatactg agccaccatg ctttaactta tgaattttta atggttttat ttttaatatt 120tatatattta taattcataa aataaaata 14910155DNAArtificial SequenceSynthetic primer 10acatgcctgt aggcaagaca cccacacaca taaaaacaaa ataaaataag gatagaaagg 60ccagggggat gaatcctcga ggtcgacggt atcgataagc ttgatatcga attccgaagt 120tcctattctc tagaaagtat aggaacttca ggtct 1551175DNAArtificial SequenceSynthetic primer 11agaaagtata ggaacttcat cagtcaggta cataatggtg gatccattaa tcagaggtag 60aagaaaactt attcc 751224DNAArtificial SequenceSynthetic primer 12gacaactttc agggagggag tcag 241321DNAArtificial SequenceSynthetic primer 13atcgcacttg tgtccgtgga c 211422DNAArtificial SequenceSynthetic primer 14gcctctctca acccagtata ac 221520DNAArtificial SequenceSynthetic primer 15cttggagtca accacctctg 201623DNAArtificial SequenceSynthetic primer 16gatctttcac tgaaacttga ctg 231722DNAArtificial SequenceSynthetic primer 17ctccaactac atctactttc tg 221823DNAArtificial SequenceSynthetic primer 18ggatcggcca ttgaacaaga tgg 231923DNAArtificial SequenceSynthetic primer 19cagaagaact cgtcaagaag gcg 23205553DNAArtificial SequenceSynthetic primer 20ctcagggatt cactgcagcc ttaccaggta ccacggatct ggttcctgag cacagcaagg 60ttaggttaca tccagtgaga cagtgagaca acatagagca acagcacaga ggtgccaggc 120acctcggaag ccacagagga gaagccactg gccagtggtt ggggcagagc acaggaagcc 180aagtacacct cactcgtgag atgggtgaaa acagaggtct ggctggctgg agagagtctg 240tgtgagagac gttctttgca ttagcaagac caaagcatca aacaagagta ctggagtgac 300ccaagtcttg atcagacaga cgcacagcac atgaagccta actccggcac tcaggaggta 360taggtaggag ggacaggagt ttgaggactg tgaaaaatgt ctaagacctt gaggccaagc 420cgtatctgaa tcagatttta gcagagccat tctggcagtg agggctgcaa agggcagggt 480cagggagact gcttatcagg ttcctgtaga gacccgggtg ggacaggatg gcagtagaga 540ggggagagtc taattctgga gaggtgtgta tgcaaattac atgtttatac tttatataat 600ataaagtaaa catgcatacg atatctactg cctcttaata tacacactca gtttctatga 660attctatgtt tgtgtagagg atgaatgtct gtgtacaaca tgtagaggca gatgagaagg 720tcaagtatct cccttcttct gaagacagtg tctctcacgg agcctggagc taggctggca 780gccagcgatc ccagtcactc ttctgtcccc atctccacag tgagggggtt cccaggtgcc 840tgtgggacca cgttggcttt tttatatggg tgctggggat ctgaacccag ccctcacgct 900tgcttggcaa gctctcttag cctgaaacat cttcccagcc cagtacttaa atataaaccc 960atgcacactt ttctgcagag ctgcacacat tacccatgtg cactaccact gagctatgcc 1020tctgagccac atatcaactt ttttggtttg gttttatttg tttctagttt tgttttgttt 1080ttgagacaac gtagccctgg ctgtcctgga gcttgctgtg tagaccaggc tggccttgaa 1140ctcacagaga tcatcctacc tctgctgccc aaatactggt attaaaggca tgaaccatca 1200tgccctggcc ccaaatcagt ttttgttttg ttttgttttt gttttttaaa agtgtgtgtg 1260tgtgtgtgtg tgtgtgtgca atgcccccaa aggccaggag agggcatccg atccctggga 1320gctggagtta caggtggtgg aaagtcaact gatgtgggtg ctgggaacct aactgggccc 1380tctgcaagca cggtacaaac tcttgactgc tgagccacct ccccaccttc taagggattt 1440attctaaaaa tgtcaaatat ttacaaaata cccctgagga acccattacc cagcctcaat 1500ggttagcatg tgagggtcga tttgtctcat ctctaccttc cttattcccc tagtgacaat 1560gactggatcc agagtctagg tgagagcaga agctgggtgg ggtggatgta gcctgtgagg 1620tctggacaca ctacgtgtga gaaacctgag ctccagtatg ccatcaaagc tggattgtgg 1680acgccatgtt ccttcctgga gcgtggcacg gcctggcctc ccaagtctca ggccacagca 1740cagggtctcc atcttctccg tcctcccagc ttaccaccgt tccactgcac agagaagtca 1800ctttgctgat gtcacagtgt tatcaatcca tcacccttat ctcctgtcac ctcccacggg 1860cctcggtgac cctcacttct gcttcttgag actcccactc cctggggctc tgaccctgcc 1920tgtgcccggc tcttggttag gcctcctacc cttcttccgg tattaccagt gggtagtggg 1980tcatttctta tcccccttgc ctctctcaac ccagtataac tccatttatg ggaatgcctc 2040catgttcagg gccacagagg ccgctcagct cagcattgct gatctctgac catatctcat 2100acctgctaaa ctcccagaag ctcctgggtc acctgcagcc gacttctcac tcaatccaac 2160gtgcattcgt tgtgagtggt gacccatggc cgccactgct gctggtgatg atcacagctg 2220ggtttggggt tgttttatgt ttgtctttca aacattgtat acttacatat ttttttctaa 2280tgtgccaaat gccacaccaa cactcgacac ttattaccac acatgatttt ctcacaccac 2340ttttctactg ccagccggtt ttactggaat acccagacag aataactttc tcaaagccac 2400tcagaaagat gtctagagct ggattcagag gtggttgact ccaagtcttt ctttgtgtgt 2460gcatatgcat gtgtgtatag atatgtgtgt gcatacgcat gtgtgtatag atgtgtgtgc 2520atatgcatgt atgtatagat atgtgtatag atgtttgtat atgcatgtgt atatagatgc 2580atgtgtgtat agatatgtgt gcatatgcat gtgtgtatag atgcatgtgc atatgcatgt 2640gtgtatagat gtgtgtgcat atgcatgtgt gtatagatgt gtgtatagat gtgtgtgcat 2700atgtgcatgt ttagatgtgt gtggaagtca gaagacaact ttcagggagg gagtcagtta 2760ccccttcctc tgtgtgggcc ctggggattg aacttgggtc atcagatttt gatctgagtc 2820acctcactgg agtcctgact ccaggtctta actctaccat gatactgcta agccgctcaa 2880agccacgggg taccttccca ctctcacact cgggccactc tcaaggaaac tgcagattct 2940aactgcccct atccatccgt tctccctttc tgacaccctt cacttcctct agggcttcct 3000cgacaccaca ctgggccttc cacaggcttc ctcttgcctg gttctggcac tgagccccac 3060agtgccagtc ccagctccta gcctctgagg agctggaagg ggcgccttcc tcagctgcag 3120gagagaaggg aaggcaagtc tgggctgggg caagcaggct gtagggacag acagccaggc 3180ctaaggacac agcggggctg cttgtttctg atcctttctc cttaaatggt gcatgtgaca 3240ccatggtttg acatttccta gtggcaagtc tgagcatctg ctgaagtgac tctgggtgga 3300gatggtttct ccagagagct aggcatggca gaacaaaggt gacgctgtct tcctcgccca 3360gtgggctatg aggacaccaa gtttccttct ggaatgactc ggcaggttga aaggtcatca 3420cagctgcatc tcagcatcgt gtcacccaag ctccaggccc ttctcagagt cctaggcaca 3480gtgtgcgcac acccactccc acagccctct caacactcca cctgcatcca cctccctttc 3540ctgaatgaag tgcccatggt gtatcatggt cagcttcctc tgctatccag gttcataaat 3600ctaaggactg aaacccaaca ctgaaactcc tcagaaacaa gtgtggggac caaagcgtga 3660atgtccagac ttgagtatta tagtcattca gccaaccaaa atgtgatgag taatgaagta 3720cccactgcgt gcagggtgct taactaagta cctgaactta gtacttagaa caacctcaca 3780agacaagtac aactgtcact cctcagtttc gggactgagg ttgagtgtat gcccaaagtc 3840acatgagagg acacagaaca gctaggtctg acaagcagcg ggggtggggg tggggtgggg 3900tggggtgtag agtctttttc cttagtcttg tcgcaatggt atatcaggtg actcaggcta 3960agtgctttct ccatagatct cagacttgtg gtcacaggag ccccagggga gggggtgcac 4020agctttctcc tcactgtgta gaaacccggc cacaagcccc tgccttctgg actccacact 4080ctggtacaca aaactgctca atggctctcg gctcacaaag ggctggagag ggtctatgac 4140agtcactttc ctgccctgtc cctccactga ccaccactgg ctggctgaca ctcttcatcc 4200cacttttcca gcctctgcct tatctgctcc attcacagat aatgtgaatc tgtcacttgg 4260ggtggccttg gacgtccttt ctagtctttg ggaagacttg gagtctggga ctattcagac 4320tcttttctct gactccttcc aaccactgaa acaagcaaga caaacggaac tttgcttctg 4380agcggagttg ccttactcta tccctttctg actatcatct tcccacagag cccagcagtt 4440aaggtgctga gaggaaaacc tgaatggtct attctttgct ccagggacac tgtgggatcc 4500agccctcccc ttccctaggt gtccttctgt gccccttctt agcaggattc ttccagctat 4560ggatgacaag ttcctgctgg atctcagcca ctcgatgccc caccctggct ctcgcgcaag 4620gtcaacccca gccaagctca actgaggcgg gatagttgct tctcctagag gcgaccgacc 4680tttcaaatgc ttctagagaa gcccaccctc ggttaagagc actggctgct cttccagagg 4740acgctaattc agttctcagt acctacaaga aggctcacaa ccacctgtaa cttcagtgtc 4800aggggtctca acactctctt ccggacccca gaggcatcgg gtatatgttt ggtacacaca 4860tgaaagcaaa ataatcagat gtataaaatt aataattcta aaaaaaaaaa aaagccaaat 4920tgaaacaaag aatactcctc tacctgtaca gcgaaggatc agcttcagag ccagagagat 4980gctcatcggg gagacgctcg ctcaccatgt ggctgagtgg atctgtagga catcacagaa 5040gtggaagagg gaaaccaagc ccaagccata agtaaataac tcatttatct actaatttat 5100tttaggtttt cgagacaggg tttctctgta ctctgtgtag ccctggctgt cctgaaactc 5160actctgtaga ccaggctgct ctgaactcaa gagatccccc tgcctctgcc tcccagtgct 5220gggattaaag gtgtgcgcca ccactgctca gcttaatctt tttttttttt cttgtaatga 5280acccctttca cccaaagaga gctcaggcct ttaccctggc tcgtcgccca ggggaacaga 5340cttgaaattt gcctcgttgc ttatcatcag tggctggaag gtaacagttt cccaagaatt 5400gactgtgtcc ctaggcctac agaacatgac tgtgtccaac tttacaacag tcacagttaa 5460tatatatata tttgtaagtg gggtatggtg gcttatatct gtaacttcaa cacttgagag 5520gtggaggcag gagagtgacc atgaatctga ggg 5553215196DNAArtificial SequenceSynthetic primer 21taactcaagt tctgggggag ctgatgctct cctctggcct cctgtggagg tacacagacc 60acatgcctgt aggcaagaca cccacacaca taaaaacaaa ataaaataag gatagaaagg 120ccagggggat gaatccagag gtagaagaaa acttattccc tggaattgtc ctctgactcc 180cctcccaaaa cctctaacac gcatctctct ctctctctct ctctctctct ctctctctct 240ctctcacaca cacatacaca cacacataca cacacacaca cacaaatgag gggtatgcat 300atatacacat gtatacacat acatacacac aaatgagatg gtatgcatac tcacacatgt 360gcacacacac agatgaggag tatgtacaca cacacacatg cacacacact aagtaaataa 420aacagagaag aacagaggca tggcactaca ccctaccttc catggggagg gattactgag 480acaacagagc ctgcggcttg tgaaacaaga accagaaatc aaaaaataat tttttgaaag 540gacaaggaat cggtagcact tacctacaat ccagtttgaa gcaggccagc atgcatcccc 600agaatccaca taaaagtgga aagagaaaat caactctaca aagatgtgtt ctgacctcca 660catacgcatc atacatacac acagtcatca tatatatata tatatatata tatatatata 720tatatatata tatatatata ttagagaaaa tcaagagatt taagtgttta aaactgctaa 780cacagtagaa ctacaaaact tgactacaaa acatgtcata gggaagccac acatgtgggt 840gtggcctgag acctgtgggg gactgtggtg gattgaacga aaatggccgc catagaccca 900tatgcttgaa tacttggtcc accgttggtg gaactgtttg ggaaggatta ggaggtgtgg 960ccccgttgga ggaggagtgt cactggggca ggctttgagg tttcaaaagc ccaggctatt 1020cccagtgtgt tctctctgcc cccagctttc agatcaagac atgagctctg acacagtgcc 1080tctgttctgc catcatgtac tctaaaccca gaaacctata gtcccaaatt aaacacttcc 1140ttttataagt tgctttggtc atggtgtctt gtccattaag agcagtaact aagacagggc 1200agggagactg tttaaagaac aggagtaatt ggttaggtgt ggtggagtac acttttaatt 1260ccagcacttg ggaagcacag gcaggggtag ctctgtgagt ctgaagccag cctggtcggt 1320atattgagct ccaggccagc cagagctaca taatgagaac ctgtttctaa agtggggggg 1380tggggggtgt gagttgctgg gcaggaatga tggctcagtg gctaagacca cctgctgctc 1440ttggttgttt ccaagcaacc acatactggc tcacaattat ctgtagctcc aggggatttg 1500acacctctgg cttctgtggg cactgaactc acatgcacat atccatgtgc agacacacac 1560atctacacat aacttacaga aaaaaaaatt acatatatat atatatatat atatacatat 1620acatatatat atatatacac atatacatat atatacatat atatatatat atatatatat 1680gtatgtaaag ttgataatta aaaatatagc taaggatagt cacgaattgg ggctggagag 1740acagctcagt ggttatgagc accaactgct cttccagagg accctggttc aagtcccagc 1800aaccacatgg tggatcacaa ccatctgtaa ctccagttct tgggcatctg acaccatttc 1860ctgacctctg tgagtacttc gtgcagaaca cagacatgca tgcagtcaat gccctcacac 1920acacaaaagc aatttgaaca aatctcaatc ttaattaacg ctgttattaa aagggaagga 1980atcgagagga gagaacccaa cggagctcca cactgtcaag cttgcccgtc agaagatgct 2040ctatgcagtc tcttctacta gagggatcta gtaacagtga cttggcactc ttctgaccct 2100caatcacatg catgtgacaa aaattaacac caacactgac atcaccccaa aaatacatac 2160caacttttta accccttcaa aataagggga aaaaaatcaa aagcttaaaa tctcaccctg 2220accacaggca gtttcaccct gcccacatga aataccagca tgccaagggc aatagcggct 2280tctaaggcca atcagggaag gtatgcagtg aaaaatttaa tttcctctct tttgattgaa 2340actcatttgc atgtcctggc taatgtgagc cctgtctgcc acaggatatg agttgtgggt 2400ttgcacttgg tatatgaggc tgagatgata aaggaacggc tgtcaaaaag aaagacctaa 2460aatagaccac gggacccagg caggttctag gtcagtcagt

cacacctaca cttggatggt 2520cagaacacac taactctttt gtttgcctcc acatagggag ccctagactc tggacttaaa 2580catccaatgc tcagcctctg attcgatcaa gggaagaaga ggtaaggcca gccataaaac 2640acgcgtggat taagggaact tgaggattaa gtaagtcgtg aggcttgtga ctggagcagt 2700tactgaggcc accagccaca gtcagtgctc agtggagtgt gttcgccaca gatggtgaag 2760gtggaactgt taactctctc tcccatgatg cacatgggct ctgcttttgc cctcagccag 2820tggatgggtt atctctaact ggcatctggc aatgggcaat aagtacctca tggataaatg 2880cctaacaaaa ccatatatag ctgtgtcttg cattcttggg gagccgggaa gaaatgctgg 2940cctgggctgg agagatggct cagtggttaa gagcactgat tgctcttcca gaggtcctga 3000gttcaattcc cagcaaccac atggctcaca accttctgta atggggtctg gtgtgtctga 3060agacagctac agtgcactca tacacataaa atagagacag gtggatctct gtaagtttga 3120ggccagcctg gtctacagag tgagttctag gacatccagg gctatacaga gaaaccctgt 3180ctcaaaaaac caaaagcaag tttaaagtag atttccccct cctgctgtac tagggcttga 3240acccatggcc ttgtgcacag cagggaagca ctgtactatg aggtatacct ctagacctcc 3300tggtctccca tcctaaagct atgatgtttt caccttagct ctctagactg cctttgtgtg 3360tgtgcacaag catgtatgta catgtgcctt gacactacca tgtgaagccc agagatctgg 3420gtctttctta atcactatct tacttttttt ttttttaata aatgttagtg tgtgtgcatg 3480tgtgcacata atgtgtgcac ctgtgtggca tggtgcgcac gttgaagtat gtgagtatct 3540gagatggaac tcaggccttg aggttgtgca gcaagtgtct tttacccgct gagccatctc 3600cccggcatgc ggtcccctcc cttctgatta gctcttcctg ccagtttctt cccagtgcaa 3660gctgcggaag ggacctgttc cttcttgggg agaacctcct gtgtactgtg aggaagcatc 3720cgtcacctgt caataaagct ggcggttggt ctattaactg aggcaggaaa tggggtggaa 3780gttccagtag ggagagagga actctgggat aggcaggttt aggaagacat tctcccggga 3840ttctgagact caagaaactg aggcaaggta actagccgtg tagcactcac agaatagaat 3900aaatggttaa ttaaggtact gggtagtcaa ggagtgggcc aaagcttgtg gtctaggtgt 3960ttattcataa acagtaagtc tcagagtcac tgttttgggc actagggtgt cagtggaaaa 4020gcctgaggtt aaaccttctt gttctgtatt ggccagtgtt aaagcagaca aagtctgctt 4080tcatgctgtg ggcccagtgc atgcccagtg tccatttgac actacatttg aaagacatgt 4140ttgtcccctt ggtctaagag tggaagctgg catatctacc tattgtctct ccagcccacg 4200actggcgtgt ctgttctcag gggcacaata tacaccacaa gcgggaaggt ggtcagtaga 4260gtcctgctgc agcgcaggag agacacattt taatttttat gatttttgtg tgtgcatgtg 4320tgtggtgtgt gtgcaaatgt gtgcatttgc ttgtgtgggc ccacgtacac agtccagagg 4380ataacattgg gtgtcctgtg ctatcattct gccttatgtc tttttttttt tttttttggt 4440tttttgagac agggtttctc tgtgtagccc tggctgtcct ggaactcact ttgtagacca 4500ggctggcctc gaactcagaa atccgcctgc ctctgcctcc tgagagctgg gattaaaggc 4560gtgcgctacc acgcccggct gccttatgtc tttaagacag gatctttcac tgaaacttga 4620ctgtgtctgc cccagcagtg ctggggtcac aggcaccctt gaccatgctc agcctttaga 4680tgagtgctgg agatgtgaat tcaggtcctg atgccacctt agtaagtgtt cttgccccac 4740tgagccatct ccccaacccc aagatatact caagagggca ccagggcact taaacattgc 4800tagtggcaaa gaaagctggg gcagctactg caggaactgt atggagggtt ctgaagaatt 4860aaatattata tctctgttgg actgagaacc caacagagat gcttgtacat tcacaatcat 4920tgtggcactg cacagtcatg aatatatgga accaaggcat caacagataa ctagataaag 4980aatatgttgt gcatagacac aatggaattg tgcttaggcg tgaagaatga agtcatgttt 5040acagaaagta gatgtagttg gagattatgt taaataaagc cagattgaga aaaaaaacaa 5100ttttttccat tctatgcaga atctggattt aaatctgtat atttagagca tgtgtgcatg 5160gttaaagtga gagggaatta agagaggcag gagagg 51962210573DNAArtificial SequenceSynthetic primer 22cttccagaat aaattcatag ggaggcccag gcacagtggc tcacgcctgt aatcccagca 60ctttgggagg ccgaggcagg cggatcacga ggtcaggaga tggagaccat cctggctaac 120acggtgaaac cccgtctcta ctaaaaatac aaaaaattag ccgggcgtgg tggcaggtgc 180ctgtagtccc agctactcgg ggaggctgag gcaggagaac ggcatgaacc caggaggcgg 240agcttgcagt gaactaagat cacgccactg cactccagcc tgggtgacag agcaagattc 300catctcaaaa aaaaaaaaaa aaaaaagaaa ttcataggga aaagaaggtc agagaccaag 360ggaagggaag gttctgggag aaaagcaggg ggcaggcagg gcccaagaat cctgctgccc 420atgagccctt actgggaggt ggggtggcct tgcacagggc ccaggcacct gagtgagtgg 480tggggtcctt acgttcactg ctggggtgag gcaatgagca ccttattgtg tccacatgaa 540ttcaataaaa aacaagcagg gcgggtggtg gggcactgac taggagggct gatttgtaag 600ttggtaagac tgtagctctt tttcctaatt agctgaggat gtgtttaggt tccattcaaa 660aagtgggcat tcctggccag gcatggtggc tcacacctgt aatctcagag ctttgggaga 720ctgaggtagg aggatcactt gagcccagga atttgagatg agcctaggca acatagtgag 780actcttatct ctatcaaaaa ataaaaataa aaatgagcca ggcatggtgc ggtggcacgc 840acctactgct aggggggctg aggtgggagg atcacttgag cctgggaggt tgaggctgca 900gtgatccctg atcacaacat tgcatttcag cctgggtgac agagtgagac cctgtctcag 960aaaaaaaaaa aaaaaagtca ttcctgaaac ctcagaatag acctaccttg ccaagggctt 1020ccttatgggt aaggacctta tggacctgct gggacccaaa ctaggcctca cctgatacga 1080cctgtccttc tcaaaacacc taaacttggg agaacattgt cccccagtgc tggggtagga 1140gagtctgcct gttattctgc ctctatgcag agaaggagcc ccagatcagc ttttccatga 1200caggacagtt tccaagatgc cacctgtact tggaagaagc caggttaaaa tacttttcaa 1260gtaaaacttt cttgatatta ctctatcttt ccccaggagg actgcattac aacaaattcg 1320gacacctgtg gcctctccct tctatgcaaa gcaaaaagcc agcagcagcc ccaagctgat 1380aagattaatc taaagagcaa attatggtgt aatttcctat gctgaaactt tgtagttaat 1440tttttaaaaa ggtttcattt tcctattggt ctgatttcac aggaacattt tacctgtttg 1500tgaggcattt tttctcctgg aagagaggtg ctgattggcc ccaagtgact gacaatctgg 1560tgtaacgaaa atttccaatg taaactcatt ttccctcggt ttcagcaatt ttaaatctat 1620atatagagat atctttgtca gcattgcatc gttagcttct cctgataaac taattgcctc 1680acattgtcac tgcaaatcga cacctattaa tgggtctcac ctcccaactg cttccccctc 1740tgttcttcct gctagcatgt gccggcaact ttgtccacgg acacaagtgc gatatcacct 1800tacaggagat catcaaaact ttgaacagcc tcacagagca gaaggtgagt acctatctgg 1860caccatctct ccagatgttc tggtgatgct ctcagtattt ctaggcatga aaacgttaac 1920agctgctaga gaagttggaa ctggtggttg gtggcagtcc agggcacaca gcgaggcttc 1980tcccctgcca ctcttttttc tgagggtttg taggaagttt cctcagttgg agggagtgag 2040agctgctcat caaggacttc tctgtccggt tggaggttaa ctctgtctct tgctctctca 2100tttctgcctg gaccaagact ctgtgcaccg agttgaccgt aacagacatc tttgctgcct 2160ccaaggtaag aagccgtccc acggtctgtt ttagcaaatg gggagatcca tccccaaatg 2220tctgaacaag aaacttgtct aatggaaaac gagcgggccc aaattaactc taaggtgtta 2280gatgttttca aagaacgaga agtctgatct ttactcttaa gcatgttttg gtctttctgg 2340tttcacttga tttagaagac atgtaataga aagcttacat gctgtagtcc tgactcagat 2400cctggtcaaa gaaaagccct cttgggtttt acttagcttt ggcatagtgc ctggaacgta 2460ggaggcactc aataaatgcc tgttgaatga gagaattttt ctggcccata catttctgaa 2520aaaccaaata ctctcacaga aacagatatt gagatgacag gttgagggag ctttcatttt 2580gtctaagaga cttcctatgg caacagaaaa ggtatcgcca gagcccctcc tcttccacag 2640cctggccacc taacagccct ctgggttccg gggctggccg tccagagctc ctcagcttgc 2700tctggccggc cgaactcccc tccagctcgg tctggaacca tcctgctggg cagcgtccag 2760cacatccctg cttcgggctg cctgggcacc tcgcctctct gcctcctgtg ctgcctcacc 2820cccacccctc tatctgtagt gggaggagat agatttgaca gctgatagtg cattttctct 2880gacaaacaca tgactacagc cgtatcaata gttttgtgca tttcagttcc tgttttcatg 2940gaaacacacg gctgagaatg aaagccccaa agcctcaatt tcacagtggt ctcctaacta 3000cctgctttcc atgcaaacta gggagatgat atggccagga gtgaagccct gtgtgttggg 3060cagggtcaca ctccagcacc cagaccatag aacagggccc atcctgcttc atgagggaaa 3120ctgctcttcg ggcctttagc tggactatct catttcatta gttatcccgg gagtccgata 3180caggatgaga ttctgaaggg caaatacaca cttttttttt tttttgagat agggtcttgt 3240tctgtcaccc aggctggagt gcagtggtgc gatttcagct catagcagcc tccacctccc 3300aggctcaagc tatcttccta cctcagcctc ccaagtagcc gggacgacag gtgtgcacca 3360ccacgcctgg ctaatttttg tatttttttg tagagatgga gtcttgccat tttgcccagg 3420cttgtctcga acttctgggc tcaagcaatc cgtccacctc ggcctctcaa agtgctggga 3480ttagccactg cacctgggca acagtttatg tgtgtgtgtg tgtgtgtgtg tgtgtgtata 3540tatgtgtgtg tgtatatata tgtgtgtatg tatatatgtg tatgtatatg tgtgtgtgtg 3600tgtgtgtgtg tgtgtataaa atctccaagt ccatccaacc gagatggctc ctactagaag 3660ccaagagtcc accgggttga gcactgggtc tctggaggcc tgtggcactg ctgagaaggc 3720tctaacaaag ccaagggaag ggccacctca ctagaagcca ggcctggagg aagggtgagg 3780gctgagggcc tggaggtaag actgcctgtg gttttagacc cagctctgcc actgactagc 3840tgtgtggctg gccttcagca catcttcaca cctctctgca cctcagtttc cacatgtgaa 3900gatatgaaag tgattctgaa ggtgattgca aggttgattg gaatccagct cttgagttag 3960tgcaaagtgt tattgtgaga tgatataacc acgattaaaa gcaagaacag gtgcagagaa 4020gcgatgattc taagaaggag gggaccgggt tggaaaggat caaaccatcc aggatgccga 4080gtctggggca atccatctgg gctgtttctg gaagaccccc gggtgcaggc caggacactg 4140ctgccctccc gtccttaact cccctcttca ctcagtcctc actcacctcc ctctcacaca 4200cacaaacatc tcctagaata atccccactg cctgccttca ctcttacccg tctcatttgc 4260ctcccctgaa cttcatcctc ctggagttca cgatctcact cttcactctt ttcttcccct 4320cgaagattca gcactgctta cttacatgtt aagatatttc agaacagtga aatgttgcta 4380ttttcaaaaa cctacaaagg tggtatgcag aggaaaaggt acttctttgt gttcccaaag 4440aaaacatctt tccaaaatcc agcctattga ttttatttct tcgggggaac aagaatttta 4500gtatctctaa gttgggtagc attctactct tggcagttgc tggaaagaag gcactggtct 4560aggtcctggg cttcacaggt aacacctgtc agggtgtcta tgaagtcaag gctgtctgag 4620gaacagcaaa gtgggaagaa gcaagctggc tggctgatga agggtttctt gggtggacaa 4680gtagttggag ctatttccta tttaccaaag agagctaaag ttcataattc tacagagagt 4740tccataatga acctcaaata cctctgtttt ttgaaggagt ttctcatata cagcactagc 4800tgactatcct gggcaggatg ggagataatg aatgcagtgc caatcgggct ggatttatat 4860ggtcctcagt gaggctggtc aagaaccgag ttagaactct cacagagtca ctgccacaga 4920agaaatctcc caagtggctg tttcctgaca ttcccgggag ggacaggcct ccttctgagt 4980cactccctaa gcagttctga actgtgaggt cagccaggct gtccaagtgc actccctgag 5040ccactggcag acacactcag cagccagagc tagacaggca ggtggtagga gtccagggcc 5100acggcaggga tggagtgtcg ccccctcgct gcgataccag agcaactaaa acgttaaggc 5160cttgcactaa agctgccctt aggatgcatt cttttaaagt ttttccattt aatgcagact 5220cttttcaatt cttattttat ccttgtttcc tttagaaagt cctttcaaaa atatctttag 5280agggtttttt cctatactat gtggccatat acgggtcaaa attaagttta atttccaggc 5340tccaagccag cgtttcagaa aaatctcacc aaggtttgtg gtaaaagaag caaagggctg 5400actttttggt tttcttgaat ctcactgttc cctctgcagc agcatgcatg tctgcccacc 5460tccagacaca caggcaccat ctgccgcccc ccatcagccc gtgtcccttc cacctcgact 5520cgcctacaaa gcccagagag gtctgtttct tggcccccag agcccaaaga tactgacaca 5580ctcttacatt tccaactaga atcaggaacg aggagtgact ctcagtcagt tcattaagta 5640aatgtctttc taaccgctct gcccatggga catcacgccc cacaggggaa aggggaagct 5700tctgtagcct gggattctgg tgcctcagtc tgggtctaga ctttcctgaa aaaacgttaa 5760aatatgaact gcattcctag aatttagcct acataaataa gagatgaaca caaagatttc 5820tatagtttac tcactgccgc ttatttacag aagcaaaaat ctgccacgat aggggcctga 5880caaatgacag taccactgtg caatgcgttt ctacgcagct ctcaatccca tgttctctaa 5940taccaccgaa gggcttagga aatgcttatg gtatatgtaa agagtaaaga agttacaaaa 6000cagtatcaac agttgacccc tattttaaaa agtattttta aaagtgtgac gatatttacc 6060aaaatattaa cagcaatagt tacctctggc tggtgggatg agtgaatgta tttttgttga 6120atatatgtta cctttatagt aaatatatgt tatcttgatc atcagaaaaa aaaatatgta 6180agaacttgaa agctgcttgg acagcgctgc tgatagaaac ccctgagcat cttgtcactg 6240ttcttctgat tcagagggtc tgggtggggc aggggtggtc tgagattctg tatttctaag 6300aagctcccag tgatgtccat gctgctggtc catggaccac actttgagta tcaagggacc 6360agagcatgtc gggggagagg ctggggatag ctttctttat ctgaactgga taaaggaact 6420gggctcaagc taagaaccct ctccaggttc tgcatctttg ttcttcagtg aaaaatgaga 6480ggacacacca ggccaggttc agactgagac acaatccctc tcctgggttc ccaatgactt 6540gtctcttgtc cattcccttc tctaaggcta agggccccca ggaagagcca tgtggccaga 6600ccctcacagt tgctggcatt ccaaggagat tctcactccg catcatttgg ggccaaaagg 6660ccccttacag aagctctgcc caaggctcag atcaatggca cctgctccca gagcctcctc 6720tgatctccca ggacaccttt ccctgatctg tgcacttatc tcttgctgcc tggcaaaatg 6780tcttagctcc tcacttgggc catgtgctgc tctcctctcc catggggaga gccacacgga 6840gagtgctggc caaagcagca gagttcaggc caaaggatgt gcactcattt attcaacagg 6900catgcaggat ttccagggaa agctggattt taaaacctct gggaacaaga gcagaacctg 6960actgagagct catgtgggca cttttcatag cagaatagct catgaggtat agagacacgg 7020acgcagaacg tgggctgtag cgacagatgg tcctgcattc tagtccccac tgtgcctttt 7080cctcatggga tgactttatt caggtaccct ttcggcaaaa tcctccaaga gaaaggaaac 7140tgggaggttc tggggagaag gctgctgcgt ttgcaattgg gagaggttgt tgacagaggt 7200ttatgtctgt ggcaagcagc cttccttcag tggaatactt gaagacaggt ctgtagttga 7260gcaaactcac ctccatttgt cctcctggaa agaagaaatc aagaggaaaa atctctctcc 7320catcctccaa atggagctgg cacattgcta tctgtggcat ttgtctttcc agaacacaac 7380tgagaaggaa accttctgca gggctgcgac tgtgctccgg cagttctaca gccaccatga 7440gaaggacact cgctgcctgg gtgcgactgc acagcagttc cacaggcaca agcagctgat 7500ccgattcctg aaacggctcg acaggaacct ctggggcctg gcgggcttgg taagctgcac 7560tgtattcctg gcaagccggc cgcgtggctc ctggtggaca gcagcctcac ttctaaacac 7620tccttaggag ctgcagcacc cttggtcaac ccattcattc attcactcat tcaataagta 7680tttgctgaag ttccacaagt gctgggtgtg gttctaggtg ctgaggacgt gtcactaaag 7740acacgcaggc cgagtccctg ttctcatgga atgttctaat gggagagtta gaaaaacaaa 7800catgtaaaat gatggccagc agtgatacgt gctacaaaga aaaacataga aataaagaac 7860ataagagtca tgggggaggg ggctgactta ggagctggtg acattatctg agcagatatt 7920tgaattgagg gagcaggcca catgactaac tagggagacc attccaggca gaaggaggag 7980gtatgcaaag gccttaggat ggaaatgaac taacttcctg tatttaaaga ccagtaggaa 8040ggccagtgtg gctggatcag agtgagtgag gggtagtttc caggacagca gatcacacaa 8100ggcctttaga ttccaccacg agtatggcag ggaacacctg cagagctttg ggcaggacaa 8160agactgtaca atctgattta cgtgatttaa aagggtcagt ctggctactg tgtggtaaat 8220aggctgaaag ggggaaagca tagaagcaag atggcctgtt gggaggctac cacagtaaac 8280caggctagag atgatggtgg cgtggacaga atgaagcaag atggcctgtt gggaggctac 8340cacagtaaac caggctagag acgatggtgg cgtggacaga atgaagcaag atggcctgtt 8400gggaggctac cacagtaaac caggctagag atgatggtgg cgtggacaga atgggagcag 8460ttgaggtgaa cagatttggg atatgactaa aaataaaacc agaaggattt gctgacagat 8520cggttgtagg gggtaagata caggggagga aaagatgacc tctttgttcc tgcccaaacc 8580cctctggcga tggtcagtac tgtttacaga gagatgaaag actggcggca aggcagggct 8640ggaggttcag cagaagatca agagttcaat tttgtacatc gtacatgtaa ggtggctctt 8700ggatagccaa gtgaaggtgt tgagaagatg gttagaaaag tctggaactt aggggagagg 8760tcagaacttg caatacaaaa aggagagtcc ttagatagat actgctgaaa atctgaatga 8820cagaaaggga gagatcaaag gactgagcct gagatcaaca catggaggtc aggagaggag 8880gatccagcca aggggcctga ggaggagtga ccagtgaggc aggagaacac tggagagtgg 8940gcggtacccc aggaagccgt tgaggacact caaggaggga gggttgactg tgtcaaatgt 9000actgaaagga caggtcaggt gaggaccaag aaaggcccct gggtttggct gatggaggcc 9060atgggtgagg ctgatgtaaa tggagaggca ggaaggaaag cccagctgga gtgggctcac 9120cgaggatagg gtggcgagag gagacaaaga aggaacagtg agggcagaac actctttgaa 9180gatgtttagc tataaggctg cagagaaact gacccacagc tgcagggtgg ttatggagtg 9240agggaagctc ttttaaggtt gggggtatac ccagcatgtt aatgcacctg ggggaatggt 9300ccagtggagc aggaagaact gaagagagca gaaagaggaa gaatcattag ggggcagaag 9360tccttgtagc ccagagtgga tgttatctaa tatcgagtgg aggaattaat tggctttaga 9420ggagaacaag gacatgtatc ccctctctgg gcctatcacc ttgtagacaa tgggataggt 9480catgggatag gaacttggca caacacatgt tctctctttt aattctctcc attatcttat 9540gaagcaggca agtaggcaaa caattgtccc aactttacaa aagaaactga agcttttata 9600aattaagtag tacatcctaa gcaatacaat taataaatgg tagagctgag attcaaactg 9660aagcagtggc ctgggggtag catctggaat ccttcccacc tttagggctg ctgtgctgcg 9720gtgctgctgt ttaatggcac aggagggcca catgactgaa tctctctcag cagtccaggc 9780agtcatgcag aaggcccagt agagcaccgg gcaggtctga gccagcatct tcaagttcca 9840ccctctgagc aagcacctag ctgtgacaca cctctccaga gactgcactc ccccccgcgc 9900cacccacccc aaaagcagat aggtaatggt atacagtaac catttctaga agtgtaagta 9960gtatgcaccc aaaataggca aaacctgctg gcctagtgat agagacaact cccagtcagg 10020ctagactgga ggccttggtt ttataagtgt tcaggtgaca agtgccacag taggcttgat 10080caagtagaca ggcaggcaag acaaatgctt accaatgcaa gctaatgaaa tgtttctttt 10140gcagaattcc tgtcctgtga aggaagccaa ccagagtacg ttggaaaact tcttggaaag 10200gctaaagacg atcatgagag agaaatattc aaagtgttcg agctgaatat tttaatttat 10260gagtttttga tagctttatt ttttaagtat ttatatattt ataactcatc ataaaataaa 10320gtatatatag aatctaacag caatggcatt taatgtattg gctatgttta cttgacaaat 10380gaaattatgg tttgcaactt ttagggaaat caatttagtt taccaagaga ctataaatgc 10440tatgggagca aaacaggaaa gaccacttcc ccctcgaggg gttccctctc gagttaggga 10500cataacacac aagataatta aagaacacaa ggccatacaa gatgtaaata agacaccttg 10560ggtccaagag tgc 1057323145DNAArtificial SequenceSynthetic primer 23tggggtatgg tggcttatat ctgtaacttc aacacttgag aggtggaggc aggagagtga 60ccatgaatct gagggcttcc agaataaatt catagggagg cccaggcaca gtggctcacg 120cctgtaatcc cagcactttg ggagg 14524196DNAArtificial SequenceSynthetic primer 24ggggttccct ctcgagttag ggacataaca cacaagataa ttaaagaaca caaggccata 60caagatgtaa ataagacacc ttgggtccaa gagtgcgtcg acggtatcga taagcttgat 120atcgaattcc gaagttccta ttctctagaa agtataggaa cttcaggtct gaagaggagt 180ttacgtccag ccaagc 19625150DNAArtificial SequenceSynthetic primer 25tgcggaaccc ttcgaagttc ctattctcta gaaagtatag gaacttcatc agtcaggtac 60ataatggtgg atcctaactc aagttctggg ggagctgatg ctctcctctg gcctcctgtg 120gaggtacaca gaccacatgc ctgtaggcaa 1502624DNAArtificial SequenceSynthetic primer 26ctgtgatcat ggttccttat ctgg 242723DNAArtificial SequenceSynthetic primer 27cctccccgag tagctgggac tac 232825DNAArtificial SequenceSynthetic primer 28ccactagggg tccacagcta gtcat 252925DNAArtificial SequenceSynthetic primer 29cttcagtgaa acctcctgag cctgg 253025DNAArtificial SequenceSynthetic primer 30agtcagagct acagaagtgg agggt 253125DNAArtificial SequenceSynthetic primer 31ctgctctgca ggaagtaagg gttcc 253224DNAArtificial SequenceSynthetic primer 32ccacatcact gaaagacttc ctgg 243325DNAArtificial SequenceSynthetic primer 33gatcaagtag acaggcaggc aagac 253424DNAArtificial SequenceSynthetic primer 34ctgtgatcat ggttccttat ctgg 243520DNAArtificial SequenceSynthetic primer 35agggacagat gcaggctggg

203625DNAArtificial SequenceSynthetic primer 36gagatgcgtg ttagaggttt tggga 253726DNAArtificial SequenceSynthetic primer 37tcagcgatat taagaacgtt gatccg 263827DNAArtificial SequenceSynthetic primer 38tgaagaattg ccggtcctat ttactcg 27395122DNAMouse 39cttctcgcag gaaagccccg cgcggcgcgt ggagcctgaa ctcgcaggtt ctggctggac 60ttctcgaagc tgaggagaag cagagggacc tggcttctga ttttggatct gcgtgcttgc 120tggttctggc gcctgctggt cttgttcctg taacctagga ctcggggctt gcacatgctt 180tttttttgaa gttgctggag agggagccca ggaccttgtg caggcacctt ttgtgtcccc 240aatggggcgg ctttgcacca agttcctgac ctctgtgggc tgtctgattt tgctgttggt 300gactggatct gggagcatca aggtcctggg tgagcccacc tgcttctctg actacatccg 360cacttccacg tgtgagtggt tcctggatag cgctgtggac tgcagttctc agctctgcct 420acactacagg ctgatgttct tcgagttctc tgaaaacctc acatgcatcc cgaggaacag 480tgccagcact gtgtgtgtgt gccacatgga aatgaatagg ccggtccaat cagacagata 540ccagatggaa ctgtgggctg agcacagaca gctgtggcag ggctccttca gccccagtgg 600taatgtgaag cccctagctc cagacaacct cacactccac accaatgtgt ccgacgaatg 660gctgctgacc tggaataacc tgtacccatc gaacaactta ctgtacaaag acctcatctc 720catggtcaac atctccagag aggacaaccc tgcagaattc atagtctata atgtgaccta 780caaggaaccc aggctgagct tcccgatcaa catcctgatg tcaggggtct actatacggc 840gcgtgtgagg gtcagatccc agatactcac tggcacctgg agtgagtgga gtcctagcat 900cacgtggtac aaccacttcc agctgcccct gatacagcgc cttccactgg gggtcaccat 960ctcctgcctc tgcatcccgt tgttttgcct gttctgttac ttcagcatta ccaagattaa 1020gaagatatgg tgggaccaga ttcccacccc agcacgcagt cccttggtgg ccatcatcat 1080tcaggatgca caggtgcccc tctgggataa gcagacccga agccaggagt caaccaagta 1140cccgcactgg aaaacttgtc tagacaagct gctgccttgc ttgctgaagc acagagtaaa 1200gaagaagaca gacttcccga aggctgcccc aaccaagtct ctccagagtc ctggaaaggc 1260aggctggtgt cccatggagg tcagcaggac cgtcctctgg ccagagaatg ttagtgtcag 1320tgtggtgcgc tgtatggagc tgtttgaggc cccagtacag aatgtggagg aggaagaaga 1380tgagatagtc aaagaggacc tgagcatgtc acctgagaac agcggaggct gcggcttcca 1440ggagagccag gcagacatca tggctcggct cactgagaac ctgttttccg acttgttgga 1500ggctgagaat gggggccttg gccagtcagc cttggcagag tcatgctccc ctctgccttc 1560aggaagtggg caggcttctg tatcctgggc ctgcctcccc atggggccca gtgaggaggc 1620cacatgccag gtcacagagc agccttcaca cccaggccct ctttcaggca gcccagccca 1680gagtgcacct actctggctt gcacgcaggt cccacttgtc cttgcagaca atcctgccta 1740ccggagtttt agtgactgct gtagcccggc cccaaatcct ggagagctgg ctccagagca 1800gcagcaggct gatcatctgg aagaagagga gcctccaagc ccggctgacc cccattcttc 1860agggccacca atgcagccag tggagagctg ggagcagatc cttcacatga gtgtcctgca 1920gcatggggca gctgctggct ccaccccagc ccctgccggt ggctaccagg agtttgtgca 1980ggcagtgaag cagggtgccg cccaggatcc tggggtgcct ggtgtcaggc cttctggaga 2040ccccggttac aaggccttct cgagcctgct cagcagcaat ggcatccgcg gggacacagc 2100agcagcgggg actgacgatg ggcatggagg ctacaagccc ttccagaatc ctgttcctaa 2160ccagtcccct agctccgtgc ccttatttac tttcggacta gacacggagc tgtcacccag 2220tcctctgaac tcagacccac ccaaaagccc cccagaatgc cttggtctgg agctggggct 2280caaaggaggt gactgggtga aggcccctcc tcctgcagat caggtgccca agccctttgg 2340ggatgacctg ggctttggta ttgtgtactc gtccctcact tgccacttgt gtggccacct 2400gaagcaacac cacagccagg aggaaggtgg ccagagcccc atcgttgcta gccctggctg 2460tggctgctgc tacgatgaca gatcaccatc cctggggagc ctctcggggg ccttggaaag 2520ctgtcctgag ggaataccac cagaagccaa cctcatgtca gcacccaaga caccctcaaa 2580cttgtcaggg gagggcaagg gccctggtca ctctcctgtt cccagccaga cgaccgaggt 2640gcctgtgggc gccctgggca ttgctgtttc ttaggtgagt gagtgtgctg ttgttgctga 2700ggtctgtgct gaggccaggg ttcctccaag ccagggaagt acttcctggg agacagccca 2760gctggcaggt ttcccagaaa tccagagaat ggtgaattga agatgtaaac ttggcctgac 2820cctggacgct cggagcctgg ctgtctcctc ttccactggc ctgggctctc ctccctccca 2880agggatacag gggctcactg tgcttggtcc cacagcagtg ctgacgttcc taagtcctgg 2940gctttcctag ctgatgttgt cctacctact cagtcccatt ttgtccaccg aatagacctg 3000tcactcaagg ctctcagcgg tcctgccata gctgctggac gctcccagct ggaagctggg 3060cctagaaact cacagatggc ctggcagtgg catgggaggc cctaaaaatt agtggaaatt 3120ttgagagagg acaggtattg ccccacagag gccattcatt gaacagccag gactgggact 3180agaggcagag cctgctgtcc tccgctcagt tgtagaaagc aacaaggaca caaacttgat 3240tgcccaaagt cactgccagt tacccacata tgaccagaag ccagggctcc tgggatgtgg 3300aagataaaca aacacagttg ccgggtggca gggcccagcg ggcacgataa ctggcagtca 3360aggcgatacc tcgagggaac tgtggggctg gtcctggttg gtggtcaggt ggtagggata 3420gcagatggca gactttggtg agtgagtgag tctgactgtg ttctggaaga tgggaccggg 3480ctcagcactg tctgctcacg tccccactgt tgcaacacct agtctgtttg caaggaggac 3540aggacaggtc acatggagct ttatgtcaat aaagtcttta tcttgtcagg tttcctttac 3600tatacacacg ccgagcccac agtgcacgaa agctgaaatg tgcaggcagg gggttgggga 3660agtggggaga caaggcccca gcagttggtt taagggaatg acttgggaat ggggcagagc 3720tgtggctact ccatctctca tccttaccct cctgctacca cagcttgtgc ccatgtgtgc 3780ctgctcaggg gaggggtcct cctctttccc tctcctttag gcagagtgct agaggtgttg 3840gatgctcctt agcacgcaga gggcgtgaca ctggctgcta agtgttgttg aatgtgtcaa 3900ggcattgaca tcagtaggcc ccacatctta ggcaggggat ttggggtgtg gcaacctgcc 3960aggccagcag agaacttgaa tatgatgttc agaaggaaag acagagacag gtcacttagg 4020gaggcttgaa ggaggccact tttgagatga gcccgtggta tctgcaaaaa agcctggaag 4080aactcagaac atgcagcaga tggctcatgt ctgaagtttg aggcacaggg atctggtgtg 4140gtcacagcat cagagagaag ggtcctggca tgatggggac tggtgtgcag ttggatactg 4200gtattctgcc ttagttctgc ctcttgagag gtggtccagt agaagccagc ctcagacttt 4260cacatctcag aacagtcttt tcaaagacaa ggtctcagtg tggctcagac gggcctctag 4320catcactcgg tctcctgagt gctgggatca caggtgtgtg ctactacacc cagcttccca 4380gagcagtctt accacagccc ctaagcaggg gaggagagga aacggagcca tccaagatct 4440acctcctgtg ggcatccctc ctgcctggct gaaggagaag tctgggttta aaatccccat 4500tgctcagatt tgtatcctgg agggaagtga ctcccacagg aatgaactga ctgtcccagg 4560aaattcttcc cgctgtgcct ttagaacttc ctctgtaaag tacctccctt cagctggtgg 4620tgcctgtctt taacagtggc tgacagccag ggagtttgag gctagcctag gctttgacag 4680tgagatcctg ttcaataaac gcagcttccc agtccggcac ttgaccttca agctctgacc 4740tctggattag cccaggattt cttcactggt cctccacctt ccaccccctt ccactgctta 4800tacccgctcc catcatttcc aacccaggaa gctgctaaga tctatgtggt agccctggct 4860ccattaccac cctctcccca tctcctgggt gactgaggga actaaggtac aagcccgagg 4920aacttgggat tccctgccct gctgttacct tgcctcctgt ctgccatcat taacatagga 4980ggtgccagcc tgtgccgtcc tggctcccat actcactacc taaggctacc tgttgggaat 5040tccaaccctc accagtccca tctcttcatt ctattaaaat tacttatcaa gcaaaaaaaa 5100aaaaaaaaaa aaaaaaaaaa aa 512240810PRTMouse 40Met Gly Arg Leu Cys Thr Lys Phe Leu Thr Ser Val Gly Cys Leu Ile1 5 10 15Leu Leu Leu Val Thr Gly Ser Gly Ser Ile Lys Val Leu Gly Glu Pro 20 25 30Thr Cys Phe Ser Asp Tyr Ile Arg Thr Ser Thr Cys Glu Trp Phe Leu 35 40 45Asp Ser Ala Val Asp Cys Ser Ser Gln Leu Cys Leu His Tyr Arg Leu 50 55 60Met Phe Phe Glu Phe Ser Glu Asn Leu Thr Cys Ile Pro Arg Asn Ser65 70 75 80Ala Ser Thr Val Cys Val Cys His Met Glu Met Asn Arg Pro Val Gln 85 90 95Ser Asp Arg Tyr Gln Met Glu Leu Trp Ala Glu His Arg Gln Leu Trp 100 105 110Gln Gly Ser Phe Ser Pro Ser Gly Asn Val Lys Pro Leu Ala Pro Asp 115 120 125Asn Leu Thr Leu His Thr Asn Val Ser Asp Glu Trp Leu Leu Thr Trp 130 135 140Asn Asn Leu Tyr Pro Ser Asn Asn Leu Leu Tyr Lys Asp Leu Ile Ser145 150 155 160Met Val Asn Ile Ser Arg Glu Asp Asn Pro Ala Glu Phe Ile Val Tyr 165 170 175Asn Val Thr Tyr Lys Glu Pro Arg Leu Ser Phe Pro Ile Asn Ile Leu 180 185 190Met Ser Gly Val Tyr Tyr Thr Ala Arg Val Arg Val Arg Ser Gln Ile 195 200 205Leu Thr Gly Thr Trp Ser Glu Trp Ser Pro Ser Ile Thr Trp Tyr Asn 210 215 220His Phe Gln Leu Pro Leu Ile Gln Arg Leu Pro Leu Gly Val Thr Ile225 230 235 240Ser Cys Leu Cys Ile Pro Leu Phe Cys Leu Phe Cys Tyr Phe Ser Ile 245 250 255Thr Lys Ile Lys Lys Ile Trp Trp Asp Gln Ile Pro Thr Pro Ala Arg 260 265 270Ser Pro Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Val Pro Leu Trp 275 280 285Asp Lys Gln Thr Arg Ser Gln Glu Ser Thr Lys Tyr Pro His Trp Lys 290 295 300Thr Cys Leu Asp Lys Leu Leu Pro Cys Leu Leu Lys His Arg Val Lys305 310 315 320Lys Lys Thr Asp Phe Pro Lys Ala Ala Pro Thr Lys Ser Leu Gln Ser 325 330 335Pro Gly Lys Ala Gly Trp Cys Pro Met Glu Val Ser Arg Thr Val Leu 340 345 350Trp Pro Glu Asn Val Ser Val Ser Val Val Arg Cys Met Glu Leu Phe 355 360 365Glu Ala Pro Val Gln Asn Val Glu Glu Glu Glu Asp Glu Ile Val Lys 370 375 380Glu Asp Leu Ser Met Ser Pro Glu Asn Ser Gly Gly Cys Gly Phe Gln385 390 395 400Glu Ser Gln Ala Asp Ile Met Ala Arg Leu Thr Glu Asn Leu Phe Ser 405 410 415Asp Leu Leu Glu Ala Glu Asn Gly Gly Leu Gly Gln Ser Ala Leu Ala 420 425 430Glu Ser Cys Ser Pro Leu Pro Ser Gly Ser Gly Gln Ala Ser Val Ser 435 440 445Trp Ala Cys Leu Pro Met Gly Pro Ser Glu Glu Ala Thr Cys Gln Val 450 455 460Thr Glu Gln Pro Ser His Pro Gly Pro Leu Ser Gly Ser Pro Ala Gln465 470 475 480Ser Ala Pro Thr Leu Ala Cys Thr Gln Val Pro Leu Val Leu Ala Asp 485 490 495Asn Pro Ala Tyr Arg Ser Phe Ser Asp Cys Cys Ser Pro Ala Pro Asn 500 505 510Pro Gly Glu Leu Ala Pro Glu Gln Gln Gln Ala Asp His Leu Glu Glu 515 520 525Glu Glu Pro Pro Ser Pro Ala Asp Pro His Ser Ser Gly Pro Pro Met 530 535 540Gln Pro Val Glu Ser Trp Glu Gln Ile Leu His Met Ser Val Leu Gln545 550 555 560His Gly Ala Ala Ala Gly Ser Thr Pro Ala Pro Ala Gly Gly Tyr Gln 565 570 575Glu Phe Val Gln Ala Val Lys Gln Gly Ala Ala Gln Asp Pro Gly Val 580 585 590Pro Gly Val Arg Pro Ser Gly Asp Pro Gly Tyr Lys Ala Phe Ser Ser 595 600 605Leu Leu Ser Ser Asn Gly Ile Arg Gly Asp Thr Ala Ala Ala Gly Thr 610 615 620Asp Asp Gly His Gly Gly Tyr Lys Pro Phe Gln Asn Pro Val Pro Asn625 630 635 640Gln Ser Pro Ser Ser Val Pro Leu Phe Thr Phe Gly Leu Asp Thr Glu 645 650 655Leu Ser Pro Ser Pro Leu Asn Ser Asp Pro Pro Lys Ser Pro Pro Glu 660 665 670Cys Leu Gly Leu Glu Leu Gly Leu Lys Gly Gly Asp Trp Val Lys Ala 675 680 685Pro Pro Pro Ala Asp Gln Val Pro Lys Pro Phe Gly Asp Asp Leu Gly 690 695 700Phe Gly Ile Val Tyr Ser Ser Leu Thr Cys His Leu Cys Gly His Leu705 710 715 720Lys Gln His His Ser Gln Glu Glu Gly Gly Gln Ser Pro Ile Val Ala 725 730 735Ser Pro Gly Cys Gly Cys Cys Tyr Asp Asp Arg Ser Pro Ser Leu Gly 740 745 750Ser Leu Ser Gly Ala Leu Glu Ser Cys Pro Glu Gly Ile Pro Pro Glu 755 760 765Ala Asn Leu Met Ser Ala Pro Lys Thr Pro Ser Asn Leu Ser Gly Glu 770 775 780Gly Lys Gly Pro Gly His Ser Pro Val Pro Ser Gln Thr Thr Glu Val785 790 795 800Pro Val Gly Ala Leu Gly Ile Ala Val Ser 805 810413710DNAhuman 41gggtctccgc gcccaggaaa gccccgcgcg gcgcgggcca gggaagggcc acccaggggt 60cccccacttc ccgcttgggc gcccggacgg cgaatggagc aggggcgcgc agataattaa 120agatttacac acagctggaa gaaatcatag agaagccggg cgtggtggct catgcctata 180atcccagcac ttttggaggc tgaggcgggc agatcacttg agatcaggag ttcgagacca 240gcctggtgcc ttggcatctc ccaatggggt ggctttgctc tgggctcctg ttccctgtga 300gctgcctggt cctgctgcag gtggcaagct ctgggaacat gaaggtcttg caggagccca 360cctgcgtctc cgactacatg agcatctcta cttgcgagtg gaagatgaat ggtcccacca 420attgcagcac cgagctccgc ctgttgtacc agctggtttt tctgctctcc gaagcccaca 480cgtgtatccc tgagaacaac ggaggcgcgg ggtgcgtgtg ccacctgctc atggatgacg 540tggtcagtgc ggataactat acactggacc tgtgggctgg gcagcagctg ctgtggaagg 600gctccttcaa gcccagcgag catgtgaaac ccagggcccc aggaaacctg acagttcaca 660ccaatgtctc cgacactctg ctgctgacct ggagcaaccc gtatccccct gacaattacc 720tgtataatca tctcacctat gcagtcaaca tttggagtga aaacgacccg gcagatttca 780gaatctataa cgtgacctac ctagaaccct ccctccgcat cgcagccagc accctgaagt 840ctgggatttc ctacagggca cgggtgaggg cctgggctca gtgctataac accacctgga 900gtgagtggag ccccagcacc aagtggcaca actcctacag ggagcccttc gagcagcacc 960tcctgctggg cgtcagcgtt tcctgcattg tcatcctggc cgtctgcctg ttgtgctatg 1020tcagcatcac caagattaag aaagaatggt gggatcagat tcccaaccca gcccgcagcc 1080gcctcgtggc tataataatc caggatgctc aggggtcaca gtgggagaag cggtcccgag 1140gccaggaacc agccaagtgc ccacactgga agaattgtct taccaagctc ttgccctgtt 1200ttctggagca caacatgaaa agggatgaag atcctcacaa ggctgccaaa gagatgcctt 1260tccagggctc tggaaaatca gcatggtgcc cagtggagat cagcaagaca gtcctctggc 1320cagagagcat cagcgtggtg cgatgtgtgg agttgtttga ggccccggtg gagtgtgagg 1380aggaggagga ggtagaggaa gaaaaaggga gcttctgtgc atcgcctgag agcagcaggg 1440atgacttcca ggagggaagg gagggcattg tggcccggct aacagagagc ctgttcctgg 1500acctgctcgg agaggagaat gggggctttt gccagcagga catgggggag tcatgccttc 1560ttccaccttc gggaagtacg agtgctcaca tgccctggga tgagttccca agtgcagggc 1620ccaaggaggc acctccctgg ggcaaggagc agcctctcca cctggagcca agtcctcctg 1680ccagcccgac ccagagtcca gacaacctga cttgcacaga gacgcccctc gtcatcgcag 1740gcaaccctgc ttaccgcagc ttcagcaact ccctgagcca gtcaccgtgt cccagagagc 1800tgggtccaga cccactgctg gccagacacc tggaggaagt agaacccgag atgccctgtg 1860tcccccagct ctctgagcca accactgtgc cccaacctga gccagaaacc tgggagcaga 1920tcctccgccg aaatgtcctc cagcatgggg cagctgcagc ccccgtctcg gcccccacca 1980gtggctatca ggagtttgta catgcggtgg agcagggtgg cacccaggcc agtgcggtgg 2040tgggcttggg tcccccagga gaggctggtt acaaggcctt ctcaagcctg cttgccagca 2100gtgctgtgtc cccagagaaa tgtgggtttg gggctagcag tggggaagag gggtataagc 2160ctttccaaga cctcattcct ggctgccctg gggaccctgc cccagtccct gtccccttgt 2220tcacctttgg actggacagg gagccacctc gcagtccgca gagctcacat ctcccaagca 2280gctccccaga gcacctgggt ctggagccgg gggaaaaggt agaggacatg ccaaagcccc 2340cacttcccca ggagcaggcc acagaccccc ttgtggacag cctgggcagt ggcattgtct 2400actcagccct tacctgccac ctgtgcggcc acctgaaaca gtgtcatggc caggaggatg 2460gtggccagac ccctgtcatg gccagtcctt gctgtggctg ctgctgtgga gacaggtcct 2520cgccccctac aacccccctg agggccccag acccctctcc aggtggggtt ccactggagg 2580ccagtctgtg tccggcctcc ctggcaccct cgggcatctc agagaagagt aaatcctcat 2640catccttcca tcctgcccct ggcaatgctc agagctcaag ccagaccccc aaaatcgtga 2700actttgtctc cgtgggaccc acatacatga gggtctctta ggtgcatgtc ctcttgttgc 2760tgagtctgca gatgaggact agggcttatc catgcctggg aaatgccacc tcctggaagg 2820cagccaggct ggcagatttc caaaagactt gaagaaccat ggtatgaagg tgattggccc 2880cactgacgtt ggcctaacac tgggctgcag agactggacc ccgcccagca ttgggctggg 2940ctcgccacat cccatgagag tagagggcac tgggtcgccg tgccccacgg caggcccctg 3000caggaaaact gaggcccttg ggcacctcga cttgtgaacg agttgttggc tgctccctcc 3060acagcttctg cagcagactg tccctgttgt aactgcccaa ggcatgtttt gcccaccaga 3120tcatggccca cgtggaggcc cacctgcctc tgtctcactg aactagaagc cgagcctaga 3180aactaacaca gccatcaagg gaatgacttg ggcggccttg ggaaatcgat gagaaattga 3240acttcaggga gggtggtcat tgcctagagg tgctcattca tttaacagag cttccttagg 3300ttgatgctgg aggcagaatc ccggctgtca aggggtgttc agttaagggg agcaacagag 3360gacatgaaaa attgctatga ctaaagcagg gacaatttgc tgccaaacac ccatgcccag 3420ctgtatggct gggggctcct cgtatgcatg gaacccccag aataaatatg ctcagccacc 3480ctgtgggccg ggcaatccag acagcaggca taaggcacca gttaccctgc atgttggccc 3540agacctcagg tgctagggaa ggcgggaacc ttgggttgag taatgctcgt ctgtgtgttt 3600tagtttcatc acctgttatc tgtgtttgct gaggagagtg gaacagaagg ggtggagttt 3660tgtataaata aagtttcttt gtctctttaa aaaaaaaaaa aaaaaaaaaa 371042825PRThuman 42Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val1 5 10 15Leu Leu Gln Val Ala Ser Ser Gly Asn Met Lys Val Leu Gln Glu Pro 20 25 30Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met 35 40 45Asn Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu 50 55 60Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly65 70 75 80Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala 85 90 95Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys 100 105 110Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn 115 120 125Leu Thr Val His Thr Asn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser 130

135 140Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala145 150 155 160Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn 165 170 175Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys 180 185 190Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr 195 200 205Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser 210 215 220Tyr Arg Glu Pro Phe Glu Gln His Leu Leu Leu Gly Val Ser Val Ser225 230 235 240Cys Ile Val Ile Leu Ala Val Cys Leu Leu Cys Tyr Val Ser Ile Thr 245 250 255Lys Ile Lys Lys Glu Trp Trp Asp Gln Ile Pro Asn Pro Ala Arg Ser 260 265 270Arg Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Gly Ser Gln Trp Glu 275 280 285Lys Arg Ser Arg Gly Gln Glu Pro Ala Lys Cys Pro His Trp Lys Asn 290 295 300Cys Leu Thr Lys Leu Leu Pro Cys Phe Leu Glu His Asn Met Lys Arg305 310 315 320Asp Glu Asp Pro His Lys Ala Ala Lys Glu Met Pro Phe Gln Gly Ser 325 330 335Gly Lys Ser Ala Trp Cys Pro Val Glu Ile Ser Lys Thr Val Leu Trp 340 345 350Pro Glu Ser Ile Ser Val Val Arg Cys Val Glu Leu Phe Glu Ala Pro 355 360 365Val Glu Cys Glu Glu Glu Glu Glu Val Glu Glu Glu Lys Gly Ser Phe 370 375 380Cys Ala Ser Pro Glu Ser Ser Arg Asp Asp Phe Gln Glu Gly Arg Glu385 390 395 400Gly Ile Val Ala Arg Leu Thr Glu Ser Leu Phe Leu Asp Leu Leu Gly 405 410 415Glu Glu Asn Gly Gly Phe Cys Gln Gln Asp Met Gly Glu Ser Cys Leu 420 425 430Leu Pro Pro Ser Gly Ser Thr Ser Ala His Met Pro Trp Asp Glu Phe 435 440 445Pro Ser Ala Gly Pro Lys Glu Ala Pro Pro Trp Gly Lys Glu Gln Pro 450 455 460Leu His Leu Glu Pro Ser Pro Pro Ala Ser Pro Thr Gln Ser Pro Asp465 470 475 480Asn Leu Thr Cys Thr Glu Thr Pro Leu Val Ile Ala Gly Asn Pro Ala 485 490 495Tyr Arg Ser Phe Ser Asn Ser Leu Ser Gln Ser Pro Cys Pro Arg Glu 500 505 510Leu Gly Pro Asp Pro Leu Leu Ala Arg His Leu Glu Glu Val Glu Pro 515 520 525Glu Met Pro Cys Val Pro Gln Leu Ser Glu Pro Thr Thr Val Pro Gln 530 535 540Pro Glu Pro Glu Thr Trp Glu Gln Ile Leu Arg Arg Asn Val Leu Gln545 550 555 560His Gly Ala Ala Ala Ala Pro Val Ser Ala Pro Thr Ser Gly Tyr Gln 565 570 575Glu Phe Val His Ala Val Glu Gln Gly Gly Thr Gln Ala Ser Ala Val 580 585 590Val Gly Leu Gly Pro Pro Gly Glu Ala Gly Tyr Lys Ala Phe Ser Ser 595 600 605Leu Leu Ala Ser Ser Ala Val Ser Pro Glu Lys Cys Gly Phe Gly Ala 610 615 620Ser Ser Gly Glu Glu Gly Tyr Lys Pro Phe Gln Asp Leu Ile Pro Gly625 630 635 640Cys Pro Gly Asp Pro Ala Pro Val Pro Val Pro Leu Phe Thr Phe Gly 645 650 655Leu Asp Arg Glu Pro Pro Arg Ser Pro Gln Ser Ser His Leu Pro Ser 660 665 670Ser Ser Pro Glu His Leu Gly Leu Glu Pro Gly Glu Lys Val Glu Asp 675 680 685Met Pro Lys Pro Pro Leu Pro Gln Glu Gln Ala Thr Asp Pro Leu Val 690 695 700Asp Ser Leu Gly Ser Gly Ile Val Tyr Ser Ala Leu Thr Cys His Leu705 710 715 720Cys Gly His Leu Lys Gln Cys His Gly Gln Glu Asp Gly Gly Gln Thr 725 730 735Pro Val Met Ala Ser Pro Cys Cys Gly Cys Cys Cys Gly Asp Arg Ser 740 745 750Ser Pro Pro Thr Thr Pro Leu Arg Ala Pro Asp Pro Ser Pro Gly Gly 755 760 765Val Pro Leu Glu Ala Ser Leu Cys Pro Ala Ser Leu Ala Pro Ser Gly 770 775 780Ile Ser Glu Lys Ser Lys Ser Ser Ser Ser Phe His Pro Ala Pro Gly785 790 795 800Asn Ala Gln Ser Ser Ser Gln Thr Pro Lys Ile Val Asn Phe Val Ser 805 810 815Val Gly Pro Thr Tyr Met Arg Val Ser 820 825435119DNAArtificial SequenceSynthetic primer 43cttctcgcag gaaagccccg cgcggcgcgt ggagcctgaa ctcgcaggtt ctggctggac 60ttctcgaagc tgaggagaag cagagggacc tggcttctga ttttggatct gcgtgcttgc 120tggttctggc gcctgctggt cttgttcctg taacctagga ctcggggctt gcacatgctt 180tttttttgaa gttgctggag agggagccca ggaccttgtg caggcacctt ttgtgtcccc 240aatggggcgg ctttgcacca agttcctgac ctctgtgggc tgtctgattt tgctgttggt 300gactggatct gggagcatca aggtcctgca ggagcccacc tgcgtctccg actacatgag 360catctctact tgcgagtgga agatgaatgg tcccaccaat tgcagcaccg agctccgcct 420gttgtaccag ctggtttttc tgctctccga agcccacacg tgtatccctg agaacaacgg 480aggcgcgggg tgcgtgtgcc acctgctcat ggatgacgtg gtcagtgcgg ataactatac 540actggacctg tgggctgggc agcagctgct gtggaagggc tccttcaagc ccagcgagca 600tgtgaaaccc agggccccag gaaacctgac agttcacacc aatgtctccg acactctgct 660gctgacctgg agcaacccgt atccccctga caattacctg tataatcatc tcacctatgc 720agtcaacatt tggagtgaaa acgacccggc agatttcaga atctataacg tgacctacct 780agaaccctcc ctccgcatcg cagccagcac cctgaagtct gggatttcct acagggcacg 840ggtgagggcc tgggctcagt gctataacac cacctggagt gagtggagcc ctagcatcac 900gtggtacaac cacttccagc tgcccctgat acagcgcctt ccactggggg tcaccatctc 960ctgcctctgc atcccgttgt tttgcctgtt ctgttacttc agcattacca agattaagaa 1020gatatggtgg gaccagattc ccaccccagc acgcagtccc ttggtggcca tcatcattca 1080ggatgcacag gtgcccctct gggataagca gacccgaagc caggagtcaa ccaagtaccc 1140gcactggaaa acttgtctag acaagctgct gccttgcttg ctgaagcaca gagtaaagaa 1200gaagacagac ttcccgaagg ctgccccaac caagtctctc cagagtcctg gaaaggcagg 1260ctggtgtccc atggaggtca gcaggaccgt cctctggcca gagaatgtta gtgtcagtgt 1320ggtgcgctgt atggagctgt ttgaggcccc agtacagaat gtggaggagg aagaagatga 1380gatagtcaaa gaggacctga gcatgtcacc tgagaacagc ggaggctgcg gcttccagga 1440gagccaggca gacatcatgg ctcggctcac tgagaacctg ttttccgact tgttggaggc 1500tgagaatggg ggccttggcc agtcagcctt ggcagagtca tgctcccctc tgccttcagg 1560aagtgggcag gcttctgtat cctgggcctg cctccccatg gggcccagtg aggaggccac 1620atgccaggtc acagagcagc cttcacaccc aggccctctt tcaggcagcc cagcccagag 1680tgcacctact ctggcttgca cgcaggtccc acttgtcctt gcagacaatc ctgcctaccg 1740gagttttagt gactgctgta gcccggcccc aaatcctgga gagctggctc cagagcagca 1800gcaggctgat catctggaag aagaggagcc tccaagcccg gctgaccccc attcttcagg 1860gccaccaatg cagccagtgg agagctggga gcagatcctt cacatgagtg tcctgcagca 1920tggggcagct gctggctcca ccccagcccc tgccggtggc taccaggagt ttgtgcaggc 1980agtgaagcag ggtgccgccc aggatcctgg ggtgcctggt gtcaggcctt ctggagaccc 2040cggttacaag gccttctcga gcctgctcag cagcaatggc atccgcgggg acacagcagc 2100agcggggact gacgatgggc atggaggcta caagcccttc cagaatcctg ttcctaacca 2160gtcccctagc tccgtgccct tatttacttt cggactagac acggagctgt cacccagtcc 2220tctgaactca gacccaccca aaagcccccc agaatgcctt ggtctggagc tggggctcaa 2280aggaggtgac tgggtgaagg cccctcctcc tgcagatcag gtgcccaagc cctttgggga 2340tgacctgggc tttggtattg tgtactcgtc cctcacttgc cacttgtgtg gccacctgaa 2400gcaacaccac agccaggagg aaggtggcca gagccccatc gttgctagcc ctggctgtgg 2460ctgctgctac gatgacagat caccatccct ggggagcctc tcgggggcct tggaaagctg 2520tcctgaggga ataccaccag aagccaacct catgtcagca cccaagacac cctcaaactt 2580gtcaggggag ggcaagggcc ctggtcactc tcctgttccc agccagacga ccgaggtgcc 2640tgtgggcgcc ctgggcattg ctgtttctta ggtgagtgag tgtgctgttg ttgctgaggt 2700ctgtgctgag gccagggttc ctccaagcca gggaagtact tcctgggaga cagcccagct 2760ggcaggtttc ccagaaatcc agagaatggt gaattgaaga tgtaaacttg gcctgaccct 2820ggacgctcgg agcctggctg tctcctcttc cactggcctg ggctctcctc cctcccaagg 2880gatacagggg ctcactgtgc ttggtcccac agcagtgctg acgttcctaa gtcctgggct 2940ttcctagctg atgttgtcct acctactcag tcccattttg tccaccgaat agacctgtca 3000ctcaaggctc tcagcggtcc tgccatagct gctggacgct cccagctgga agctgggcct 3060agaaactcac agatggcctg gcagtggcat gggaggccct aaaaattagt ggaaattttg 3120agagaggaca ggtattgccc cacagaggcc attcattgaa cagccaggac tgggactaga 3180ggcagagcct gctgtcctcc gctcagttgt agaaagcaac aaggacacaa acttgattgc 3240ccaaagtcac tgccagttac ccacatatga ccagaagcca gggctcctgg gatgtggaag 3300ataaacaaac acagttgccg ggtggcaggg cccagcgggc acgataactg gcagtcaagg 3360cgatacctcg agggaactgt ggggctggtc ctggttggtg gtcaggtggt agggatagca 3420gatggcagac tttggtgagt gagtgagtct gactgtgttc tggaagatgg gaccgggctc 3480agcactgtct gctcacgtcc ccactgttgc aacacctagt ctgtttgcaa ggaggacagg 3540acaggtcaca tggagcttta tgtcaataaa gtctttatct tgtcaggttt cctttactat 3600acacacgccg agcccacagt gcacgaaagc tgaaatgtgc aggcaggggg ttggggaagt 3660ggggagacaa ggccccagca gttggtttaa gggaatgact tgggaatggg gcagagctgt 3720ggctactcca tctctcatcc ttaccctcct gctaccacag cttgtgccca tgtgtgcctg 3780ctcaggggag gggtcctcct ctttccctct cctttaggca gagtgctaga ggtgttggat 3840gctccttagc acgcagaggg cgtgacactg gctgctaagt gttgttgaat gtgtcaaggc 3900attgacatca gtaggcccca catcttaggc aggggatttg gggtgtggca acctgccagg 3960ccagcagaga acttgaatat gatgttcaga aggaaagaca gagacaggtc acttagggag 4020gcttgaagga ggccactttt gagatgagcc cgtggtatct gcaaaaaagc ctggaagaac 4080tcagaacatg cagcagatgg ctcatgtctg aagtttgagg cacagggatc tggtgtggtc 4140acagcatcag agagaagggt cctggcatga tggggactgg tgtgcagttg gatactggta 4200ttctgcctta gttctgcctc ttgagaggtg gtccagtaga agccagcctc agactttcac 4260atctcagaac agtcttttca aagacaaggt ctcagtgtgg ctcagacggg cctctagcat 4320cactcggtct cctgagtgct gggatcacag gtgtgtgcta ctacacccag cttcccagag 4380cagtcttacc acagccccta agcaggggag gagaggaaac ggagccatcc aagatctacc 4440tcctgtgggc atccctcctg cctggctgaa ggagaagtct gggtttaaaa tccccattgc 4500tcagatttgt atcctggagg gaagtgactc ccacaggaat gaactgactg tcccaggaaa 4560ttcttcccgc tgtgccttta gaacttcctc tgtaaagtac ctcccttcag ctggtggtgc 4620ctgtctttaa cagtggctga cagccaggga gtttgaggct agcctaggct ttgacagtga 4680gatcctgttc aataaacgca gcttcccagt ccggcacttg accttcaagc tctgacctct 4740ggattagccc aggatttctt cactggtcct ccaccttcca cccccttcca ctgcttatac 4800ccgctcccat catttccaac ccaggaagct gctaagatct atgtggtagc cctggctcca 4860ttaccaccct ctccccatct cctgggtgac tgagggaact aaggtacaag cccgaggaac 4920ttgggattcc ctgccctgct gttaccttgc ctcctgtctg ccatcattaa cataggaggt 4980gccagcctgt gccgtcctgg ctcccatact cactacctaa ggctacctgt tgggaattcc 5040aaccctcacc agtcccatct cttcattcta ttaaaattac ttatcaagca aaaaaaaaaa 5100aaaaaaaaaa aaaaaaaaa 511944809PRTArtificial SequenceSynthetic primer 44Met Gly Arg Leu Cys Thr Lys Phe Leu Thr Ser Val Gly Cys Leu Ile1 5 10 15Leu Leu Leu Val Thr Gly Ser Gly Ser Ile Lys Val Leu Gln Glu Pro 20 25 30Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met 35 40 45Asn Gly Pro Thr Asn Cys Ser Thr Glu Leu Arg Leu Leu Tyr Gln Leu 50 55 60Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly65 70 75 80Gly Ala Gly Cys Val Cys His Leu Leu Met Asp Asp Val Val Ser Ala 85 90 95Asp Asn Tyr Thr Leu Asp Leu Trp Ala Gly Gln Gln Leu Leu Trp Lys 100 105 110Gly Ser Phe Lys Pro Ser Glu His Val Lys Pro Arg Ala Pro Gly Asn 115 120 125Leu Thr Val His Thr Asn Val Ser Asp Thr Leu Leu Leu Thr Trp Ser 130 135 140Asn Pro Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn His Leu Thr Tyr Ala145 150 155 160Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn 165 170 175Val Thr Tyr Leu Glu Pro Ser Leu Arg Ile Ala Ala Ser Thr Leu Lys 180 185 190Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr 195 200 205Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser Ile Thr Trp Tyr Asn His 210 215 220Phe Gln Leu Pro Leu Ile Gln Arg Leu Pro Leu Gly Val Thr Ile Ser225 230 235 240Cys Leu Cys Ile Pro Leu Phe Cys Leu Phe Cys Tyr Phe Ser Ile Thr 245 250 255Lys Ile Lys Lys Ile Trp Trp Asp Gln Ile Pro Thr Pro Ala Arg Ser 260 265 270Pro Leu Val Ala Ile Ile Ile Gln Asp Ala Gln Val Pro Leu Trp Asp 275 280 285Lys Gln Thr Arg Ser Gln Glu Ser Thr Lys Tyr Pro His Trp Lys Thr 290 295 300Cys Leu Asp Lys Leu Leu Pro Cys Leu Leu Lys His Arg Val Lys Lys305 310 315 320Lys Thr Asp Phe Pro Lys Ala Ala Pro Thr Lys Ser Leu Gln Ser Pro 325 330 335Gly Lys Ala Gly Trp Cys Pro Met Glu Val Ser Arg Thr Val Leu Trp 340 345 350Pro Glu Asn Val Ser Val Ser Val Val Arg Cys Met Glu Leu Phe Glu 355 360 365Ala Pro Val Gln Asn Val Glu Glu Glu Glu Asp Glu Ile Val Lys Glu 370 375 380Asp Leu Ser Met Ser Pro Glu Asn Ser Gly Gly Cys Gly Phe Gln Glu385 390 395 400Ser Gln Ala Asp Ile Met Ala Arg Leu Thr Glu Asn Leu Phe Ser Asp 405 410 415Leu Leu Glu Ala Glu Asn Gly Gly Leu Gly Gln Ser Ala Leu Ala Glu 420 425 430Ser Cys Ser Pro Leu Pro Ser Gly Ser Gly Gln Ala Ser Val Ser Trp 435 440 445Ala Cys Leu Pro Met Gly Pro Ser Glu Glu Ala Thr Cys Gln Val Thr 450 455 460Glu Gln Pro Ser His Pro Gly Pro Leu Ser Gly Ser Pro Ala Gln Ser465 470 475 480Ala Pro Thr Leu Ala Cys Thr Gln Val Pro Leu Val Leu Ala Asp Asn 485 490 495Pro Ala Tyr Arg Ser Phe Ser Asp Cys Cys Ser Pro Ala Pro Asn Pro 500 505 510Gly Glu Leu Ala Pro Glu Gln Gln Gln Ala Asp His Leu Glu Glu Glu 515 520 525Glu Pro Pro Ser Pro Ala Asp Pro His Ser Ser Gly Pro Pro Met Gln 530 535 540Pro Val Glu Ser Trp Glu Gln Ile Leu His Met Ser Val Leu Gln His545 550 555 560Gly Ala Ala Ala Gly Ser Thr Pro Ala Pro Ala Gly Gly Tyr Gln Glu 565 570 575Phe Val Gln Ala Val Lys Gln Gly Ala Ala Gln Asp Pro Gly Val Pro 580 585 590Gly Val Arg Pro Ser Gly Asp Pro Gly Tyr Lys Ala Phe Ser Ser Leu 595 600 605Leu Ser Ser Asn Gly Ile Arg Gly Asp Thr Ala Ala Ala Gly Thr Asp 610 615 620Asp Gly His Gly Gly Tyr Lys Pro Phe Gln Asn Pro Val Pro Asn Gln625 630 635 640Ser Pro Ser Ser Val Pro Leu Phe Thr Phe Gly Leu Asp Thr Glu Leu 645 650 655Ser Pro Ser Pro Leu Asn Ser Asp Pro Pro Lys Ser Pro Pro Glu Cys 660 665 670Leu Gly Leu Glu Leu Gly Leu Lys Gly Gly Asp Trp Val Lys Ala Pro 675 680 685Pro Pro Ala Asp Gln Val Pro Lys Pro Phe Gly Asp Asp Leu Gly Phe 690 695 700Gly Ile Val Tyr Ser Ser Leu Thr Cys His Leu Cys Gly His Leu Lys705 710 715 720Gln His His Ser Gln Glu Glu Gly Gly Gln Ser Pro Ile Val Ala Ser 725 730 735Pro Gly Cys Gly Cys Cys Tyr Asp Asp Arg Ser Pro Ser Leu Gly Ser 740 745 750Leu Ser Gly Ala Leu Glu Ser Cys Pro Glu Gly Ile Pro Pro Glu Ala 755 760 765Asn Leu Met Ser Ala Pro Lys Thr Pro Ser Asn Leu Ser Gly Glu Gly 770 775 780Lys Gly Pro Gly His Ser Pro Val Pro Ser Gln Thr Thr Glu Val Pro785 790 795 800Val Gly Ala Leu Gly Ile Ala Val Ser 805454264DNAArtificial SequenceSynthetic primer 45gaggagattt ccacccagct ttcccccacc acactgctgc ccccacctcc ctgtaccaga 60catggattga gtccttataa ctatgtagca agtgcttttc ccactgagca tctcctcagc 120tcctccctgt gttgctttct tacacatttg tagagagtta caggcagggt actggccctg 180gtactggttc tcaggtcact cctggcatct ccatttcctt agagacttgc tcatatctag 240gcttcaggca tgtgggacac aatagagacc aaggacatcc ctcagcacgg gaccccttgt 300gactccttca ccagcagtct tgggcctcct gcctaccctc atggcccaag cacccaattc 360taacatatga ttttaagagc agtgtgtaca gagtctttta acattcccct gttgcaggtg 420cctggacatg attctgttag ctaggctgct ctgaggagct ggaggtacgt ggccccgggt 480ggagctggtg tggtctgggc tgttgaggga agtgatggta cctggcctca ttcctcttgt 540aggaatgact ccagccacag agtgtggagg gtggcccagt ccacatcagc agcccaagac 600atcttgcagc tccagaatga gttttcaaaa ctgagcctat taaagaccct tgatggatgt 660cattccctat aggagggggt

ctttactact gtgtccacac ctgggggtca gaggacccct 720aaaatggaat ggaaaacaaa gtgtctgcct tgctcacgag ggcttggctt gtacccatgc 780tcacatgagt ctgtgaccct tctgacagta aggatttggg cttctggaag tcaggggctc 840caccatgaga acaaattcaa atcccagggc cacctataca gctgtgcaac ctccggcaag 900tcactcaacc tccctgtgcc tttgtccctt catctgaaaa tgagggaact gactgtacct 960ccatgataaa gctgggttaa gtggtgggaa gggacagagg gagagatgat cagaagaaaa 1020cttagcatgg cgttaatagc agtgctagtg aggattgcca gagggcagag gtcctccggg 1080acatcaggcc accagagtgt tctccaagag atgcacaggt tccacttggg cgatgtcacc 1140ccctggtcct ggggctcgga ttctgttaat aggaccatga agaccactgt tcctaaacaa 1200ttgctctgcc atcagccacc tcttcctccc tccctccctc cctcccccct ccctcccttc 1260ttcttctctc tctctctctc tctctctctc tctctctctc tctctttctt agtctagaat 1320agagtttatt aagggcatgg ggaaggaagc taagaaggta gtagaggcag agagagagag 1380aagagagaag tagaggggta gaggcaggcc atgagcacat gaagagagag ggggaaggga 1440atggggagag gggaaggggt gaggggacag agtgggagca agaaggcaag aaaccaagag 1500agaaacaaga gggcaagagg ctcgaagccc tttcttggcc ttcccacagc ttagccaggc 1560ttcagtgcgt cacacaggct gttgacattt gaagttgaca ggtacaaggg ttattttcat 1620ggctcatttt atttcattgt taggttctgg ctggacttct cgaagctgag gagaagcaga 1680gggacctggc ttctgatttt ggatctgcgt gcttgctggt tctggcgcct gctggtcttg 1740ttcctgtaac ctaggactcg gggcttgcac atgctttttt tttgaagttg ctggagaggg 1800agcccaggac cttgtgcagg tgagccaggt atcctgacac tgcaggattg ttcatggcat 1860gagacactgg tggctggaac tggatgcgtg tatgactggg tgtccacctt gggcattact 1920tcctctctgc agctctgccg ggcccagcct gacagacagg tccacgaagc agaaggaggc 1980agaactccag aggctgaaga acccagctgt agactcacag ctgctcggag gcaaaatggg 2040tctgggactg acccatttag atacagcctc cagataatga ggggagcggg ggactgagga 2100ctgacccatt gcaggggggt cactgaggac tgacccattg ctgcaggggg ggggtactgg 2160ggtctgaccc attgctgcag gggtcactgg ggattgtccc actgctgcgg gtggggcact 2220ggggactgac ccattgcttt gggaaggcac ttgggactga cccactgctg ttcatagagt 2280agcacacagc tccccatttg cagggcttct ctctctccca tcctccctcc caatttcttt 2340tctttgcttt gatcctcttc ctcccgaatg ctgggggttg gaacccagga cctggaacat 2400accaggcaag cctactgaat ttgtgtgttc atttaagctt ccagaacctc taagatgggg 2460ctgtcagtgg atggccactt agcagacagg gaaactgagg ccctgggtac agcttgctca 2520ggttcacagc tggtatggga gggagccagg atttgaactt gagaaagtta tgaatgtatg 2580ccacagctct cctgggacag gcgaatagag gtatgctcgc gtgctctgtg tgcagagggc 2640acagagacat gggggaggga ggccatgaca caaatgaaat ggaccccgct gacccaggat 2700cagcatctgc ccactcttct ttctgcaggc accttttgtg tccccaatgg ggcggctttg 2760caccaagttc ctgacctctg tgggctgtct gattttgctg ttggtgactg gatctggtaa 2820gtcactcatt cattcatcaa ccactaatga catgtcaagg tcctgcagtc ctcacggaat 2880agaacaaagc tcttgtgggg attggggggg aggggggagg aagcagccga tgacaaatca 2940ccatgactaa gttattttca ttgacactaa ttcaaattgg aaattggggc tgtccgggaa 3000ttgagtagaa aataatttga gttatatgcc aaaggttatc aaaagatggt gggaaaacgc 3060agctcaggac tgagtaaggg aggcttgggg gaggggtgga gagggagagg gcattgcact 3120ttaaagcagg gtgcacaggg acactggtgt caacttagag gaaggcaggg gagggcacac 3180agtgtctgtg gtgtgtcttt ggtgaggccc ccagggaggg cctgcctggc atgggttggg 3240aggacgtgtc gcagggtcat tgagtcagaa gaccttgcag agtctgatca gaggaattga 3300gtgaaggcca gggtcgctca gtgatctgga aagagagttt ctagatcaat ctagaaggtc 3360agcatgaaga cacgtgggaa ttgacctgaa ggtttctcat ccatgtggcc aggtgtggct 3420tacacctgtc agcctagcac ttgggagact gaggcaggag gattgtgagt ttgagcccag 3480cctgggctac aagttactgt aaaaaaacaa aacaaaagaa aaaaacaaaa aaaaaaaaaa 3540ccaaatggac acacaaattc taccccaagc ccagacaagg gtggattatt ctgtatgacg 3600ggaaaaccca gggtagctgt tgaaggagag ggttcaggag gttagaattt tgtttgctgt 3660atgaaaactt cctgcatggt ggaccacacc cgctggcatg tgtattgtga ctttgtctgg 3720acagtggcca tgtagggctt gggtcaaaaa gctcttgagg gtcccacact cactggctgt 3780tacacacaca cacacacaca cacacacaca cacacatact cacacacaca ccacatctga 3840attcctggca gggagctgat gggtaagtgt ctgtcccctc ctccttccct tccctctttc 3900ctgctcttcc attggctaag cctcttgggt accaaagggt cagagcctga gcctgagccg 3960tacagattgc agagtcaatc tcttgcctca gtggagaagg gaggtaggga aggagatgtg 4020ggagtggggg gcgggatggg gggtggggcg cggtggacaa cactgtagga cactgcccct 4080tcctccctgt gctttaacgt ggtgcttctg agggagagtg gcacctaggt aggtgtgtgc 4140gtgtgcaggc atgtgtgtca tgatcctgat gcactagcct ccctctgacc ttagtggtgg 4200gagcccctga ccatgccacc actgatctgg ccgttctgtc tctgcaggga gcatcaaggt 4260cctg 4264464525DNAArtificial SequenceSynthetic primer 46tgcgcagtag cacgcatgcg taatcctgat ggagcaatta ggagaagccg gtggccggct 60agcctgtgca gactgtgaaa acagagcatc tgaagctgtg tgaaaggcta gctcgcctgt 120gctcctcaga tcctgacata cgccatctcg agcaaaagat taacaatcga ttaaaaccca 180cacatgaaaa caaagaaacc agttagctcc agataaaagc aaatgccagg aaggaaagga 240ggcagagggc aggggtggag gagcaggtgg gagaggatga cgtcagagaa atctcagtgg 300ccgtgaacaa ggaagatatt ccagggcttg ttctgaatgt gtgatctttt aaaccatgtg 360gccctcttag cctgtgagaa aagtccattt ctagccctgc tgaacagaga agcccagggg 420agcaaccaac tggccttcgg tcactcagca aggacctagt agaggctggc tttgaacttg 480agccctccag ggcacttgac ctctgagctt tgggaaaaca agcgaggcag cttcacgctg 540ttgtgatctg gacttggatc agggccagtg aggacaggga cagggatggg tgtgataagg 600acttggtgtg taagagggag agttggggag acaggtggga gggctatctg tagccaggta 660accaagggtc ccaggggaac ccccagtgtg gacgcggact gcacatgaca cagggcggcc 720tccccattca tgactgtttt tctccttgca gacttccagc tgcccctgat acagcgcctt 780ccactggggg tcaccatctc ctgcctctgc atcccgttgt tttgcctgtt ctgttacttc 840agcattacca agtgagttcc tgctttggct ggtgtctctg gctggccctt cagcagtgct 900ctcagaggtc acagtcattg tgctggctga gaaaaggctg gaagtctgcc tcgaagggca 960gggcagggca gcctgtggct ggaggcttct ccacagtggc tgtgctctgg aataaagaga 1020gctaggttgg gggacagtaa tggtggctgc tctggagaga aggagctggc ttggggctag 1080ggaggggttg tttggggcat agagaagatg ggagctctgt ccctctcaat gcccatggta 1140tggtcccggc actgggcatg tggtaggagc agaggaattt gagcagatgc tgtgaatata 1200gtagggtttg agtgagaccc ggaagtggcg cttgctggca ggtagctggt cagctacctg 1260ggctcctctg tgggctcaga gtgaccatga gaacaagacc tccagaggag tctgggcaga 1320gccggtagag ggggaagatt cacttctggt gagttctacc ttggagtctc agacccagag 1380ccatgttctt ttagaagtga gattagaagt cacttcgaat tagggctgtg caggtacccg 1440gtacccgccg cctggctaag tgcagggagt gccagagcct ctcctctccg tggcttgcca 1500ctcataacct cgtcagtcac ccggggctag gcactgctgt tctgatgcag agtttctctg 1560tgtcctgttg cccttccctt ctcccacgtg atgcccctgc accccgcagc ccgcagttct 1620caaaggcaca tggcacgttc ttccatagag tcttagccat gcccccaccc taaacgttct 1680gccccttcac ttttctgggc tgttccctgc aacccccaac tctgcttctc tctcctacct 1740tcctctctta tacattgtgg tcttcttgtt tgtcttttta attgttttga aacccagggt 1800cttacacatg ccaccctatc tctgagctcc actctagcct cctgcttact tttgcttgga 1860gacagggtct tgccaagtta cctaggctgg tctccaactc tcctcagctc aggaaggtct 1920ggcactttga ctcttcctgc cctgtctcct gagtggcagg gatgatttcc ggtcaccatc 1980cctggctgtg gtttttgctc atcagcactt atggtggtga atgagtgtca tgtgatcatg 2040cctgcatttg cttcttgttt ctcttgaagg cgacccacag gggcttctcc actggctgtt 2100tttctggctt agtccctgtg gccctagtac cttgatgctc agaaaatact atgggaagac 2160ggaagagtga aagtcatagg ggccagttag cccaggaaga aaggaaatgc ccgcttccac 2220acggcccctg acctctcaag cctgttaaag ccagcatggt caaaagggac ttttgaggga 2280gcgggatcct tgctgtatag cttgaggctt tggtagtgtg gataggtcaa gcagccctgc 2340cagcatcagg ctctccctgg gcacctctcc caattcagaa tggtagtggt gatggacagt 2400taggaccaag attgttccat atccaaaatg ggatcagtgt ggccttcgtg ccctgggagg 2460agtgacggcc aggacacttg gaagagcacc ttggccactg tacattcgga gaccaaccta 2520cttcctttcc accctcagga ttaagaagat atggtgggac cagattccca ccccagcacg 2580cagtcccttg gtggccatca tcattcagga tgcacaggta agagggtaca ggcgttgcat 2640agtgtgactc ccagccatgg gtagggtggg tgggtgcgag gagcaaacgg ccaggggtaa 2700gcagggtggt gcacgggtgg taacaaagcc catgcccaca tctgtgtggg cttggtatta 2760ttcctgccat gttctgctag tcacagtgaa gtcctgtgtc cagatcagag agtccaagat 2820ggcaccggca agctggagca tctgagggca tttgctatct ctgatgccct ttcactggtg 2880tggggggcag ggagggcaga ggctgagact cccagagctc tgtggggcct gtgtggatct 2940cagctctcta ggcatggttg ctgtctgtga aaacactgag ccttctgtga ctgggttgtg 3000ctggcacccc ccacccccac tgattggctg tctattttag gtgcccctct gggataagca 3060gacccgaagc caggagtcaa ccaagtaccc gtatgtatct gaacttgaac ttgggtcata 3120gtttttgtgg gtttgtgtga gaatgtgata gttactcggc tggtggcaat caattcccct 3180tccttgcccc ccccccccac acacaattgc cttctctctt cctggcccca aggggggaga 3240agggggcaat catgggtggc tgagtctcca ttctgcagca agaacaggct aagtgttgat 3300tttaagagtc agaaccacat ctataacacc ccaatatcaa tacagggtga ttccgtgtgg 3360gattctaaag gatgaatagt tttttaaatg tggcaaaact taaccctcag ctttacctct 3420gcaggcactg gaaaacttgt ctagacaagc tgctgccttg cttgctgaag cacagagtaa 3480agaagaagac agacttcccg aaggctgccc caaccaagtc tctccagagt cctggaaagg 3540caggctggtg tcccatggag gtcagcagga ccgtcctctg gccagagaat gttagtgtca 3600gtgtggtgcg ctgtatggag ctgtttgagg ccccagtaca gaatgtggag gaggaagaag 3660atgagatagt caaagaggac ctgagcatgt cacctgagaa cagcggaggc tgcggcttcc 3720aggagagcca ggcagacatc atggctcggc tcactgagaa cctgttttcc gacttgttgg 3780aggctgagaa tgggggcctt ggccagtcag ccttggcaga gtcatgctcc cctctgcctt 3840caggaagtgg gcaggcttct gtatcctggg cctgcctccc catggggccc agtgaggagg 3900ccacatgcca ggtcacagag cagccttcac acccaggccc tctttcaggc agcccagccc 3960agagtgcacc tactctggct tgcacgcagg tcccacttgt ccttgcagac aatcctgcct 4020accggagttt tagtgactgc tgtagcccgg ccccaaatcc tggagagctg gctccagagc 4080agcagcaggc tgatcatctg gaagaagagg agcctccaag cccggctgac ccccattctt 4140cagggccacc aatgcagcca gtggagagct gggagcagat ccttcacatg agtgtcctgc 4200agcatggggc agctgctggc tccaccccag cccctgccgg tggctaccag gagtttgtgc 4260aggcagtgaa gcagggtgcc gcccaggatc ctggggtgcc tggtgtcagg ccttctggag 4320accccggtta caaggccttc tcgagcctgc tcagcagcaa tggcatccgc ggggacacag 4380cagcagcggg gactgacgat gggcatggag gctacaagcc cttccagaat cctgttccta 4440accagtcccc tagctccgtg cccttattta ctttcggact agacacggag ctgtcaccca 4500gtcctctgaa ctcagaccca cccaa 45254710537DNAArtificial SequenceSynthetic primer 47caggagccca cctgcgtctc cgactacatg agcatctcta cttgcgagtg gaagatgaat 60ggtcccacca attgcagcac cgagctccgc ctgttgtacc agctggtttt tctgctctcc 120gagtaagcct gcgctggagc tggaggtttg gggaggttgt gcccaaaggg tttgccccaa 180gagtgagctg ggtccaggtg gtgcgctgga gtgcaggatg ctgagtatgg tttgctgctg 240tttatatggt gttagagggg aggtcccatc tccagggaca tgttatgtaa gatacagtgg 300agcgcatggt gggagtgttg gtccacgtgg cacatggata cggctggaat actggactag 360accagcagtt ctcacacttt ttggtctcag gacccttttt cacacttaaa aatgagtgag 420gacccaaagg gctttggtgt aggtaacaca tcattctatg tttacctaat tagaacttgc 480aatgaagaaa tggtgtaatt tttaaaaaat taaaacaatt aaaaattttt tttcttactg 540aaatggaggt ctcactgtgt tgcccaggct gctctcaaac tcctgggctc cagtgatcct 600cctgcctccg cctcccaaag tgctgggatt acaagcgtga gccgctgtat ccggcccaaa 660atggagaaat tttaagtccc aacaacatgc aagcccgcat tcaacaaatc ttcagatcaa 720ttacatgatc acaggtcatg tagcctctag aaaattccac tgtacgccag tgagagagag 780tgaaaaggca aataacgtcc ctgtattatg atgaaaagag ttttacctgg tgggcccaga 840ccacactttg agaaccactg gactagaccc ttgattgagg agtacggtgt tgagagtgga 900gtcctctgtg atggtggatg gaccaggaca catggcatag gagtcaggtg gttccctggg 960ctactccatg gtgcacagga tgcttcgtta cactggtgcc caggacataa tcacgtacac 1020aagacacaca gttacggggc agactgggga tatacggcac accagcatgc agcgttcacc 1080agtaaaggtg gtattccatg attattctaa ggtagatggg ctgtgctttg tttccattgg 1140cttagtccag ggattggcaa actatggccc gtgagccaaa tccggcccac tgcttgtttt 1200tgtaaataaa gttttattgg aacacactgg ctgctgtagt tgtaacagaa actgcatggc 1260cctcctttat gttttttgtt tgtttgtttg tttgtttgtt ttctttgaga cagagtttcg 1320ctcttgttgc ccaggctgga gtgcagtggc acaatctcgg ctcactgcaa cctctgcctc 1380ccgggttcaa gcgattctcc tgtctcagcc tcccgagtag ttgggattaa tggtgcctgc 1440caccacaccc ggctaatttt tcgtattttt agtagagacc ggttttcatc atgttggcca 1500agctggtctc gaactcctga actcaggtga tccacccgcc tcagcgtccc aaagtgctgg 1560gattacaggc atgagccact gagcccggcc tcctccttta tcttaattga aataattcag 1620aaatggaaag tcaaatactg catgttctca cttataagta agagttaaat aatgtgtaca 1680catgggcatt attccatgta ccatggaata acagacattg aagacttggg agggtgggag 1740aggggtgaag gaagagaagt tacttaatgg gcatagtgta caccatttgg gtgacggacc 1800caccagaacc ccagacttca ccactaggca gcatatccag tgagaacaga tctgaggctt 1860gccatcaaaa ttgcacttgt aaggccgggc actgtggtgg ctcgcggctg taatcccagc 1920cctttgggag gccgaggtgg gcagatcact tgaggtcagg agttcgagac cggcctggcc 1980aacatggtga agctccatct ctactaaaaa tacaacaatt aactgggtgt agtggcgcac 2040acctgtaatc ccagctacta gggaggctga ggcgggagaa ttgcttgagc ccaggaggtg 2100gaggttgcag tgagccgaga tcacatcact gtactctagc ctgggtgaca gtgagacttt 2160gtctcaggaa aaaaaaacaa aaacaaaaaa caaaaaactc gtacccccta aatttataca 2220aataaccaaa aaaaaaaaaa aaaaaggaaa ttgtgtggcc tttgaagtcc aaaatattaa 2280ctatctggcc tgttacagaa aaagtttgca gacccctggc ctagcccgtg agatgtgggt 2340tggctgttaa ggtggaacat tggaattatc ttacgatggc caaactgtgc gatgcagagc 2400ttatgttgtt ctaaattaat tagtgccacc ggttcttccc tttcatgggc tttcaggaac 2460aagctaagtc ccaggaccag ggccggcagc taggcaggtg tgaggagcat ccttggtgca 2520tgtggtaaga ggctgtggcc agcaagagag gcaaccctag tcggctgccc cagcacaccc 2580tggccgctcc caagccccca gatctgtcct cacatccgtg atcgggaagc tggaagagtc 2640tgatgcggtt cctggaggca tgtcccggac acagctgtgg ggcccagcca gcctacaggt 2700gaccagccta acccagcccc tgtgtctgca gagcccacac gtgtatccct gagaacaacg 2760gaggcgcggg gtgcgtgtgc cacctgctca tggatgacgt ggtcagtgcg gataactata 2820cactggacct gtgggctggg cagcagctgc tgtggaaggg ctccttcaag cccagcgagc 2880atggtgagca gggcggagtg cggcaggggt ggctgggtgt gttcccacag ctgcctgggc 2940tgagggtggg gtgggcaggg gaggaggtgg ggtcatagca acagcaggag gaagccgcct 3000gtattttccc aaatctgatg ggattcctgc ccctgcctgg gcctcagtcc tcccaccttt 3060gaaacggagc tggtcgcagt agaccaccaa gcccccttca gcccagctgt ttccacccct 3120gaacttaagt gcccaggaag gcgtattgag atgaggtgtg cttgctggaa ggcatgcctg 3180ctgctgattg aaaaccgaac tgggaacatt ccttccattc tgtgtccact ggtcagctgc 3240tgcggctttg gatggtcttg accgtggaag gctgaccttc ttctggtacc cggagtccct 3300gcaggaatcc cccttgagct tgctgggctg tggtgacagg agtttaaaac atgcgttgta 3360ttccagtgat gcatgatatg acatgcatca caggaataaa aacctgaggt ctcatggata 3420tgattgcttc aaaggagacc aagttttaaa acagatgaat caaaataaag aaaaatactc 3480agtaaatcat cataaagtac agagatgtgg ccaaaggtgt gaaggatgca gctgtaaaag 3540ctgaagtttg aggccgggtg tggtggttca tgcctataat cccagcactt tgggaggccg 3600agcccagcgg atcaccggag gtcaggagtt cgagaccagc ctggacaaca tggtaaaacc 3660ccgtctctac taaaaataca aaaaattagt ctggcatggt ggcaggcgcc tgtaatccca 3720gctacttggg aggctgaggt aggagaatgg cttgaaccca ggagaaggag gttgcagtga 3780gcttagatca tgctactgcc ctccagcctg ggcgacagag tgagattacg tctcaaaaaa 3840ataaaaataa ataaaaataa aaagattttt taaaaggctg aagtttgggt tactttggct 3900catacacttt gccttcactg tagaaaggtg gttagtaaag accaggcgcg gtggctcatg 3960cctggaatcc cagcactttg ggagcccagc gcaggcagat cacttgagcc ctgggctatt 4020gaggctgcag tgagctggga ttgtgccact gcactccagc ctgggcaaca gagtgggacc 4080ctgtctcaaa aaagaagaaa aaaagggtaa ttaataaaca ctaaagttct atgtagaatt 4140ttagcaacat tattgttatt ataatcttct ttgctatggc tctgaatctg tgtggtgctc 4200cagaagtatg ctatggaggt tttgtcgacc aaaaatctgg gtggtggctg tggtttgtag 4260gccggggctg ggctgggtga tgggggagtc actgcataga tcctcacata gaggccgctt 4320ctcccgcagt gaaacccagg gccccaggaa acctgacagt tcacaccaat gtctccgaca 4380ctctgctgct gacctggagc aacccgtatc cccctgacaa ttacctgtat aatcatctca 4440cctatgcagt caacatttgg agtgaaaacg acccggcaga tgtgagtggg catgctttga 4500cgtttttctg tgacctctgg ggaacagggt gggtgaccag cagaggccca gtccctggag 4560ccaggagcct gggaggcaag ccctggggct ggatagcaaa tcccaggagc tagagacctg 4620gcttctcacc tggctctgca ctaggcaagt ccctttgctt cctggccccc cacccctcac 4680atcagagaag gggagttatc tctgcatgcc gctcctcctc tgtaaaggta gggctgtggg 4740ccacatctgt gtttcccagt ttgggggaca caagtgatcg taggtggcac attgacagct 4800cacttgaata accctattat tgaagagaat aatactgact caagagacag tgacccgtgt 4860cagttccctt ttgaggccaa cgggttaagg aggaagtccc catacagctg actcgtttac 4920taattcctct taatgaagag agcagaggcc acaccccagg cttagacttt cccaagaaaa 4980caagatcagt ttgttggttg ttccccatgg aagctggtcc tgacattccc ttcacagtag 5040tgttggtgga gtttttgttg ttgtttgttt tgagacagag tctcactctg tcacccaggg 5100tggaacacag tggcgtgatc ttggctcact gcaacctccg cctcctgggt tctagcgatt 5160ctcctgcctc agcctcctga gcagccggga ctacaggcac ctgccaccgt gcccagctaa 5220tttttgtata tttagtagag atggggtttc actgcgttgg ccaggctggt ctcaaactcc 5280tgacctcaga tgatccactc gccttggcct cccaaagtgc tgggattaca ggtgtgagcc 5340accgcacctg gccagtggag ttccttctta agtacatgta ttgacatctt taaaaagggc 5400gagaggattt acaggaaact atcaggtcag taatggcagg ggccgtccac agtgggtggc 5460tgagtccccc tatttttctg ctggtgtgca gggaggtcat ttcctgccac ccatgtttcc 5520ccaccctgaa tccaccttcc tcacattccc attggaggga caatctctgg acatatggga 5580cctggggtcc cacagggctg caatccaatg cctgctgtgc cactcgccag ctgtgtgatg 5640ttgggcatat cccataacct ctttgtgcct cagtttcctc atctgtaaca caggagtgac 5700aagagcaccc gcccacaggg ctatgacagt acaaggtgtg tgatacagat gagctcccct 5760gtttggccca catgtgtcct aaaagccatg tgccctttct cttgagtgcc ccaggccaca 5820gagatcccca tctgcccgct gtcccacaca ctggtctgtc atttgttcct tgaggtttgt 5880gagggccggc tctgtgcatc ccaggggccc aggctgggcc tggttggctc tcagggagca 5940ggcacccgcc accttaagct cccatgctgg tgtctgtcac tgcttcctct caatctggcc 6000aagccagggg tgtcgattta tatctctcag gtctggtttc ccctttggca ctgggccagg 6060tatggggaaa gagcaggaat ggggcagttg gctcacacag cagaggctca gaaagcgggg 6120ggcatggggg gaaggagtgc acagatgcta gagagtgggg caagttttgt ttggtcaata 6180aatctccttc tcatgcccca ggcctgtgca agacctacag agagtcccaa ggatgggctg 6240gggggaagag aaaggtacca ccttcagagt ccaaagatat gttatttaat attttcatat 6300ttctagatct gccttcaggc atggctggat ccagcttcta ggaacctgtc cagctctgcg 6360ccctgcttta ttctgtactg gcttcgtttt taggcaggct cttccctcat gtagtggcag 6420atatgcctac tagttgctcc aggcctacat cccaaagcca cagtgggaaa agggtttttt 6480ttcttgacgg ttctaataag agtcctaagg ctgctgctca gtggcctggc ttcgatgctg 6540tgccagcctc tgaaccaatc actggctgtg ggtggagaga gggtgctggt ggagggccct 6600gcttgtccag ggaggagtca catacctgcc tctagggctg caggtgggct cagctccatc 6660caaaccagat gaactgaaaa taaggcagga gtggcttccc caggggaaac tggggaagag 6720gaagcaggac tgtgctggct aaaatgccag ccaggtttaa gacgtggcac cagatgccag

6780tcatgggatt ggattggtca gcatgcctgg gctatggctt aggggtatgt tggtgctcag 6840ggatgccaca ggcctccaga taccaggtct gaggcagaag aatgaagtcc agcttctctt 6900gtgggtggaa cagtggcaac tgagataccc catctctccc ttcccaagaa cagagctgaa 6960cataaagaat ttagtgattg gccagagctt ggccacatgc tcccctctga tgaatgatag 7020gccaggtgat gggattggca caattggctt agactaatga gggttggccc tggagttgca 7080ggcagtggag ttctgtccta agcagtgggc acctaaaccc gatggcataa aagctgggcg 7140ggtgtccacc tgcatctgcc acagcactat aggcaccaac tgtggctcat actgagtggg 7200ataaattcca gaaagaaaca ttaggaactt actatagaat tttggggcta gagctactca 7260ttcattcccc tagataattt ctaggcaagg ttccatagtg gagggggagt tttggcttgg 7320gcattgaagg atgcatagga gttttctaga tggggaaaga agggaacggt agaccaggca 7380gagggaactg catgataaaa ggtttatggg tgtgaaaatt catggaatgt ttgaggatta 7440tggggttggg ggatgtggga atatgtgtag cgataaagca ccaaacaaag ccaaaagttt 7500agttagagcc ctgaatgcct gcctcataat ggtttccata ttttatatgc ctactatgtg 7560ccaggcacat tgctcagggt cacacagctg gaaatggcag ggctgagttt ttgttgttgt 7620tgttgttgtt gagacagagt ctcactctat cacccaggct ggaatgcagg ggcgtgatca 7680tggctcactg catccttgac ttcctgggat caggtgattc tcccacctct gcctcccagg 7740tagctgggac tacaggcaca ggccaccacg ccaggctaat tttttgtatt tttagtagcg 7800acagggtctc gccatgttgt ccgggctggt ctggatctcc tggcttcaag tgatccccct 7860ggctcagcct cccaaggtgc tgggattaca ggcttgagcc accgcatcca gcccagatct 7920gagatttgca cccagtattt gaactcccaa gcctgtgctc tttttcctcc catggacatt 7980tctctcagag atggtctccc aaacacctgt ccttcttgtt aaaaaacaga caaaccgcaa 8040gtagttcttt ggaagctcag atttctcttt tgtttcttag taaaacattt cccagttccc 8100agctcccttc cagggtgtaa gatttcttcg gtaacttaca tctagctgtt gcttcttgtt 8160tgctcatgtt tagaaagaaa gacaaaagag agtgagaatt ttctctccct tccccagtct 8220ccccacaact cacaccccac cctcagctcc ctctgtaata ggaaaatctc tgaactctct 8280gtagttgctc cagcaatctt ttggaacttt gcttctttct tgtgaaaaaa cctccccttg 8340gctcactttg caccaggttt ccccaaatgt gcttccaacc acaagcagaa atggagctgc 8400cagtaaccag gaagaaactg ccgggggctg aggaagagga gagggaggtg catagccctg 8460gatctcgcag ggagaggggt gacaggatga gaactcaggt tgctcacttg ccatcagggt 8520cagtcatgaa tatagcgttc atgtatcact ttttaaagct tttttggagg gtaaaagtaa 8580tagttacaca aaataaaaat acaaatggta caaaaggact tagaatggaa acatgtttct 8640ctcccgactc cagcctcctg tttttcttcc cagagactga ccactgctgt ctgtctcttg 8700ccagaaggga aagggaggca aggttagggc aggcagaggg catgtgcatc ctttagagag 8760agcttatgtc tatacaagca aatgtgtgtg ttcagtcatc gctgtcttag ttttctattg 8820ctgcataata atggtactac cagcttcaca gctttaaaca acacccattt attatctcat 8880agtttctgtg gttgggagtc tggacatagc ttagccaggt tctctgcttt agagtctcgt 8940gaggctataa tcaaggtgtg ggatggggct gcagtttcat ctgaggctca attggggaag 9000ggtcacttct aagctcatac aatattggtg acattcagtc cctggcaggc tgttgaactg 9060agagcctcag tttcgtgctg gctgttggtt gtagttaacc ctgaattcct tcccatgtgc 9120cctttgcaaa gccatcaagg cagagagact tgcctagcaa gtaggatatt acagtcttct 9180gtaatataat cacatccatg aaatcctcta tatatcccat cacctttacc atattctgtg 9240ggttagaaac aagtagcagg tcctgcccac actcgagaag accagatgac acaaagatgt 9300gattcaaagt ggggatcatc ggggccatct taggtttgtc tgcagtgatc actgtgccat 9360ctctctctct ctcttttttt tttttttttt ttccgagacg aagtcgtcac tctgtcaccc 9420aggctggagt gcagtggcat gatctcagct taccacaatc tctgcctccc aggttcaaat 9480gattcttctg cctcagcctc ctgagtagct gggattacag gtgcccgcca ccacacccag 9540ctaatttttg tatttttagt agagacagag tttcaccatg ttggccaggc tggtcttgaa 9600ctcctcacct caagtgatcc acccacttcg gcctcccaaa gtgctgggat tacaggcatg 9660agccaccatg cccagcccca tctctcttta aaaaacaaac aaacaaacaa aaaacataaa 9720aagaagcaga gaacacatac acatctgcat cttcccttgt ttacttaaca atagatcttg 9780gaagtcactt ctcagtagag gctaggttgg gcagagcatt ggattctagg ccagtgagtt 9840tggacttgac catggagaca ctaggaagcc catgaaggac agagagagat gcctcgaccc 9900tgccagtcct ttagaaagat cacccagtgc tttttgtata ccaaacccta tttgaaatac 9960ttacgtatat taacccattt ccttatcacc acaaccctgc gggaagggag ataggcactt 10020ttattatctt cattttgcag atgaggacat tgaggtccag agaggttatg tcacttactt 10080aaggtcacac agccaggaag tggtagtagg gactcttacc cttgttttac agatgagatt 10140gaattatctc acgaaaactc agaaaggtta aacaacttgc ctaagtaaca tacagctaat 10200tagtcgagga gcctgacgca tgttgctgta gcctggtcac agttacagag gtggcaagca 10260atggcctgaa caggacgaac aaccaaatac ccaggctggt ggctcttaaa catggtgggg 10320tcagctaacg acagcaacca gggtgggcac tggtgcccct cgcccccggc tggtgcccta 10380acatctccct tttctctacc agttcagaat ctataacgtg acctacctag aaccctccct 10440ccgcatcgca gccagcaccc tgaagtctgg gatttcctac agggcacggg tgagggcctg 10500ggctcagtgc tataacacca cctggagtga gtggagc 1053748183DNAArtificial SequenceSynthetic primer 48cctccctctg accttagtgg tgggagcccc tgaccatgcc accactgatc tggccgttct 60gtctctgcag ggagcatcaa ggtcctgcag gagcccacct gcgtctccga ctacatgagc 120atctctactt gcgagtggaa gatgaatggt cccaccaatt gcagcaccga gctccgcctg 180ttg 18349183DNAArtificial SequenceSynthetic primer 49cagcaccctg aagtctggga tttcctacag ggcacgggtg agggcctggg ctcagtgcta 60taacaccacc tggagtgagt ggagccctag catcacgtgg tacaaccgtg agtatcaggg 120tcgtaggctg tgaggatctc tacagccgtg tatattctct gttcagaaat tccctctggc 180tga 1835087DNAArtificial SequenceSynthetic primer 50aggcccggag tttaaatccc cagagcccac gtaaaagcct gatatcgaat tccgaagttc 60ctattctcta gaaagtatag gaacttc 8751183DNAArtificial SequenceSynthetic primer 51gaagttccta ttctctagaa agtataggaa cttcatcagt caggtacata atggtggatc 60caagctttgc gcagtagcac gcatgcgtaa tcctgatgga gcaattagga gaagccggtg 120gccggctagc ctgtgcagac tgtgaaaaca gagcatctga agctgtgtga aaggctagct 180cgc 1835221DNAArtificial SequenceSynthetic primer 52cgcattgtct gagtaggtgt c 215324DNAArtificial SequenceSynthetic primer 53gctgttcaat gaatggcctc tgtg 245421DNAArtificial SequenceSynthetic primer 54gtggccaccg tttctgggaa c 215522DNAArtificial SequenceSynthetic primer 55tcaacaatct aagcacggac ct 225621DNAArtificial SequenceSynthetic primer 56gtgctgaggc cagggttcct c 215724DNAArtificial SequenceSynthetic primer 57gctgttcaat gaatggcctc tgtg 245824DNAArtificial SequenceSynthetic primer 58gacacacgtg tctgccagct ctgt 245925DNAArtificial SequenceSynthetic primer 59cacggtcagc cagagggaat ttctg 256024DNAArtificial SequenceSynthetic primer 60gtggggtcag ctaacgacag caac 246125DNAArtificial SequenceSynthetic primer 61cgtgtcaaaa gcagaaacgc aggag 256225DNAArtificial SequenceSynthetic primer 62gcgtatgtca ggatctgagg agcac 256325DNAArtificial SequenceSynthetic primer 63gacaagcgtt agtaggcaca tatac 256424DNAArtificial SequenceSynthetic primer 64gctccaattt cccacaacat tagt 246523DNAArtificial SequenceSynthetic primer 65aacatagact ggcgttcacc tgg 236623DNAArtificial SequenceSynthetic primer 66tccgcacttc cacgtgtgag tgg 236723DNAArtificial SequenceSynthetic primer 67gtggttcctg gatagcgctg tgg 236823DNAArtificial SequenceSynthetic primer 68atccaggaac cactcacacg tgg 236923DNAArtificial SequenceSynthetic primer 69tatgttgtgc tgtatgcttg tgg 237023DNAArtificial SequenceSynthetic primer 70ctcagctctg cctacactac agg 237123DNAArtificial SequenceSynthetic primer 71agaacatcag cctgtagtgt agg 237223DNAArtificial SequenceSynthetic primer 72ctactatacg gcgcgtgtga ggg 237323DNAArtificial SequenceSynthetic primer 73gatgtcaggg gtctactata cgg 237423DNAArtificial SequenceSynthetic primer 74tatagtagac ccctgacatc agg 237523DNAArtificial SequenceSynthetic primer 75acgtgtgtcg gttcccagcc tgg 237623DNAArtificial SequenceSynthetic primer 76ctgacatcag gatgttgatc ggg 237723DNAArtificial SequenceSynthetic primer 77gttgatcggg aagctcagcc tgg 237823DNAArtificial SequenceSynthetic primer 78atgtgaccta caaggaaccc agg 237923DNAArtificial SequenceSynthetic primer 79agtctataat gtgacctaca agg 2380644DNAArtificial SequenceSynthetic primer 80ttgttagcat ctcttgataa acttaattgt ctctcgtcac tgacggcaca gagctattga 60tgggtctcac ctcccaactg cttccccctc tgttcttcct gctagcatgt gccggcaact 120ttgtccacgg acacaagtgc gatatcacct tacaggagat catcaaaact ttgaacagcc 180tcacagagca gaagactctg tgcaccgagt tgaccgtaac agacatcttt gctgcctcca 240agaacacaac tgagaaggaa accttctgca gggctgcgac tgtgctccgg cagttctaca 300gccaccatga gaaggacact cgctgcctgg gtgcgactgc acagcagttc cacaggcaca 360agcagctgat ccgattcctg aaacggctcg acaggaacct ctggggcctg gcgggcttga 420attcctgtcc tgtgaaggaa gccaaccaga gtacgttgga aaacttcttg gaaaggctaa 480agacgatcat gagagagaaa tattcaaagt gttcgagctg atactgagcc accatgcttt 540aacttatgaa tttttaatgg ttttattttt aatatttata tatttataat tcataaaata 600aaatatttgt ataatgtaac agaaaaaaaa aaaaaaaaaa aaaa 6448121DNAArtificial SequenceSynthetic primer 81gggagggtga gtggagtccc a 218222DNAArtificial SequenceSynthetic primer 82cagcgcaggc ttactcggag ag 22

Claims

1. A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric interleukin-4 receptor (IL4R).

2. The animal of claim 1, wherein the sequence encoding the human or chimeric IL4R is operably linked to an endogenous regulatory element at the endogenous IL4R gene locus in the at least one chromosome.

3. The animal of claim 1, wherein the sequence encoding a human or chimeric IL4R comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human IL4R (NP_000409.1; SEQ ID NO: 42).

4. The animal of claim 1, wherein the sequence encoding a human or chimeric IL4R comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 44.

5. The animal of claim 1, wherein the sequence encoding a human or chimeric IL4R comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to amino acids 30-216 of SEQ ID NO: 42.

6. The animal of any one of claims 1-5, wherein the animal is a mammal, e.g., a monkey, a rodent or a mouse.

7. The animal of any one of claims 1-5, wherein the animal is a mouse.

8. The animal of any one of claims 1-7, wherein the animal does not express endogenous IL4R.

9. The animal of claim 1, wherein the animal has one or more cells expressing human or chimeric IL4R.

10. The animal of claim 1, wherein the animal has one or more cells expressing human or chimeric IL4R, and the expressed human or chimeric IL4R can bind to endogenous IL4.

11. The animal of claim 1, wherein the animal has one or more cells expressing human or chimeric IL4R, and the expressed human or chimeric IL4R can bind to human IL4.

12. A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous IL4R with a sequence encoding a corresponding region of human IL4R at an endogenous IL4R gene locus.

13. The animal of claim 12, wherein the sequence encoding the corresponding region of human IL4R is operably linked to an endogenous regulatory element at the endogenous IL4R locus, and one or more cells of the animal express a chimeric IL4R.

14. The animal of claim 12, wherein the animal does not express endogenous IL4R.

15. The animal of claim 12, wherein the replaced locus is the extracellular region of IL4R.

16. The animal of claim 12, wherein the animal has one or more cells expressing a chimeric IL4R having an extracellular region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the extracellular region of human IL4R.

17. The animal of claim 16, wherein the extracellular region of the chimeric IL4R has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 150 contiguous amino acids that are identical to a contiguous sequence present in the extracellular region of human IL4R.

18. The animal of claim 12, wherein the animal is a mouse, and the replaced endogenous IL4R region is exon 4, exon 5, exon 6, and/or exon 7 of the endogenous mouse IL4R gene.

19. The animal of claim 12, wherein the animal is heterozygous with respect to the replacement at the endogenous IL4R gene locus.

20. The animal of claim 12, wherein the animal is homozygous with respect to the replacement at the endogenous IL4R gene locus.

21. A method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous IL4R gene locus, a sequence encoding a region of an endogenous IL4R with a sequence encoding a corresponding region of human IL4R.

22. The method of claim 21, wherein the sequence encoding the corresponding region of human IL4R comprises exon 4, exon 5, exon 6, and/or exon 7 of a human IL4R gene.

23. The method of claim 21, wherein the sequence encoding the corresponding region of IL4R comprises at least 100, 200, or 300 nucleotides of exon 4, exon 5, exon 6 and/or exon 7 of a human IL4R gene.

24. The method of claim 21, wherein the sequence encoding the corresponding region of human IL4R encodes a sequence that is at least 90% identical to amino acids 30-216 of SEQ ID NO: 42.

25. The method of claim 21, wherein the locus is located within the extracellular region of IL4R.

26. The method of claim 21, wherein the animal is a mouse, and the locus is exons 4, exon 5, exon 6 and/or exon 7 of the mouse IL4R gene.

27. A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric IL4R polypeptide, wherein the chimeric IL4R polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4R, wherein the animal expresses the chimeric IL4R.

28. The animal of claim 27, wherein the chimeric IL4R polypeptide has at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4R extracellular region.

29. The animal of claim 27, wherein the chimeric IL4R polypeptide comprises a sequence that is at least 90%, 95%, or 99% identical to amino acids 30-216 of SEQ ID NO: 42.

30. The animal of claim 27, wherein the nucleotide sequence is operably linked to an endogenous IL4R regulatory element of the animal.

31. The animal of claim 27, wherein the chimeric IL4R polypeptide comprises an endogenous IL4R transmembrane region and/or an endogenous cytoplasmic region.

32. The animal of claim 27, wherein the nucleotide sequence is integrated to an endogenous IL4R gene locus of the animal.

33. The animal of claim 27, wherein the chimeric IL4R has at least one mouse IL4R activity and/or at least one human IL4R activity.

34. A method of making a genetically-modified mouse cell that expresses a chimeric IL4R, the method comprising: replacing, at an endogenous mouse IL4R gene locus, a nucleotide sequence encoding a region of mouse IL4R with a nucleotide sequence encoding a corresponding region of human IL4R, thereby generating a genetically-modified mouse cell that includes a nucleotide sequence that encodes the chimeric IL4R, wherein the mouse cell expresses the chimeric IL4R.

35. The method of claim 34, wherein the chimeric IL4R comprises: an extracellular region of human IL4R; a transmembrane region of mouse IL4R; and/or a cytoplasmic region of mouse IL4R.

36. The method of claim 35, wherein the nucleotide sequence encoding the chimeric IL4R is operably linked to an endogenous IL4R regulatory region, e.g., promoter.

37. The animal of any one of claims 1-20 and 27-33, wherein the animal further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4, IL33, IL13, programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte Activating 3 (LAG-3), B And T Lymphocyte Associated (BTLA), Programmed Cell Death 1 Ligand 1 (PD-L1), CD27, CD28, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT), T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), Glucocorticoid-Induced TNFR-Related Protein (GITR), CD137, TNF Receptor Superfamily Member 4 (OX40), CD47, or Signal regulatory protein .alpha. (SIRPa)).

38. The animal of claim 37, wherein the additional human or chimeric protein is IL4.

39. The method of any one of claims 21-26 and 34-36, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4, IL33, PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, TIGIT, TIM-3, GITR, CD137, OX40, CD47 or SIRPa).

40. The method of claim 39, wherein the additional human or chimeric protein is IL4.

41. A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric IL4.

42. The animal of claim 41, wherein the sequence encoding the human or chimeric IL4 is operably linked to an endogenous regulatory element at the endogenous IL4 gene locus in the at least one chromosome.

43. The animal of claim 41, wherein the sequence encoding the human or chimeric IL4 is operably linked to a human regulatory element at the endogenous IL4 gene locus in the at least one chromosome.

44. The animal of claim 41, wherein the sequence encoding a human or chimeric IL4 comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to human IL4 (NP_000580.1; SEQ ID NO: 4).

45. The animal of claim 41, wherein the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8 or SEQ ID NO: 9.

46. The animal of claim 41, wherein the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10 or SEQ ID NO: 11.

47. The animal of claim 41, wherein the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 23.

48. The animal of claim 41, wherein the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 24.

49. The animal of claim 41, wherein the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 25.

50. The animal of any one of claims 41-49, wherein the animal is a mammal, e.g., a monkey, a rodent or a mouse.

51. The animal of any one of claims 41-49, wherein the animal is a mouse.

52. The animal of any one of claims 41-51, wherein the animal does not express endogenous IL4.

53. The animal of claim 41, wherein the animal has one or more cells expressing human IL4.

54. The animal of claim 41, wherein the animal has one or more cells expressing human or chimeric IL4, and the expressed human or chimeric IL4 can bind to endogenous IL4R.

55. The animal of claim 41, wherein the animal has one or more cells expressing human or chimeric IL4, and the expressed human or chimeric IL4 can bind to human IL4R.

56. A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous IL4 with a sequence encoding a corresponding region of human IL4 at an endogenous IL4 gene locus.

57. The animal of claim 56, wherein the sequence encoding the corresponding region of human IL4 is operably linked to an endogenous regulatory element at the endogenous IL4 locus, and one or more cells of the animal expresses a human IL4.

58. The animal of claim 56, wherein the sequence encoding the corresponding region of human IL4 is operably linked to a human regulatory element at the endogenous IL4 locus, and one or more cells of the animal expresses a human IL4.

59. The animal of claim 56, wherein the animal does not express endogenous IL4.

60. The animal of claim 56, wherein the replaced locus comprises a sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

61. The animal of claim 56, wherein the animal is a mouse, and the replaced endogenous IL4 region is exon 1, exon 2, exon 3 and/or exon 4 of the endogenous mouse IL4 gene.

62. The animal of claim 56, wherein the animal is heterozygous with respect to the replacement at the endogenous IL4 gene locus.

63. The animal of claim 56, wherein the animal is homozygous with respect to the replacement at the endogenous IL4 gene locus.

64. A method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous IL4 gene locus, a sequence encoding a region of an endogenous IL4 with a sequence encoding a corresponding region of human IL4.

65. The method of claim 64, wherein the sequence encoding the corresponding region of human IL4 comprises exon 1, exon 2, exon 3 and/or exon 4 of a human IL4 gene.

66. The method of claim 64, wherein the sequence encoding the corresponding region of IL4 comprises at least 30, 50, 75, 100, or 150 nucleotides of exon 1, exon 2, exon 3 and/or exon 4 of a human IL4 gene.

67. The method of claim 64, wherein the sequence encoding the corresponding region of human IL4 encodes a sequence that is at least 90% identical to SEQ ID NO: 4.

68. The method of claim 64, wherein replaced locus comprises a sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

69. The method of claim 64, wherein the animal is a mouse, and the locus is exon 1, exon 2, exon 3 and exon 4 of the mouse IL4 gene.

70. A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a human or chimeric IL4 polypeptide, wherein the chimeric IL4 polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4, wherein the animal expresses the chimeric IL4.

71. The animal of claim 70, wherein the chimeric IL4 polypeptide has at least 100 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human IL4.

72. The animal of claim 70, wherein the nucleotide sequence is operably linked to an endogenous IL4 regulatory element of the animal.

73. The animal of claim 70, wherein the nucleotide sequence is operably linked to a human IL4 regulatory element of the animal.

74. The animal of claim 70, wherein the nucleotide sequence is integrated to an endogenous IL4 gene locus of the animal.

75. The animal of claim 70, wherein the chimeric IL4 has at least one mouse IL4 activity and/or at least one human IL4 activity.

76. A method of making a genetically-modified mouse cell that expresses a human or chimeric IL4, the method comprising: replacing, at an endogenous mouse IL4 gene locus, a nucleotide sequence encoding a region of mouse IL4 with a nucleotide sequence encoding a corresponding region of human IL4, thereby generating a genetically-modified mouse cell that includes a nucleotide sequence that encodes the human or chimeric IL4, wherein the mouse cell expresses the human or chimeric IL4.

77. The method of claim 76, wherein the nucleotide sequence encoding the human or chimeric IL4 is operably linked to an endogenous IL4 regulatory region, e.g., promoter.

78. The method of claim 76, wherein the nucleotide sequence encoding the human or chimeric IL4 is operably linked to a human IL4 regulatory region, e.g., promoter.

79. The animal of any one of claims 41-63 and 70-75, wherein the animal further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4R, IL33, IL13, programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte Activating 3 (LAG-3), B And T Lymphocyte Associated (BTLA), Programmed Cell Death 1 Ligand 1 (PD-L1), CD27, CD28, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT), T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), Glucocorticoid-Induced TNFR-Related Protein (GITR), CD137, TNF Receptor Superfamily Member 4 (OX40), CD47, or SIRPa).

80. The animal of claim 79, wherein the additional human or chimeric protein is IL4R.

81. The method of any one of claims 64-69 and 76-78, wherein the animal or mouse further comprises a sequence encoding an additional human or chimeric protein (e.g., IL4R, IL33, IL13, PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, TIGIT, TIM-3, GITR, CD137, OX40, CD47, or SIRPa).

82. The method of claim 81, wherein the additional human or chimeric protein is IL4R.

83. A method of determining effectiveness of an IL4-IL4R pathway inhibitor for treating an allergic disorder, comprising: administering the IL4-IL4R pathway inhibitor to the animal of any one of claims 1-20, 27-33, 37-38, 41-63, 70-75, and 79-80; and determining the inhibitory effects of the IL4-IL4R pathway inhibitor.

84. The method of claim 83, wherein the allergic disorder is asthma.

85. The method of claim 83, wherein the allergic disorder is atopic dermatitis.

86. The method of claim 83, wherein the allergic disorder is chromic sinusitis.

87. The method of claim 83, wherein the IL4-IL4R pathway inhibitor is an anti-human IL4 antibody.

88. The method of claim 83, wherein the IL4-IL4R pathway inhibitor is an anti-human IL4R antibody.

89. The method of claim 83, wherein the inhibitory effects are evaluated by serum IgE levels; pathological lung histology features; number of leukocytes (CD45+ cells), eosinophils (Eos) or neutrophils in bronchoalveolar lavage fluid (BALF); or ratio of eosinophils or neutrophils cells in CD45+ cells in bronchoalveolar lavage fluid (BALF).

90. A method of determining effectiveness of an IL4-IL4R pathway inhibitor for reducing inflammation, comprising: administering the IL4-IL4R pathway inhibitor to the animal of any one of claims 1-20, 27-33, 37-38, 41-63, 70-75, and 79-80; and determining the inhibitory effects of the IL4-IL4R pathway inhibitor.

91. A method of determining effectiveness of an IL4-IL4R pathway inhibitor for treating autoimmune disorder, comprising: administering the IL4-IL4R pathway inhibitor to the animal of any one of claims 1-20, 27-33, 37-38, 41-63, 70-75, and 79-80; and determining the inhibitory effects of the IL4-IL4R pathway inhibitor.

92. A method of determining effectiveness of an IL4-IL4R pathway inhibitor for treating cancer, comprising: administering the IL4-IL4R pathway inhibitor to the animal of any one of claims 1-20, 27-33, 37-38, 41-63, 70-75, and 79-80; and determining the inhibitory effects of the IL4-IL4R pathway inhibitor.

93. The method of claim 92, wherein determining the inhibitory effects of the treatment involves measuring the tumor volume in the animal.

94. A method of determining toxicity of an anti-IL4R antibody or an anti-IL4 antibody, the method comprising administering the anti-IL4R antibody or the anti-IL4 antibody to the animal of any one of 1-20, 27-33, 37-38, 41-63, 70-75, and 79-80; and determining weight change of the animal.

95. The method of claim 94, the method further comprising performing a blood test (e.g., determining red blood cell count).

96. A protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following: (a) an amino acid sequence set forth in SEQ ID NO: 44; (b) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44; (c) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 44 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and (d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 44.

97. A nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following: (a) a sequence that encodes the protein of claim 96; (b) SEQ ID NO: 8, 9, 10, 11, 23, 24, 25, 43, 48, 49, 50, or 51; or (c) a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, 9, 10, 11, 23, 24, 25, 43, 48, 49, 50, or 51.

98. A cell comprising the protein of claim 96 and/or the nucleic acid of claim 97.

99. An animal comprising the protein of claim 96 and/or the nucleic acid of claim 97.