Novel Inflammation to vivo model

Abstract

The present invention relates to a non-human animal deficient in the N-terminal domain of the IL-33 gene. Also provided herein is the use of said non-human animal as an in vivo model of inflammatory diseases, especially with regard to screening methods for anti-inflammatory compounds, and methods for evaluating and optimising the pharmacological properties of a given anti-inflammatory compound.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of International Application No. PCT/EP2011/068696 having an international filing date of Oct. 26, 2011, the entire contents of which are incorporated herein by reference, and which claims benefit under 35 U.S.C. §119 to European Patent Application No. 10189446.7 filed Oct. 29, 2010.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing submitted via EFS-Web and hereby incorporated by reference in its entirety. Said ASCII copy, created Apr. 17, 2013, is named P4670C1_Sequence_listing.txt, and is 28.132 bytes in size.

FIELD OF THE INVENTION

[0003] The present invention relates to a non-human animal deficient in the N-terminal domain of the IL-33 gene. Also provided herein is the use of said non-human animal as an in vivo model of inflammatory diseases, especially with regard to screening methods for anti-inflammatory compounds, and methods for evaluating and optimising the pharmacological properties of a given anti-inflammatory compound.

BACKGROUND OF THE INVENTION

[0004] The interleukin 33 (IL-33) cytokine is the newest member of the interleukin 1 (IL-1) family. Because of its nuclear localization, it was originally described as "Nuclear Factor from High Endothelial Venules". IL-33 is primarily expressed by fibroblasts and epithelial, endothelial and airway smooth muscle cells. IL-33 is the ligand for the IL-1 receptor-related protein ST2. The ST2 receptor is expressed in almost all innate immune cells (mast cells, basophils, eosinophils, neutrophils, natural killer (NK) cells and macrophages) and in NK T and T helper (Th) 2 cells. The interaction of IL-33 with ST2 on targeted cells can trigger the expression and secretion of pro-inflammatory, Th1, Th2 and Th17 cytokines and expression of chemokines involved in Th1, Th2 and innate immune effector functions (published papers and Hicks et al. manuscript in preparation). IL-33 binds to its specific surface receptor through its pro-inflammatory cytokine domain. In addition, IL-33 also has a N-terminal domain that contains a typical DNA-binding helix-turn-helix motif. In its nuclear uncleaved form, IL-33 interacts with histones 2A and 2B in heterochromatin promoting chromatin compaction and functioning as a potential transcriptional repressor. There is strong support showing that IL-33 is similar to other chromatin-associated cytokines (IL-1α and HMGB1) that appears to exert a dual-function, regulating transcriptional repression in the nucleus and signaling via a classic receptor acting as a potent pro-inflammatory cytokine. Thus, it has been proposed that similarly to HMGB1, IL-33 may function as an `alarmin` belonging to the larger family of damage-associated molecular pattern (DAMP) molecules.

[0005] The IL-33/ST2 axis plays pivotal roles in the patho-physiology of human inflammatory diseases as confirmed by their high levels of expression in diseased tissues. Elevated levels of either IL-33 and/or its soluble receptor ST2 are observed in rheumatoid arthritis (RA), inflammatory bowel disease (IBD), psoriatic and ulcerative colitis, acute eosinophilic pneumonia, severe asthma, idiopathic pulmonary fibrosis, liver fibrotic diseases, atopic dermatitis, systemic sclerosis, autoimmune and trauma patients. Similar to its role in humans, the IL-33/ST2 axis has been shown to be critical in murine inflammatory models. IL-33 exacerbates collagen-induced arthritis (CIA), allergic conjunctivitis and experimental autoimmune encephalomyelitis (EAE), Interruption of IL-3/ST2 signaling with antibodies has been shown to be beneficial for the resolution of allergic airway inflammation and bleomycin-induced lung injury. It has also been shown recently that IL-33 is up-regulated in an IBD mouse model of chronic intestinal inflammation (Oboki et al, PNAS 2010 107 (43) 18581-18586).

SUMMARY OF THE INVENTION

[0006] The non-human animal may be any non-human animal. Preferably, the non-human animal is a mammal, more preferably a rodent such as rat or a mouse, most preferably, the non-human animal is a mouse. In a preferred embodiment, said non-human animal is a mouse and the N-terminal deletion of the IL-33 gene comprises a deletion of the full DNA binding domain at the N-terminus of the IL-33 gene. Preferably, said N-terminal deletion of the IL-33 gene in the mouse comprises a deletion of amino acids 1-67 of the expression product of the IL-33 gene.

[0007] The non-human animal may be heterozygous or homozygous for the N-terminal IL-33 deletion. Preferably, the non-human animal is heterozygous for the N-terminal IL-33 deletion.

[0008] The non-human animal deficient in the N-terminus of the IL-33 gene according to the present invention displays the typical characteristics of inflammatory diseases. For instance, a mouse according to the present invention has such characteristics as normal birth rate and growth until about 3-4 months of age, then hemorrhagic lesions in ears and repeatedly observed large coagula in the thoracic cavity, smaller size and general ill picture. On pathological examination these mice reveal multiorgan inflammation, including chronic, multifocal myocarditis; chronic, suppurative, strong ileitis with enlarged intestinal walls; chronic utreteritis with marked hydronephrosis and kidney atrophy, moderate multifocal and perivascular infiltrates in the lung, splenic hyperplasia with strong expansion of eosinophils and macrophages in immune organs, lung and intestine.

[0009] Since the non-human animal according to the present invention exhibits an inflammatory phenotype, it is useful as an in vivo inflammation model. This non-human animal with an N-terminal IL-33 deletion can serve to practically prove the causal relationship between IL-33 dysfunctions and inflammatory disorders and allow the design of directed therapeutic strategies aimed to reduce or abolish the abnormal overproduction of IL-33 in patients. As massive production and secretion of IL-33 into the extracellular compartment is the cause of this severe inflammatory condition, this novel and unique non-human animal model can also be used to evaluate the in vivo efficacy and potency of IL-33 drug candidates if cross-reactivity with the non-human IL-33 or its receptor ST2 is given. Hence in a second object of the invention, said non-human test animal is used as an in vivo model of inflammatory diseases, especially with regard to screening for anti-inflammatory compounds. In one embodiment a method for screening for anti-inflammatory compounds is provided, comprising administering a candidate compound to a non-human animal with a N-terminal IL-33 deletion according to the present invention. In one embodiment, said method comprises a) providing the non-human animal with a N-terminal IL-33 deletion, b) administering to said non-human animal a candidate compound, c) comparing the inflammation symptoms of said non-human animal to those of a non-human animal with a N-terminal IL-33 deletion not administered said compound; wherein the compound that alleviates said inflammation symptoms is selected as an anti-inflammatory compound.

[0010] Candidate compounds include, but are not limited to small molecules, (poly) peptides, (glyco) proteins, antibodies or antibody fragments, (poly) or (oligo) nucleotides, nucleosides, lipids, combinations thereof and modified derivatives thereof.

[0011] Methods of administration of a candidate compound to be screened include, but are not limited to, oral administration and parenteral administration (e.g. intravenous administration, intraperitoneal administration and intranasal administration). In case of oral administration a candidate agent may be blended in a feed for administration.

[0012] The candidate agent may be administered in combination with a pharmaceutically acceptable conventional excipient (such as carrier and diluent) or additives. Further, the candidate agent may be encapsulated in, or bound (or attached) to, liposomes (e.g. positively charged liposomes) or nano-particles and administered.

[0013] Evaluation can be conducted for example using mitigation or recovery of inflammation symptoms, increase in body weight, recovery from hypertrophy of the ileon, or the like, as an indication, by observation with naked eye, measurement of body weight, histopathological observation (e.g. microscopic observation after tissue staining) and FACS analysis (see Examples below).

[0014] Through use of the subject animals with a N-terminal IL-33 deletion or cells derived there from, one can identify ligands or substrates that bind to, modulate, antagonize or agonize cellular IL-33. Of particular interest are screening assays for anti-inflammatory compounds that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including in vivo studies, determination of the localization of drugs after administration, labeled in vitro protein-protein binding assays, protein-DNA binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like. Depending on the particular assay, whole animals may be used, or cells derived there from. Cells may be freshly isolated from an animal, or may be immortalized in culture.

[0015] In another embodiment of the invention, said in vivo model is used for evaluation of the pharmacological effects such as in vivo efficacy and potency of IL-33 drug candidates. Therefore in another embodiment of the invention a method for evaluation of the pharmacological effects of a IL-33 drug candidate is provided, said method comprising administering said IL-33 drug candidate to a non-human animal with a N-terminal IL-33 deletion according to the present invention.

[0016] The present invention also relates to descendants of the non-human animals with an N-terminal IL-33 deletion as provided by the invention, obtained by breeding with the same or with another genotype. Preferably, the descendant is obtained by breeding with the same genotype. Said descendant comprises an N-terminal IL-33 deletion as the non-human animal with an N-terminal IL-33 deletion described above. A further object of the invention is the use of said descendants as an in vivo model of inflammatory diseases. In one embodiment, said descendants are used as an in vivo model for screening of anti-inflammatory compounds. In another embodiment, said descendants are used as an in vivo model for evaluation of the pharmacological effects of an anti-inflammatory compound.

[0017] Furthermore, the present invention relates to a cell line or primary cell culture derived from a non-human animal with an N-terminal IL-33 deletion or its descendants as described above.

[0018] In addition, the present invention also provides a tissue or an organ explant or culture thereof, derived from a non-human animal with an N-terminal IL-33 deletion or its descendants as described above.

[0019] The present invention also provides a tissue or cell extract derived a non-human animal with an N-terminal IL-33 deletion or its descendants as described above.

[0020] In another embodiment of the invention, above-mentioned cell line or primary cell culture, tissue or an organ explant or culture thereof, tissue or cell extract derived from a non-human animal with an N-terminal IL-33 deletion or its descendants is used as a model of inflammatory diseases. In one embodiment, said model of inflammatory diseases is used for screening of anti-inflammatory compounds, in another embodiment, said model of inflammatory diseases is used for or evaluation of the pharmacological effects of an anti-inflammatory compound, as outlined above.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

[0021] Methods for producing a non-human animal with a deletion in a gene, such as the N-terminus of the IL-33 gene, are well known in the art. Suitable methods are described i.e. in Hogan B et al: Manipulating the mouse embryo, A laboratory manual, 2nd Edition (1994), Cold Spring Harbor Laboratory Press.

[0022] The term "IL-33 gene" as used herein relates in particular to the interleukin 33 gene also known as DVS27; NF-HEV; NFEHEV; C9orf26; DKFZp586H0523; RP11-575C20.2; IL33, IL-1F11, 9230117N10Rik, RGD1311155 and the like. Said gene comprises a N-terminal domain with a putative DNA-binding helix-turn-helix motif and a C-terminal cytokine domain. The IL33 gene is conserved in humans, chimpanzee, dog, cow, mouse, and rat. By way of an example the DNA sequence of the mouse IL-33 gene is depicted in SEQ ID. 1. The term "expression product of the IL-33 gene" refers to the translated protein of the IL-33 gene, i.e. the IL-33 protein.

[0023] "N-terminal IL-33 deletion" as used herein means that all or part of the N-terminus of the IL-33 gene is modified (for example substituted, deleted, added and/or inserted) or disrupted, whereby the expression product of the IL-33 gene lacks the whole or parts of the N-terminal domain and/or does not exhibit the function of the N-terminal domain. The N-terminal domain as used herein comprises a putative helix-turn-helix motif which is responsible for DNA binding and nucleon localization of IL-33. In a preferred embodiment the non-human animal is a mouse and amino acids 1-67 of the mouse expression product of the IL-33 gene (IL-33 protein) are modified or disrupted.

[0024] Such non-human animal is depicted herein as a non-human animal with a N-terminal IL-33 deletion, or a non-human animal deficient in the N-terminus of the IL-33 gene or a N-terminal IL-33 gene knock-out non-human animal and the like. In one special preferred embodiment said modification of the N-terminus of the IL-33 gene is achieved through knock-in of a DsRed cassette, thus deleting the N-terminal part of the IL-33 gene. Said non-human animal is accordingly also referred to as DsRed-IL33/COOH non-human animal. The term "wild type" as used herein refers to a non-human animal having a full-length IL-33 gene.

[0025] "Non-human animal" as described herein refers to any animal that is not a human. Preferably, the non-human animal is a mammal, more preferably a rodent such as rat or a mouse, most preferably, the non-human animal is a mouse.

[0026] The non-human animal with a N-terminal IL-33 deletion as described above can be used as a model for treatment of inflammatory diseases. It allows to investigate the effect of an potentially non-inflammatory compound on the inflammatory disease in a non-human in vivo model. Since the transgenic animal is immunotolerant for the transgenic human maB11 antibody, the effect of chronic treatment with a therapeutic antibody such as for example Mab 11 can be determined. Also, the effect on extracellular IL-33 levels and the process and kinetics of an inflammatory disease can be followed.

[0027] The term "inflammatory disease" as used herein relates to any impairment of health or a condition of abnormal functioning characterized by inflammation. In particular, the term "inflammatory disease" as used herein relates to diseases connected to an increased level of IL-33, for example, but not limited to inflammatory bowel disease, rheumatoid arthritis, urticuria, atherosclerotic vascular disease, psoriatic colitis, ulcerative colitis, acute eosinophilic pneumonia, severe asthma, idiopathic pulmonary fibrosis, liver fibrotic diseases, atopic dermatitis, systemic sclerosis, autoimmune diseases and the like.

[0028] "Anti-inflammatory compounds" as used herein means any molecule with the capability of reducing inflammatory responses, in detail, affecting the biological action of IL-33. As such, "Anti-inflammatory compound" includes, but is not limited to small molecules, (poly) peptides, (glyco) proteins, antibodies or antibody fragments, (poly) or (oligo) nucleotides, nucleosides, lipids, combinations thereof and modified derivatives thereof "Anti-inflammatory compound" also includes molecules that mediate RNA interference such as shRNA, microRNA, siRNA, antisense oligonucleotides, spiegelmers, LNA or PNA oligomers, or combinations thereof. Putative anti-inflammatory compounds as used herein are also depicted as "IL-33 drug candidates" or "candidate compounds".

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1a: Schematic drawing of the targeted mutation of IL-33. The DsRed cassette was inserted at the Start codon and fused with IL-33 at its amino acid position 68.

[0030] FIG. 1b: Targeting strategy. RFP: red fluorescent protein; tm1: targeted mutation 1; wt: wild-type; Marker: marker X (Roche); kb: 1 kilo base pairs; EcoRI: Restriction endonuclease site; NeoR. Neomycin selection cassette; triangles: lox sites

[0031] FIG. 2: Phenotype of heterozygous DsRedIL-33/COOH knock-in mice. Growth retardation, erected fur.

[0032] FIG. 3: Phenotype of heterozygous DsRedIL-33/COOH knock-in mice. Organ morphology.

[0033] FIG. 4: Phenotype of heterozygous DsRedIL-33/COOH knock-in mice. Organ morphology compared to wild-type mouse. SI: small intestine; He: heart; Th: thymus; Ki: kidney; Lu: lung; Sp: spleen; Li: liver Co*: blood coagulum in thoracic cavity

[0034] FIG. 5: Phenotype of heterozygous DsRedIL-33/COOH knock-in mice. Ileon hypertrophy. Intestine preparation opened longitudinally.

[0035] FIG. 6: Phenotype of heterozygous DsRedIL-33/COOH knock-in mice (DsRedIL-33/COOH-KI) via FACS analysis. Cell suspensions of the indicated organs were stained with antibodies specific for defined surface marker: CD45(labeled APC-Cy7), total leukocytes; CD11b (labeled allophycocyanin, APC), macrophages; SiglecF (labeled phycoerythrin PE), eosinophils; Gr1 (labeled PE-Cy7), granulocytes; F4/80 (labeled Alexa 488), monocytes. Stained cells were acquired using a FACS Canto I device (BD Corp.) and analysed with FlowJo software. Y-axis: SiglecF, X-Axis: F4/80. Figures show the superposed stainings of different cell populations, DsRedIL-33/COOH knock-in mice have an increase in eosinophils as determined by high expression of SiglecF and F4/80 (see marked populations, "Eos"=Eosinophils)

[0036] FIG. 7: Phenotype of heterozygous DsRedIL-33/COOH knock-in mice. Serum IL-33 levels.

EXAMPLES

[0037] The present invention will now be described in more detail by working examples, provided that the examples should not be interpreted as those limiting the scope of the present invention.

Example 1

Generation of a Targeting Vector

[0038] The targeting vector (SEQ ID. NO. 2) was generated using recombineering technology as supplied by Gene Bridges GmbH, Heidelberg. It contains the following elements:

TABLE-US-00001 1-70 IL-33 exon 1b 71-3871 intron 3872-3882 IL-33 exon 2 3883-4557 dsRed monomer CDS 4558-4579 IL-33 exon 3 4580-4590 intronic sequence 4591-4626 lox site 4625-6040 neomycin selection cassette 6041-6074 lox site 6075-6571 intronic sequence 6572-6682 IL-33 exon 4 6683-7285 intron 7286-7603 pBluescript SKII+ 7604-8463 diphteria toxin a selection cassette 8464-10697 pBluescript SKII+

[0039] The targeting vector was used for homologous recombination in BALB/c ES cells. Positive clones were identified using PCR screening strategies (sequences of oligonucleotides see below). Following electroporation of an Cre expressing plasmid site specific recombination leads to the removal of the neomycin selection cassette in vitro. After blastocyst injection of ES cell clones chimeric animals were breed and DNA preparations of biopsies of the F1 and F2 generation were used to confirm the identity of the targeted mutation as well as genotyping using PCR (PCR AB, CD, EF and EFG in FIG. 1b).

TABLE-US-00002 Oligonucletides Oligonucletide A: (SEQ ID. NO. 3) TAG AAA GAG CCC AGT GTT AAG C Oligonucletide B: (SEQ ID. NO. 4) GGC TTG CCC TCG CCC TCG Oligonucletide C: (SEQ ID. NO. 5) CAC CTG CGA CTT CAA GAC C Oligonucletide D: (SEQ ID. NO. 6) ACG ATT CCT TAG TGA TGG GGC Oligonucletide E: (SEQ ID. NO. 7) GTT GCT TCT GAT GAC TTC AGG Oligonucletide F: (SEQ ID. NO. 8) GCA ATA GCC CTT GCC AAG GC Oligonucletide G: (SEQ ID. NO. 9) TGC TGT TCC AGC CTC TGT TGG

Example 2

Generation of N-Terminal IL-33 Knockout Mouse

[0040] Two genetically modified knock-in mouse models in a Balb/c background were generated. The first mutant targets the N-terminal intracellular transcriptional factor-like activity by in-frame replacing it with the fluorochrome DsRd-monomer (DsRed-IL33/COOH) keeping the functional cytokine domain complete (FIG. 1). The second mutant replaces the pro-inflammatory cytokine domain by in-frame knocking-in the fluorochrome DsRed keeping the DNA binding domain intact (NH2/L33-DsRed).

Example 3

Characterization of N-Terminal IL-33 Knockout Mouse

[0041] Unexpectedly, the genetic engineered heterozygous mutant mice missing the IL-33 N-terminal domain but maintaining an active the proinflammatory cytokine activity in the cytoplasm (DsRed-IL33/COOH) die at around of 4 months of age. Apparently normal at birth, they become progressively sick and finally moribund between 4 and 5 months later with an estimated phenotype penetration of around 60%. At the age of 3-4 months, these mutants start displaying bloody lesions in ears and enlarged bellies. Upon necropsy, a massive splenomegaly, hypertrophy of the ilion suggestive of intestinal inflammation, the presence of large coagula in the thoracic cavity and kidney atrophy are repeatedly observed (FIGS. 2-5). Closer examination of different organs in FACS analysis showed a strong eosinophilia in lungs, spleen, lymph nodes, Payer's Patches and peripheral blood (FIG. 6). Serum IL-33 levels are elevated in DsRed-IL33/COOH knockout mice (FIG. 7).

Discussion

[0042] We have recently found that intranasal administration of IL-33 evoked profound lung inflammation with multinucleated giant cells of macrophage origin in the interstitium (Hicks et al. manuscript in preparation). In the same manner, IL-33 also evoked a bone marrow hyperplasia with big cluster of myeloid/granulocytic cells as shown by histopathology (Hicks et al. manuscript in preparation). In addition, high levels of soluble IL-33 are found in the plasma of peripheral blood of these mice, strongly suggesting that the immunopathological effects seen in our DsRed-IL33/COOH mutant animals is the result of the continuous presence of the fully active DsRed-IL33/COOH cytokine in the cytoplasm and its possible release into the extracellular compartment, thus behaving as a potent endogenous DAMP signal as it has been hypothesized.

[0043] This "alarmin"-like effect of knocking out the N-terminal domain of IL-33 is thus similar to human immune-pathological conditions (whose diagnosis remains unknown) when a potent danger signal is released after tissue damage, necrosis or autoimmunity. As the occurrence of severe multi-organ inflammation in patients with homozygous mutations or deletions of gene encoding IL-1RA and its blockade with mAbs have demonstrated the central role of IL-1α and IL-β in a number of auto-inflammatory diseases (Weber at al, Science signaling, 3, 2010). Possibly genetic variations of IL-33 could result in over-expression and/or secretion of this possible "danger" molecule and it may contribute to the pathogenesis of auto-inflammatory or allergic diseases. Precisely, it has recently been shown a positive association between polymorphisms in the IL-33 gene and higher IL-33 levels with susceptibility of Japanese cedar pollinosis (Sakashita et al. Clin. Exp. Allergy December 2008).

Sequence CWU 1

1

919371DNAMus musculus 1atgagaccta gaatgaagta ttccaactcc aagatttccc cggcaaagtt cagcagcacc 60gcaggcgaag ccctggtccc gccttgcaaa ataagaagta agagcgctct ctttaaccac 120tattaaatct attatcaaat atttgttatt tcaacatgaa gacgggcgac cttaccaaaa 180actctcaagt atacaagagc tgaaaagaaa gagagacagc ttgttgtttt cttaaacaga 240cttcacagca tcaccgttgt tatttggtac aacataggac agttgaccac ttattcttgg 300actagtaaca agaagggtag cagagctctg gtggacagaa cggaagttag agggtgatgg 360gaaaggaaag cgatgtcccc ttctgccctt cccgaggtgc tggactgctc acacacttct 420ttcaaagcca ggatttaaaa gaaaaaaaat tgacagtggt ttctgaatgg ccagaaaaaa 480atcacagtta aattaatcct ttcatttgca gttatgtcat aatgctctgt gcagatataa 540tgactcagtg caagtgactc ggtgttccca agtttcctcc tattaaagaa tgcactgtct 600tcagatccaa gaaaacgagg ccctttatcg gcactgcctt gtggagtaga catgccagaa 660tgcctgacat tggtgcccgc gcttgcttcg aatactgaga tcttacaatt cacatgttct 720agttttcatc tccatctcac cctggaatac tatttacgat gtccagaaca caatggactt 780ttctgcatgg gacttaaatt tttcttgggc taagtaaaat gagaattaac aaataactcg 840ttaatctttt tagttccgtt gcctgaattt gcatttgact gctccctcag atggcagtgc 900tcacttactt cttcccctac caaataagac tctgtgtagc tcacactgat ctggaaactc 960gcaaccctcc tgctttagcc tcttgggtac tgggactaga ggcacgacca catctggcca 1020gtggtccttt tctttgatgg aggtagagtc taatgagtag ctttgatatt aaagatattg 1080cattaattaa ttttacaccg ctatggccaa atacccagca gaagcaacat aaaggcttca 1140tagcttagaa gatacaggcc attaggatac agcagggatg gtggctgaac cagtttgatc 1200tctggtagcc ttagcttcca gggacttctc atatggcacc gcccaagagt gggagatgaa 1260ggaccaggct ataatctgag actgtccccc acggtctagg tatatcagca aggccccaaa 1320agtttcacaa tctcccaaag agtttcacta gtttgggacc aagtgctcaa ggacaagatt 1380ctgtgggaac atttaactgt aacacacttc ttagcacaac tctgagctat gtaattttgg 1440gcagtgccat gaaccctaaa agcctcgaat tatcattgta aaactaagat aaaactttgc 1500aagataatca tgaatggcta agttaaaaga cacataaaag taattagcag agtgtttggc 1560ctcgttttta tatgcagtca atggcagttc ttttagtatc aataaccttt atggcttaca 1620gccatcctcc aaaaattatg acgagcagaa atctcctatg gatgttaacg tgcccctcag 1680ttaggcaaag attggtccat actgaatgta gacttggtcc ttcaaatgga attatgaaaa 1740caaaacacca aaaaaaaaaa aacccaaaaa aaaacaaaaa accccaaatt tctaaagacc 1800taatgtcttt agaccatgtg gcagcaaaat gcaactccag cctcttgtct atcaaatccc 1860aaggtctgga acggctcatt ttaaaaggca aattgattga attcagaacc actcaaggca 1920ttcgtgaggc atgtctttag gtatgtgtct atgacagcat tctctggaaa ggattggctt 1980agacccaccc tgaaaatgag tagctgcgac atcccctggg ctggatccag aactaattaa 2040ggagaatacg gatatagaca caccttccct ccataaagaa cctgagacac aagcaaaact 2100aatccctttc tcctttaagt tgctgcttgt caaagacttg gtcacagtaa ggagaaaaga 2160ctatttcatt ttgcatatcc tatctgtggc ttgctgtaaa attaatgaag ttaactaagt 2220agtgctgttc cagcctctgt tggaggcacg tgtatattat aggatatgac ctacataaat 2280ctacctcttt ttttcctatt gttaggatcc caacagaaga ccaaagaatt ctgccatgtc 2340tactgcatga gactccgttc tggcctcacc ataagaaagg agactagtta ttttaggaaa 2400gaacccacga aaagatattc actaaaatcg ggtaagtaca tttctggaaa gactgatggg 2460catctatttg cttttacttt ccatacacac acacacaaca cacacacaca cacacacaca 2520cacacacaca cacacacacg cacacacaca cacaattgtt tttcttacct aaattctcct 2580tgaggaaaag aatacagatg ataatgtgtt ggccatttta gaaagggttt ttctatttta 2640gaatttctaa ataaatattc aaaaagtcta ttctagaatc cactttagaa atggatacac 2700tggtgagtaa aaacagaaga cacaagatta tgtgtaaata tgtaatcatg tggtgtgata 2760ttttgtaata tagaggaaac aaactaaatg gttcatagtg gctgctttgg aaggcagtaa 2820ctgtggtgag tggctttctg tttatttgtt tgtttgtttc ttcctttggt gtgctctact 2880ggagtgcaaa tggtcttcac tttacgtcat tattaaggag gaattgatcc cattagacca 2940ttgggtggcc acagccctag gatcacttac caccgtcgcc atattggcca tcaaaaaaga 3000tagcatattg gcacaatgaa gattgctgac tcagcccaag ccccactact gtcttgcagg 3060taccaagcat gaagagaact tctctgccta tccacgggat tctaggaaga gatccttgct 3120tggcagtatc caagcatttg ctgcgtctgt tgacacattg agcatccaag gtatgactgg 3180tcatagggga tgtgtggggt gaggtgggag atagcacatg gggccttggc aagggctatt 3240gcatagagca aactcaggag tatgtcttca tatgtattag aaacgtttgt ttcctgcttg 3300ggtaccagtt cagtggttat ccaaattggg tgtggtggag ctgtgcctat ttaatctatg 3360gttagcattc cacttgtatt ttgaaatact tgctataggt tgggagcatg tcttcatcag 3420tcctgataaa tgtaatatcc ttggagagag gaaaaaaaca aagcaccagt ccccacattt 3480caaaatgttc accatgaaag aaaataaagg aactatcttt taaaataaca tgatgtggct 3540tgaagagtag tttaaagttt ttgaccattt ttgctgcgtt ctaagtaaaa tggtaattat 3600caagaagtga agagaaccca gggcgtgtag ttcagtggta gaacatttgc ctcatgtgcc 3660aggctctgca tgtagtctct atcaatgtgg gaaaaattca aagtgagggt tgggcataat 3720ggcatataat ctccatccca gaatggggga ggcaggtgaa ttccttgggt tcaggtctgc 3780ctggtctaca cagtgagtcc cagaacagtt agagttatat agtgagaaac tgtctcaaag 3840acaaacaaaa ggccaaatta aataattaac aaattagcag ccccatcact aaggaatcgt 3900gattaagaat cacatattgc cattctaata tatattattt ttatatgtga tcttatataa 3960aacaacttag aatgtattta tgtatagtgt gtatatatat tatatatata tatatataat 4020atatacttag tatatgttta atataaatac attaacctcc tggtcaatat tcagtttctc 4080acaaactgga tttaaattac tttagatgtt tagatgttta gatgtttaga tgtttagatg 4140ttcacatgca aattctttct attttgtaca gtaaatttga atgtaggacc ctcagatggg 4200tccaagtctg cttcagttta cccatatatg cttttaaaat gttatcaagt gctctgtatg 4260tgtttcgatt gtccgggcaa aagcagaact agtttacatc tgcatccccc tagaccagtg 4320actcaatctt cctaaatgct gtaacccttt aacatagttc tgcacgctgt ggtgacccca 4380accataacct tactcttgtt gctacatcat aactgcaact ttgctactgt tatgaatcct 4440aatgcaaata tctggtctgt aagatatctg atatgcaatc cctgtaaagg gatcatttaa 4500cccacaaaag gtctggacca ccaggttgag aaacactgcc ctagagtgtt ggaaatctct 4560aagtgatcat gctttataaa ggggaataca ttcacctatg tcttccaaaa catgaccatt 4620ctttgcagct gtcccctaga ctgtgggaag gtaagaaaag gagctaccct cctcgtcctc 4680accccaactg tgcaggaagg taacaccttt aatcccaccc tggtatcagc tatggtactt 4740agcttttgcc tatgttacaa tgacaaagta gagctcacac taaagaccaa aactcttcag 4800aggaagtaat tcatataaat aaccaagaga acggttcata gaatcctttg tgttttttat 4860tcttaatgac aggaacttca cttttaacac agtctcctgc ctccctgagt acatacaatg 4920accaatctgt tagttttgtt ttggagaatg gatgttatgt gatcaatgtt gacgactctg 4980gaaaagacca agagcaaggt agagtatctc ctcacgtcta tacttagtaa tgactggcaa 5040ggtttaaaaa aaaaaaatct accaagttat caacagtgga ttaactgagc tattcaacag 5100cagcaagttt caccatcttt ggtaatttta gtccttccta tgtcaagaag gtctcagtgt 5160agtcacattc ttcagggaat aaatcagaca ttgagaaata cctattgaga aatacagaaa 5220gagggactac atgtatctac aaatgctgaa taataagatc ttgttcacct tttcagtgtc 5280cccaaagcat agtcttttgt gaaagattat ccctttgtca taaacaatac cacctgcata 5340ttggggaatc catcagagca gaatatattt taagatcttt ctaagaatca ctaaagttcc 5400gcttttttgg tatatgacaa aaggagaggg agtttgtgtt ccccgcccag cccagcccca 5460ggacgggatg gaaccgaaac cttttgatac gttcctctgt cacccagtga cacccaatgt 5520tgtctttcag accaggtgct actacgctac tatgagtctc cctgtcctgc aagtcaatca 5580ggtaatgtag aggcaggggg agggggtggt gtttgcattc tcatccagaa caaagctgaa 5640tctgaagtag cccgtggtta tcatgaggtt cagaaggtta tcttcaaccc cacactcatt 5700gtaaaacctt acctcatcta cttctgaatt agtaggagaa aagcctagct gcacttataa 5760tagtacagcc ataatagtac agcccatctc agccaagact caaaagattc tgctcaaagg 5820ttgcccagaa gcagttcctt ctacatcatc agtccatcca tgcataatgg ctcaccacta 5880tggccagtca gcaagagaca ggaaggcagg tgaatacata tgtcttccgc ttcagggcac 5940aattgtgtac cacgtgtctg cttatcttca gttggtcaga acttagtctg agagcaacag 6000gaggagaaaa gttctattat ctgttatcta ttagaggaca gctctcagca gcttcccatg 6060aacagaggga agcaggtgac caaagcagta tccattggca tcagttgaat tacaaaagat 6120cagaacaaca acagaaagta aaaactaaaa cctctcctca gaaagattta gaaagactct 6180tgggctggcc ttggtggtgc acatctttaa tcccagcact caggaggcca aggcaagcag 6240atctctgagt tcaaagccag cctgacctac agagtgagtt tcaggacaga tagggccaca 6300caaagaaacc ctatcttggg ggaaggaaga aataaaagga aggaaggaag ggagggaagg 6360gaagggaagg gaagggaagg gaagggaagg gaagggaagg gaagggaagg aagaagggag 6420ggagggagga agggaaggag gaaagaagga agagagggag gaaagaagga aggaaggaag 6480gaaggaagga aggaaggaag gaaggaagga aggaaggaag gactacctgg tatttgggtg 6540caaatacttt taaatggatt agtgagctat tttcatttgc aactgaaatt ctttgagtta 6600agttaagatg agttacaaga cttagaattt ttgatttgta atttctttgt cttactttta 6660attgataggt aaaattgtat taatcatgaa ccacagatga tttttgcttt ggagcaagcg 6720atattttttt taaaaagatt ttatttctat ttaatgggta tgaatgtttt gcttgcatgc 6780atgtaagcgc ttgagatgct tatagttgtg tagaaagcta gaagacagca ctggatgtcc 6840tgtaacttat gggctctggg aactgaatgc aggtcctctt gcaagggcat caagtgctca 6900tgacgaagga agtatctctc tagtcccata gcaggagttt tttttttttt taaagattta 6960tttattgatt atatgtaagt acactgtagc tgtcttcaga cactccagaa gagggcatca 7020gatttcatta gggatggttg tgagccacca tgtggttgct gggatttgaa ctctggacct 7080tcagaagagc aatcgggtgc tcttacccac tgagccatct caccagccca tagcaggagt 7140tttgaggtat ctattgtctc tgtctaatag agagatcaaa tgaggccaag tggcagctgt 7200atcacctcac cttataattt tcattttaag gcgacggtgt ggatgggaag aagctgatgg 7260tgaacatgag tcccatcaaa gacacagaca tctggctgca tgccaacgac aaggactact 7320ccgtggaggt aacagaaata tgaccctatg acggccacac ttggttctac ctgttaacgt 7380aaaatagatg ttagatgtta gggcaatttt gaaggcagaa gtacacactg ttgaaaataa 7440actacagtca gtcacttcta atgacttgct tgggagtagg gaagggagag aagtgtggtc 7500ttgttgctac aagtctggtc tccagcaaca acacagaatc actggagaac ttgtcagaaa 7560gcaggatttc cggctccccc cacatccgaa tcagatctaa agtctccagg acaggaactt 7620catcccttca ccacagtttg agaggcactt ctgcacagag tgagtcacag cctttccatt 7680tggctatgtt cttccagaat tgttttagga ttatttattt tatgcatatg aatgtactgt 7740cactgtgttc ggtcacacca gaagagggca tcagatccca ttacagatgg ttgtgagcca 7800ccatgtggtt gctgggaatt gagctgagga cctctggaag agcagttggt gctcttaacc 7860gctgagccat ctctccagcc ctggagatat atagtttctg actccaagtc tgtcaagggg 7920aaggcagcaa atgtgtccat acatcagcag tttggggcag cattacattc tacatctggt 7980ccgttcttgc tcagcaaaca gcaggtgtac atcagccagg acgaaaaaaa aaattctgat 8040catgtaaaga acaaacaggc cagagtgaga aggaaggaag gaagggagga aggacatggg 8100agaacataat aacgaagatg atttctataa atatatacca tttcaaagca atccagccac 8160ataagacatc aacacaaaca gtccacaaag accagcactc ttctcaggag aatactctca 8220ggtgtacatg atgcaaaatc tagcagaaag aaagcctcaa aagtaacagt gggctgccat 8280ggcacatcaa gttcttgctt accatttcct aagccctgtc tctttaatcc tcacagcaaa 8340tttatatgga aaaattgcct tggtcagcta ttgtgcaaca aactatcaca aaactcattg 8400gattaaaata ataatcacat ataggtcctc agcaactgag agctcattcc gacgacctga 8460gttggctccc cagtattcat tggcaggcga ctcacaacac ctgtagctct cgctccaaga 8520cagtcaggtg cctctggcct ccatagacat ctacacttat gtgcacacac tcagacatag 8580ccacatacac ataatttaaa ataaattctt aaaaataaca caaattaaaa acaataatct 8640gtatttaaat gagcttaatt taattgaaca acttagttta acaatcaact taattaaaat 8700atgtctgtat ttaaactgac aatgaacgaa cctagagggc gtgtccacag agagttctgg 8760ctaaactgaa aagtccaggt ttactagata catactagac agacatgttc tcatggcaaa 8820aacagagggg aacaaatctg gatgttttgc ggtagtttgt tgcacagaag ctgaccaagg 8880taattattcc atataaactt gctgtgtgaa ttaaagagat ggggtttgtg acgtatgaag 8940gaaattgacc tggtcagtct ctgccaagct gcttatccac tctccacaaa tcgctttcac 9000ctcttccatg cagcctctct attccctcca ctgctcattt ctccctcctc gactcagtac 9060aaacttccta gaaagttcaa gctataagtg atcagtcccc ccatggatgc tgacccagga 9120aggcaatggt agctctggac tccccattca caccttggtt tcttctcttc ttccctcagc 9180ttcaaagggg tgacgtctcg cctccggaac aggccttctt cgtccttcac aaaaagtcct 9240cggactttgt ttcatttgaa tgcaagaatc ttcctggcac ttacatagga gtaaaagata 9300accagctggc tctagtggag gagaaagatg agagctgcaa caatattatg tttaagctct 9360cgaaaatcta a 9371210697DNAArtificial SequenceTargeting vector for mouse IL-33 2tagctctcca ccggggctca ctgcaggaaa gtacagcatt caagaccagc tatttcctgt 60ctgtattgag gtgagtgcca ggtcaatctc tctgtttctg aggggaacca atagtagcat 120ctatcagctc tcaaatgatt ctgatataaa gagagaaaag acttgttact ggagaaagtg 180gcttgtttat cagaatggga tttgaaacca gttctgggtt atacttttag ttctgcaatt 240gtgataaaga tgctgacagt cttggtcgat gtatctgtcc ttaatgctca ctccttagtt 300tcttatctgt tcaaccacgc aggctagtag tcctttagag caaagttctt ctacatgagt 360ttgctcaagc tcgtccaagg gaggacgaaa gggaagggaa ggttaggaga tcaaataatt 420aactgaagat agagaaaggc aaaggcaggc agggcgtctc agtgaggggc tgctggtatt 480gttattatgt taacatgcat gaactctaag tctccaagaa gagaggctat cgactgcaac 540acacaggttt atttaaaaga caattttaat gactagaaaa acattgtttc taggacacat 600gcaccctctg gtgaaagaga aaacagaaat cagcactgtt tcaggaagca gctcagaagc 660tcagcactag cagtgagcaa gggacctcaa ttcctcctgg tacaggcagg ctattattta 720aactgtttcc tgcccaactg aaaatctgac ctttatgtgg ttagctaatc ctattacccc 780acacagaagg cttaaaaatt aactattctg taaaagctat aactctgctt taaaggaaat 840tatgtattct gagccagaaa ggatgactga ccattgtggc ttagtagtat ggattcaagt 900taccctaaat gatacattcc taagtgaaag ccattacatt aagggtttca ggcaaagaat 960aaaggccacc atacaccaat gtattcaata actgcattag tcataacacg ttcgacacac 1020agaagacaca agaaggagga ggtctctctt gtaggttttg aatgcttgca gcttcttagc 1080tttgtgctat gcataaacag cccattctga gagtatcaaa tcagatgcca ttattaaaag 1140aaatggctgt ttacacaaaa agcctaagac agccagagat aaagtttggc tcttgatcat 1200tgaccaatga tcaagacaat gacaatattt tactcagtga acgagaagag tacgtgggat 1260tatttaaccc agccgcagct tcttcaggga actctgcctt atgttagtta acatcttccg 1320aaatttatta ttactatttc aagaatatta atatttcaag aagctggcca tttgctttga 1380gtataagaag ggctgagtgt ttgaaataaa ataccaagtt ctaatccacc cgactcccac 1440cccccaagcc gcaccccgca cctcctcgaa gttgtcaact gatgcagttg ttggatgtat 1500tggctggcat atggttgcct ttgttctttg cctgaaagcc ttcatgcgac tgctttccgc 1560ttaggagtgc attgtttagg gaaacttgaa tcaatgctac taagccatga tcagctatca 1620tcgctggctc agaataaatg gttcccttca aagcagagtt gtaactttta tagagtagtt 1680aatttttaat ccttttgaac aaaagagaga gagagagagg atgataatgg agggagagag 1740aagagataac agttttagtt tcattggggg ggtggtacag gataagagat aagttctact 1800ttcattcttc tacacatgga tatccagctc tcccagttta aaaaaatttt taaggctata 1860tttttctgat gtgtttttga tgccactagc aagaattaga cggttctatt ggcctgagta 1920tgacataatt atattttgat ttttaaaaaa tttaagaaaa aatagacaat gtgttaaggt 1980tgaaacagtg tggggaagat aacaagattt aaaactcaaa tgtaaaagaa gtagataaaa 2040ttagacagaa gagggataaa aatgttttta aaaatcaaaa taataagtaa tagttttaaa 2100tgagaggaaa ataaaaagag agacggaagc acctttccgg gtctgtgagc agagggcaga 2160gggctagagc ttgctcccac taagctggct agaatagcga ggccagttga gagcacccat 2220cctcagcctc agggctgtgt ttactctaga ctgtaaaagc gtagatgctt tcttcctttg 2280cacttatggg gccatggcat cccccgacga tgtctacatt ccttaggctt gggcaggtta 2340tcattcactg aggctctctg gagtccgcga ctgtggcaaa gtccacctag ttcacctcag 2400aatgaaggga gcaaacggct ctcctagtta tgtccagatg gaagagcaat cagcctgtcc 2460tggtttataa gtaatcttca gctacctgct tggaggcctc agtcatcccc aatcccagga 2520gctgagtagt cctcgccacc agagggcggg agcactgatc actgtgtctt ccttcctcaa 2580ctgagcaggc tccccgtaat tgaaacttct cattggttcg ttcatccgaa ccaatccctt 2640ccaggattgc ctcatagccg gtcactatgg cagcgttccc aggcatcctg atttggcacc 2700tgtctgtatt ttctgatctt cagagttgct gctggtcaca acacttttaa actaccggag 2760agtgacggtg tggcgtcttt actctaggca gcagtggaca ggaactcgcc cttagaccta 2820atttctgccc tggcttgtga gaactgtact gttaaccatg ggtttctccc gctgcgctgc 2880gagcagcgca attctattct tctggtagcc taaacccaga tgaacttgtg attgttgctc 2940cctccctcct aatccttccc agtccctctc cccatccccc accaatgcct ggaaccggga 3000ctggagctct ggactgcttc ctacttcgtg ttatcttttt gtttctccat gagttttaat 3060tgccctgttg tttttgttgt tgttttggtt ttcccccttt atgcccagtg atttctccac 3120tagatctcct tgggctttgg tttgtccttt gttaccctgg tctttgaact tgtgagaaaa 3180tcggcaacct tactccattt attcttgagg aaccatgctt aatcttgatc actgcctgcc 3240attactcttt ttaaacattc agctggtgcc aaatctgacc cgaggaaacc ctccacggtg 3300gctcccacat gcttttgaca caaactcatt tagtttttta atagctggac gttttctggc 3360ataacgtgtt ttatggtcat tattactttc ctgaccacga atctggagtc aaagttcctt 3420ccagtggaga aaatatttag aaaataaaat gtgtacacac actgcttgtt taatactgac 3480tcaaatctaa catcgcaggg catttgcctt ggcttctgag ttacgtattt atatttctta 3540cccccctaca gtgagaaatc atacaagatc agtatcgcta tttttttttt ttttttgcta 3600tatactacaa caaaacagta ctgagatttc aacacaaaca tggcattaac actaagacta 3660ctcagcctca gatttctctg tgctttcatt actttattct ttgattctat ggctagattc 3720tagcctaaat gtagagtcag aatactatgc tgaattttat tctcccccct cccccctgta 3780tggctatcag tttcatggtg agatagccaa ggttgcttct gatgacttca ggtccatata 3840gttggattaa tgttatattt caatcccaca gaaacctgaa aaatggacaa caccgaggac 3900gtcatcaagg agttcatgca gttcaaggtg cgcatggagg gctccgtgaa cggccactac 3960ttcgagatcg agggcgaggg cgagggcaag ccctacgagg gcacccagac cgccaagctg 4020caggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc ccagttccag 4080tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactacat gaagctgtcc 4140ttccccgagg gcttcacctg ggagcgctcc atgaacttcg aggacggcgg cgtggtggag 4200gtgcagcagg actcctccct gcaggacggc accttcatct acaaggtgaa gttcaagggc 4260gtgaacttcc ccgccgacgg ccccgtaatg cagaagaaga ctgccggctg ggagccctcc 4320accgagaagc tgtaccccca ggacggcgtg ctgaagggcg agatctccca cgccctgaag 4380ctgaaggacg gcggccacta cacctgcgac ttcaagaccg tgtacaaggc caagaagccc 4440gtgcagctgc ccggcaacca ctacgtggac tccaagctgg acatcaccaa ccacaacgag 4500gactacaccg tggtggagca gtacgagcac gccgaggccc gccactccgg ctcccagaaa 4560agatattcac taaaatcggg taagtacatt ataacttcgt atagcataca ttatacgaag 4620ttatcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc 4680cgtcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc 4740gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag 4800caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc 4860cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg 4920gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca 4980aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcag 5040cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg 5100tgaggaacta aaccatggga tcggccattg aacaagatgg attgcacgca ggttctccgg 5160ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacgatc ggctgctctg 5220atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 5280tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga 5340cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 5400tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 5460tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 5520tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa

gccggtcttg 5580tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 5640ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 5700tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 5760gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 5820gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgccccc gattcgcagc 5880gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaata 5940aagaccgacc aagcgacgtc tgagagctcc ctggcgaatt cggtaccaat aaaagagctt 6000tattttcatg atctgtgtgt tggtttttgt gtgcggcgcg ataacttcgt atagcataca 6060ttatacgaag ttattcttac ctaaattctc cttgaggaaa agaatacaga tgataatgtg 6120ttggccattt tagaaagggt ttttctattt tagaatttct aaataaatat tcaaaaagtc 6180tattctagaa tccactttag aaatggatac actggtgagt aaaaacagaa gacacaagat 6240tatgtgtaaa tatgtaatca tgtggtgtga tattttgtaa tatagaggaa acaaactaaa 6300tggttcatag tggctgcttt ggaaggcagt aactgtggtg agtggctttc tgtttatttg 6360tttgtttgtt tcttcctttg gtgtgctcta ctggagtgca aatggtcttc actttacgtc 6420attattaagg aggaattgat cccattagac cattgggtgg ccacagccct aggatcactt 6480accaccgtcg ccatattggc catcaaaaaa gatagcatat tggcacaatg aagattgctg 6540actcagccca agccccacta ctgtcttgca ggtaccaagc atgaagagaa cttctctgcc 6600tatccacggg attctaggaa gagatccttg cttggcagta tccaagcatt tgctgcgtct 6660gttgacacat tgagcatcca aggtatgact ggtcataggg gatgtgtggg gtgaggtggg 6720agatagcaca tggggccttg gcaagggcta ttgcatagag caaactcagg agtatgtctt 6780catatgtatt agaaacgttt gtttcctgct tgggtaccag ttcagtggtt atccaaattg 6840ggtgtggtgg agctgtgcct atttaatcta tggttagcat tccacttgta ttttgaaata 6900cttgctatag gttgggagca tgtcttcatc agtcctgata aatgtaatat ccttggagag 6960aggaaaaaaa caaagcacca gtccccacat ttcaaaatgt tcaccatgaa agaaaataaa 7020ggaactatct tttaaaataa catgatgtgg cttgaagagt agtttaaagt ttttgaccat 7080ttttgctgcg ttctaagtaa aatggtaatt atcaagaagt gaagagaacc cagggcgtgt 7140agttcagtgg tagaacattt gcctcatgtg ccaggctctg catgtagtct ctatcaatgt 7200gggaaaaatt caaagtgagg gttgggcata atggcatata atctccatcc cagaatgggg 7260gaggcaggtg aattctaacc tcgacccgct ctagaactac gatccagaca tgataagata 7320cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 7380aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttagatc 7440ctagtggatc tgcattccac cactgctccc attcatcagt tccataggtt ggaatctaaa 7500atacacaaac aattaggaat cagtagttta acacattata cacttaaaaa ttttatattt 7560accttagagc tttaaatctc tgtaggtagt ttgtccaatt atgtcacacc acagaagtaa 7620ggtttccttc acaaagagat cgcctgacac gatttcctgc acaggcttga gccatatact 7680catacatcgc atcttggcca cgttttccac gggtttcaaa attaatctca agttctacgc 7740ttaacgcttt cgcctgttcc cagttattaa tatattcaac gctagaactc ccctcagcga 7800agggaaggct gagcactaca cgcgaagcac catcaccgaa ccttttgata aactcttccg 7860ttccgacttg ctccatcaac ggttcagtga gacttaaacc taactctttc ttaatagttt 7920cggcattatc cacttttagt gcgagaacct tcgtcagtcc tggatacgtc actttgacca 7980cgcctccagc ttttccagag agcgggtttt cattatctac agagtatccc gcagcgtcgt 8040atttattgtc ggtactataa aaccctttcc aatcatcgtc ataatttcct tgtgtaccag 8100attttggctt ttgtatacct ttttgaatgg aatctacata accaggttta gtcccgtggt 8160acgaagaaaa gttttccatc acaaaagatt tagaagaatc aacaacatca tcagggtcca 8220tggcgaggac ctgcaggtcg cagatcctga acggcagagg ttacggcagt ttgtctctcc 8280cccttccggg agccaccttc ttctccaacc gtcccggtcg cgctctcggc gcttctgagg 8340agagaactgg ctgagtgacg ccctttatag attcgccctt gtgtcccgcc cttcctttcc 8400cgccctccct tgcgctacgg ggccgcccgc accggcctac acggagcgcg cgcggcggag 8460ttgttcggtc cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt 8520aatttcgagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 8580cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 8640agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 8700gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 8760gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 8820ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 8880aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 8940ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 9000gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 9060cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 9120gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 9180tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 9240cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 9300cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 9360gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 9420agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 9480cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 9540tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 9600tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 9660ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 9720cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 9780cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 9840accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 9900ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 9960ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 10020tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 10080acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 10140tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 10200actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 10260ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 10320aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 10380ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 10440cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 10500aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 10560actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 10620cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 10680ccgaaaagtg cctcgag 10697322DNAArtificial SequencePCR Primer (Oligonucleotide A) 3tagaaagagc ccagtgttaa gc 22418DNAArtificial SequencePCR Primer (Oligonucleotide B) 4ggcttgccct cgccctcg 18519DNAArtificial SequencePCR Primer (Oligonucleotide C) 5cacctgcgac ttcaagacc 19621DNAArtificial SequencePCR Primer (Oligonucleotide D) 6acgattcctt agtgatgggg c 21721DNAArtificial SequencePCR Primer (Oligonucleotide E) 7gttgcttctg atgacttcag g 21820DNAArtificial SequencePCR Primer (Oligonucleotide F) 8gcaatagccc ttgccaaggc 20921DNAArtificial SequencePCR Primer (Oligonucleotide G) 9tgctgttcca gcctctgttg g 21

Claims

1. A non-human animal with a N-terminal deletion of the IL-33 gene.

2. The non-human animal of claim 1, wherein the full DNA binding domain at the N-terminus of the IL-33 gene is deleted.

3. The non-human animal of claim 1, wherein the non-human animal is a mammal.

4. The non-human animal of claim 3, wherein the non-human animal is a rodent.

5. The non-human animal of claim 4, wherein the non-human animal is a mouse and wherein the N-terminal deletion of the IL-33 gene comprises a deletion of amino acids 1-67 of the expression product of the IL-33 gene.

6. A descendant of the non-human animal of claim 1, obtained by breeding with animals of the same or another genotype.

7. A cell line or primary cell culture derived from the non-human animal of claim 1.

8. A tissue or an organ explant or culture thereof derived from the non-human animal of claim 1.

9. The method of using the non-human animal of claim 1 as a model of inflammatory diseases.

10. The method of claim 9 for screening anti-inflammatory compounds.

11. The method of claim 9 for evaluating the pharmacological effects of an anti-inflammatory compound.

12. A method for screening for anti-inflammatory compounds, comprising administering a candidate compound to the non-human animal of claim 1.

13. A method for evaluating the pharmacological effects of an anti-inflammatory compound, comprising administering a said anti-inflammatory compound to the non-human animal of claim 1.

14. The non-human animal, the uses and methods essentially as herein described.