Method for Gene Amplification

Abstract

The present invention provides a double-stranded DNA constructed specifically for high speed gene amplification, a method for gene amplification and a method for synthesizing protein. The gene amplification system of the present invention used a site-specific recombinase such as Cre-lox system and target sequence thereof to efficiently induce a type of replication referred to as a double rolling-circle replication (DRCR). Amplification unit, whose structure is shown in FIG. 2 (a), is constructed in animal and other cells. DRCR is induced by two recombination events triggered by a site-specific recombinase (Cre) when each replication folk progresses between each pair of target sequences (lox sequences).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 12/085,476, filed on May 23, 2008, which is a national stage application of International Application No. PCT/JP2006/314168, filed on Jul. 18, 2006, and which claims benefit of Japanese Patent Application No. 2005-338119 filed Nov. 24, 2005, the disclosures of each of which are incorporated herein in their entireties.

CROSS-REFERENCE TO RELATED DOCUMENTS

[0002] This application comprises a sequence listing filed in electronic form as an ASCII .txt file entitled 1680-26-25T25.txt, created May 9, 2013, 2200 bytes (22 kilobytes). The content of the sequence listing is incorporated herein in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to a method for amplifying gene at high speed and a method for producing proteins by using the amplified gene.

PRIOR ART

[0004] Gene amplification with cultured animal cells (Reference 1 and the like) accompanies several complications such as (1) time consuming (a half to one year), (2) presence of clones without amplification, and (3) empirical procedures with unexplained mechanism. On the other hand, there is no system of gene amplification with yeast. Although plasmids are generally used for the purpose, increase in copy number beyond a certain threshold is difficult.

[0005] The system of the present invention is based on the replication referred to as DRCR (Double Rolling-Circle Replication) induced by biological potency called as BIR (Break-Induced-Replication) (Reference 2-4). It is conceivable that a chromosome breakage is rescued itself by the following steps; i.e. the broken chromosome finds homologous sequence, invades into it, forms a replication fork, and consequently starts DNA replication. All living organisms might involve such ability.

[0006] Moreover, it is reported that natural circular DNA accompanies DRCR by recombination (Reference 5).

[0007] Reference 1: Japanese Patent Gazette 8-504585 (WO94/14968)

[0008] Reference 2: WO2005/061703

[0009] Reference 3: PNAS, vol. 98, no. 15, 8255-8262 (Jul. 17, 2001)

[0010] Reference 4: Genes Dev 12, 3831-3842 (1998)

[0011] Reference 5: Cell. 1986 Aug 15; 46 (4): 541-550

PROBLEMS TO BE SOLVED BY THE INVENTION

[0012] The present invention provides a double-stranded DNA constructed specially for high speed gene amplification, a method for gene amplification thereby and protein production thereby. The present invention is characteristic in full artificially designed system of gene amplification, the potential of higher amplification efficiency by synchronous culture, short period for amplification (probably one generation) and well elucidated mechanism of amplification.

MEANS TO SOLVE THE PROBLEMS

[0013] The amplification system of the present invention utilizes a type of DNA replication referred to as double rolling-circle replication (DRCR). The type of replication is able to amplify DNA explosively in a single cell cycle. It is assumed that the amplified products are maintained intracellularly after termination of DRCR by recombination and the like. The present inventors utilized a site-specific recombinase such as Cre-lox system and its target sequence in order to induce DRCR efficiently. More specifically, the present inventors constructed a replication unit (ex. FIG. 3) in yeast and were able to succeed in inducing DRCR by utilizing a recombination generated by a site-specific Cre recombinase (hereinafter, referred to as "Cre") during progress of a replication fork between a pair of lox sequences and to accomplish the present invention.

[0014] Namely, the present invention is a double-stranded DNA represented by a-b-c-d or a-c-b-d, wherein one of a and b is a double-stranded DNA fragment comprising a first target sequence of a site-specific recombinase, and the other is a double-stranded DNA fragment comprising an inverted sequence of said first target sequence; and one of c and d is a double-stranded DNA fragment comprising a second target sequence of the site-specific recombinase and the other is a double-stranded DNA fragment comprising an inverted sequence of said second target sequence; a replication origin and at least one target gene to be amplified are inserted anywhere between a and d; and arbitrary DNA sequences may be inserted among above fragments.

[0015] Additionally, the present invention is a recombinant vector comprising the double-stranded DNA, and is also a transformant, which is introduced with the double-stranded DNA.

[0016] Moreover, the present invention is a set of double-stranded DNA comprising a double-stranded DNA fragment represented by e-a-A-b-f and a double-stranded DNA fragment represented by g-c-B-d-h, wherein one of a and b is a double-stranded DNA fragment comprising a first target sequence of a site-specific recombinase, and the other is a double-stranded DNA fragment comprising an inverted sequence of said first target sequence; and one of c and d is a double-stranded DNA fragment comprising a second target sequence of the site-specific recombinase and the other is a double-stranded DNA fragment comprising an inverted sequence of said second target sequence; each of letters from e to h is a double-stranded DNA fragment of at least 50 bp in size, which are arranged on a chromosome or an extrachromosomal element that is a host for integration of the set of double-stranded DNA in order of e, f, a replication origin of the chromosome element or the extrachromosomal element, g and h; at least one of A and B represents the target gene to be amplified; and said replication origin or a part of it may be included in f or g; and an arbitrary DNA sequence may be inserted among these.

[0017] The present invention is also a set of recombinant vectors, wherein each vector contains each of two kinds of the double-stranded DNA, and is also a transformant or transfectant, which is introduced with two kinds of the double-stranded DNA, wherein said replication origin locates on a host chromosome or an extrachromosome.

[0018] The present invention is also a method for amplifying the target gene, comprising the steps of preparing the transformant or the transfectant and affecting said transformants with the site-specific recombinase; and is a method for manufacturing a protein encoded by the target gene, comprising a step of culturing transformed or transfected cells obtained by the above method.

EFFECTS OF THE INVENTION

[0019] The amplification system of the present invention has an excellent property in establishing efficient system for producing proteins. DRCR is capable of amplifying a target gene rapidly during a single cell cycle. Since the amplification mechanism is well elucidated, reliable amplification of a target gene is prospective. Although the present example was constructed in yeast not animal cells, it is possible to produce highly amplified products at 10 to 100 times higher frequency than a conventional system of animal cultured cells. Furthermore, the present system can be applied to primary cultured cells, in which gene amplification by drug selection has not been observed. Therefore, it is possible to apply gene amplification to targeting cells of gene therapy, and to enhance and sustain the expression of introduced gene.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows a DRCR reaction. Black arrowheads show replication folks.

[0021] FIG. 2 shows the initiation of the amplification reaction by using a site-specific recombinase and its target sequences. The triangular arrowheads (letters from a to d) represent the target sequences (e.g. loxP sequence) of a site-specific recombinase and the direction thereof. X represents replication origin (and so forth). Letters from x to z and x' to z' represent genes to be amplified. Black arrows represent replication folks.

[0022] FIG. 3 shows a construct for amplification. CEN: centromere, TEL: telomere.

[0023] FIG. 4 shows a plasmid (pSH47) for Cre expression.

[0024] FIG. 5 shows a colony forming frequency. Glc: glucose, Gal: galactose.

[0025] FIG. 6 shows the Southern blot analysis. (a) shows chromosomal DNA separated by PFGE and probed with leu2d, and (b) shows chromosomal DNA digested by SmaI and then separated by FIGE. Lane numbers from #19 to 58 show DNA prepared from colonies grown on the selective medium without leucine after Cre induction by galactose. NS shows DNA from control colonies grown on non-selective medium. P shows host cell lines. In this PFGE conditions, chromosomes with longer than about 650 kb are deemed to be concentrated above the separation limit.

[0026] FIG. 7 shows amplified products on chromosome. (a) shows the structure initially generated by DRCR. Letters from a to f represent the cleavage sites by restriction enzyme SmaI and digits show fragment size (kb). Nevertheless, 5.3 kb fragments generated by d-e cleavage are not detected by the Southern blotting, since the fragments do not include leu2d. (b) shows the structure with inversion (rearrangement to reverse direction) of the sequence between lox. Letters from a' to f represent cleavage sites changed by inversion, and digits show predicted fragment size (kb). For example, a-b cleavage produces 10.9 kb fragment. In a case of inversion of the region containing a, a'-b cleavage produces 16.8 kb fragments. Similarly, a-b' cleavage produces 5.3 kb fragment and a'-b' cleavage produces 11.1 kb fragment. The 5.3 kb fragment, which does not contain leu2d gene, is undetectable by the Southern blotting.

[0027] FIG. 8 shows amplified products on a mini chromosome (FIG. 6 (ii)). Replication from the telomere side proceeds to reverse direction due to recombination between loxP, and produces mini chromosome (about 18 kb in size) with telomere at the both ends. The SmaI cleavage sites from g to i and site h' changed by inversion produce 6.3 kb fragments containing leu2d (The fragment is derived from g-h' or h-i fragment. The fragment g-i cannot be generated because of cleavage at either h or h' site).

[0028] FIG. 9 shows amplification products on a mini chromosome (FIG. 6 (ii)). Replication from the telomere side proceeds to reverse direction due to recombination between loxm2 and produces a mini chromosome (about 40 kb in size). Letters from j to n represent SmaI cleavage sites and letters from k' to m' represent cleavage sites changeable by inversion. Digits show possible fragment size (kb). The 5.3 kb fragment, which does not contain leu2d gene, is undetectable by the Southern blotting.

[0029] FIG. 10 shows the effect of Cre recombination on not amplified structure. The sequences between lox pairs can be frequently inverted. Letters from o to r represent SmaI cleavage sites, p and q' represent the cleavage sites changeable by inversion and digits show possible fragment size (kb). The 5.3 kb fragment, which does not contain leu2d gene, is undetectable by the Southern blotting.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The gene amplification method of the present invention utilizes a double rolling-circle replication (DRCR), which enables a rapid amplification, and is presumed to be functional both in budding yeasts and in animal cells. The gene amplification system is a type of DNA replication, wherein two replication folks replicate continuously a circular DNA, as shown in FIG. 1. In the beginning, folk (1) replicates w and folk (2) replicates y ((a), (b), (c)), then folk (1) and folk (2) replicates x and folk (2) replicates z ((c), (d), (e)). In this way, the replication continues endlessly, since a template for one folk is synthesized by the other folk successively

[0031] After the amplification has proceeded, the central circular form seems to be removed by recombination and the like, and the reaction seems to be terminated (f).

[0032] The gene amplification system of the present invention utilizes a site-specific recombination, which is known to be functional even in animal cells, in order to induce DRCR. This reaction is a reversal of DNA replication by recombination during progression of the replication folk between a set of target sequences. A pair of the reactions is used for the amplification system.

[0033] Namely, in the amplification system of the present invention, firstly, DNA replication starts in the amplification unit constructed as in FIG. 2 (a). Secondly, the two replication folks represented by black arrows go just between two sets of target sequences (lox sequences) of a site-specific recombinase (e.g. Cre). Lastly, the target sequences (e.g. loxP sequences) on parent DNA strand x and x' recombine with the target sequences (e.g. loxP sequences) on de novo DNA strand y and y', respectively. After the recombination events, one of the folks synthesizes y and z strands from x strand and the other folk synthesizes y' and z' strands from x' strand (FIG. 2 (c)). In this way, the progress of each replication folk is reversed and the replicated DNA strands are replicated again (FIG. 2 (d)). DRCR is carried out by these two reactions.

[0034] The double-stranded DNA used in the present invention is represented by a-b-c-d or a-c-b-d, or preferably by a-b-c-d.

[0035] One of a and b represents a double-stranded DNA fragment comprising a first target sequence of a site-specific recombinase, and the other represents a double-stranded DNA fragment comprising inverted, sequence of the first target sequence of the site-specific recombinase. One of c and d represents a double-stranded DNA fragment comprising a second target sequence of a site-specific recombinase, and the other represents a double-stranded DNA fragment comprising inverted sequence of the second target sequence of the site-specific recombinase. The first target sequence could be the same as the second target sequence, but is preferably different from the later. Additionally, arbitrary DNA sequence may be inserted between these sequences.

[0036] The above b and c may be combined and the DNA may be represented by a-b-d, wherein d and a represent the same target sequence with the same direction.

[0037] Moreover, the sequence may be represented by a-b-X-c-d or a-c-X-b-d, preferably by a-b-X-c-d., wherein X represents a replication origin. The replication origin includes On beta located at the 3' down stream of dihydrofolate reductase (DHFR) gene, latent origin (OriP) of EBV, origins located at the vicinity of c-myc gene or others, as a candidate, and may include any origin with replication initiation activity in animal cells.

[0038] Furthermore, the sequence may be represented by a-A-b-X-c-B-d or a-A-c-X-b-B-d, preferably by a-A-b-X-c-B-d, wherein at least one of A and B represents target gene. If a number of target genes are used, they can be the same as or different from each other. DRCR (FIG. 2) explained above are similarly induced in these sequences.

[0039] A site-specific recombinase catalyzes the recombination between two short consensus DNA sequences (target sequences). The site-specific recombinase can induce site-specific recombination between the target sequences, change the target site further and modify the integrated gene.

[0040] The present invention may use the following site-specific recombinase and the target sequences specific to the recombinase (i.e. see; Developmental Cell, Vol. 6, 7-28, Jan. 2004 and the like).

(1) Cre Recombinase or Derivatives thereof.

[0041] Cre recombinase of bacterial virus P1 is applied most extensively to gene transfer and knockout in mouse. Cre protein catalyzes the recombination between two 34 base pair loxP recognition sites. The loxP sequence has a unique construction, wherein core 8 base pair sequence is flanked by two 13 base pair palindrome sequences. The asymmetric 8 base pair sequence determine the orientation of loxP site. DNA cleavage and recombination between loxP sites by Cre enzyme occur at a site between the rear of the first base and the front of the last base of the 8 base pair core sequence. Derivatives of the Cre enzyme are constructed by amino acid substitutions. The derivatives include site-specific recombinases, wherein wild type Cre recombinase is changed in its function and character by introduction of amino acid substitution; and site-specific recombinases and their genes, wherein mutations are introduced into wild type Cre recombinase gene to optimize CpG content, Kozak sequence related to translation initiation efficiency and codon-usage in host cells to increase expression efficiency and level. At least 29 kinds of Cre enzyme derivatives have been constructed. Derivatives thereof have different recombination activities and recognize different target sequences. Also, a number of mutated sequences are prepared for target sequence recognized by Cre enzyme. The present invention may use all above derivatives. Target sequences like above include loxP, lox511, lox5171, lox2272, lox2372, loxm2 (referred also as m2), loxFAS, lox71, lox 66 and mutants thereof. The mutant refers to a target sequence of site-specific recombination, wherein the sequence contains mutation introduced in one or more bases in wild type loxP sequence.

[0042] Although the recombination efficiency is generally sensitive to any change in lox sequences, mutants keeping function thereof were found. In the latter case, recombination may occur efficiently between pairs of homotypic loxP sites, but not between heterotypic sites.

(2) Flp Recombinase or Derivatives Thereof

[0043] The recombinase is Flp recombinase derived from budding yeast. The activity of the recombinase is similar or slightly inferior to that of Cre/loxP. However, the activity of the recently developed active type Flp (Flpe) is improved and is similar to that of Cre. The consensus 34 base recombination sequence is referred to as FRT. Although the structure of FRT has the same structure as loxP, the sequence is different from each other.

[0044] Derivatives thereof refer to site-specific recombinases, wherein wild type Flp recombinase is changed in its function and character by introduction of amino acid substitution; and site-specific recombinases and their genes, wherein mutations are introduced into wild type Flp recombinase gene to optimize CpG content, Kozak sequence related to translation initiation efficiency and codon-usage in host cells to increase expression efficiency and level. At least 28 kinds of Flp enzyme derivatives have been constructed.

[0045] A number of derivatives have been constructed also for Flp enzyme and its recognition sequence. The target sequence includes FRT, F3, F5, FRT mutant-10, FRT mutant+10 and mutants thereof. The mutant refers to a target sequence of site-specific recombination reaction, wherein the sequence contains mutation introduced in one or more bases of wild type FRT sequence and the like.

[0046] Flp enzyme is very sensitive to the change in the sequence of FRT site, similar to Cre enzyme. Several mutant FRT pairs that lead to efficient recombination between homotypic sites are identified. However, recombination does not occur between different mutant FRT sites or between wild and mutant sites.

(3) PhiC31 Integrase or Derivatives Thereof

[0047] PhiC31 integrase is derived from bacterial virus in Streptomyses and is functionable in human cells. The target sequence of the integrase includes attP, attB and their mutants. A mutant refers to a target sequence of the site-specific recombination, wherein the sequence contains mutation in one or more bases in wild type attP sequence and the like.

[0048] The enzyme induces recombination between a pair of three nucleotides, ttg, in the attPP' and attBB'. Since the sequences at both sides of `ttg` are unique, the sequences are changed to different sequences from the original recognition sequences after recombination. Therefore, the enzyme cannot recognize the consequent sequence as a target site. Therefore, the recombination by the enzyme occurs only once.

[0049] The derivatives of PhiC31 integrase system refer to site-specific recombinases, wherein wild type PhiC31 integrase is changed in its function and character by introduction of amino acid substitution, and site-specific recombinases and their genes, wherein mutations are introduced into wild type PhiC31 integrase gene to optimize CpG content, Kozak sequence related to translation initiation yield and codon-usage in host cells to increase expression efficiency and level.

[0050] Cre/Lox system is preferable among the site-specific recombinase and target sequence thereof.

[0051] Furthermore, it is preferable that a target gene to be expressed, selective gene (drug resistant genes for Geneticin, Neomycin, Hygromycin, Zeocin, Blasticidin or the like) for selecting cells that contain the present construct in a chromosome or an extrachromosomal element, and a marker gene (dihydrofolate reductase (DHFR), glutamine synthetase (GS), aspartate transcarbamylase (CAD), metallothionein (MT), adenosine deaminase (ADA), adenylate deaminase (AMPD1,2), UMP synthetase, P-glycoprotein (P-gp), asparagine synthetase (AS), ornithine decarboxylase (ODC) or the like) for selecting cells with gene amplification may be inserted in arbitrary site within the structure. It is preferable to insert nuclear matrix attachment region (MAR) DNA, which is deemed to be important for amplification in animal cells. Additionally, arbitrary DNA sequence could be inserted between the above fragments.

[0052] The above fragments are appropriately connected by conventional method of genetic engineering.

[0053] The double-stranded DNA fragments thus obtained are transduced into appropriate cells by the methods of virus, lipofection, electroporation or the like. Furthermore, it is preferable to establish cell lines by selecting the cells that contain the above construct on a chromosome or an extrachromosomal element, by the drug corresponding a drug resistant gene (a drug resistant gene to Geneticin, Neomycin, Hygromycin, Zeocin, Blasticidin or the like). Yeast cells and animal cells can be used as the host. Pharmaceutical proteins are produced preferably in animal cells, wherein glycosylation pattern is similar to human and it reduces risk to undesirable immunological response. Animal cells include CHO (Chinese hamster ovary) cells used frequently for protein production as well as other cells derived from human, mouse, rat and other animals.

[0054] Furthermore, the double-stranded DNA of the present invention comprises one set of double-stranded DNA fragments obtained by dividing any of the above double-stranded DNA fragments into at least two, preferably 2 to 5, and more preferably two, wherein the DNA fragment comprises partial sequence of a host chromosome or an extrachromosomal element, and may contain at least 50 bp and preferably from 500 to 1 Kbp sequences at both ends for homologous recombination. The double-stranded DNA fragment for homologous recombination can produce the above double-stranded DNA on a host chromosome or an extrachromosomal element by homologous recombination.

[0055] The replication origin may be replication origin of the host chromosome or an extrachromosomal element; or an exogenous replication origin.

[0056] Moreover, the extrachromosomal element refers to replicable sequence in host cells derived from plasmid or virus, fragments of a host chromosome or an artificial chromosome.

[0057] A set of double-stranded DNA fragments thus described include the following examples:

[0058] (1) Double-stranded DNA referred to as e-a-A-b-f and double-stranded DNA referred to as g-c-B-d-h;

[0059] (2) Double-stranded DNA referred to as e-a-A-f and double-stranded DNA referred to as g-b-c-B-d-h;

[0060] (3) Double-stranded DNA referred to as e-a-f and double-stranded DNA referred to as g-A-b-c-B-d-h;

[0061] (4) Double-stranded DNA referred to as e-a-A-b-c-f and double-stranded DNA referred to as g-B-d-h;

[0062] (5) Double-stranded DNA referred to as e-a-A-b-c-B-f and double-stranded DNA referred to as g-d-h;

[0063] (6) Double-stranded DNA referred to as e-a-A-b-B-f and double-stranded DNA referred to as g-d-h;

[0064] (7) Double-stranded DNA referred to as e-a-A-f and double-stranded DNA referred to as g-B-d-h;

[0065] (8) Double-stranded DNA referred to as e-a-f and double-stranded DNA referred to as g-A-b-B-d-h.

[0066] In the above sets of double-stranded DNA, letters from a to d, A and B are similar to the above description. However, d in (6) to (8) refers to the same target sequence with the same orientation as "a".

[0067] Letters from e to h refer to the double-stranded DNA fragments comprising nucleotide sequences with size at least 50 bp, and preferably from 500 to 1 Kbp, wherein these DNA fragments are aligned in the order of e, f, replication origin, g, and h on a cellular chromosome or on an extrachromosomal element; and arbitrary sequence may be inserted between these fragments; and replication origin or a part of it may be included in f or g.

[0068] These fragments are connected as above.

[0069] At least two double-stranded DNA fragments thus obtained are introduced into appropriate cells by methods such as virus, lipofection, electroporation and the like. Furthermore, it is preferable to establish cell lines by selecting the cells that contain the above construct on a chromosome or an extrachromosomal element, by the drug corresponding a drug resistant gene (a drug resistant gene corresponding to Geneticin, Neomycin, Hygromycin, Zeocin, Blasticidin or the like). Yeast cells and animal cells can be used as the host. Pharmaceutical proteins are produced preferably in animal cells, wherein glycosylation pattern is similar to human and it reduces risk to undesirable immunological response.

[0070] Owing to the arrangement from e to h in the order and homologous recombination of these fragments with corresponding region in a host chromosome or an extrachromosomal element, similar construction to the above is generated on a host chromosome or on an extrachromosomal element.

[0071] The transformed or transfected cells thus obtained are subjected to the action of a site-specific recombinase. At the time of the action, it is preferable that site-specific recombinase works in the cells that are actively proliferating and progressing the cell cycle, or are synchronized in S phase, since enrichment of cells in replication phase (S phase) in cell cycle is preferable.

[0072] Methods for introducing the above site-specific recombinase include, for example, a method comprising the following steps:

(1) Introducing a Plasmid Constructed to Express Said Site-Specific Recombinase;

[0073] Various expression vectors are inserted with the site-specific recombinase gene under the control of promoter functional in a host cell. The vector is transfected into the above transformed or transfected cells by lipofection, electroporation method or the like. It is preferable to use inducible promoters to induce site-specific recombinase to actively proliferating cells.

(2) Transforming the Transformants or Trasfectants Further to Express Said Site-Specific Recombinase;

[0074] A construct, containing the site-specific recombinase gene under the control of promoter functional in a host cell and any of drug resistant genes against Geneticin, Neomycin, Hygromycin, Zeocin, Blasticidin or the like for selecting cells that contain the above construct on a chromosome or an extrachromosomal element, is prepared. The construct is introduced into the above transformed cells by lipofection, electroporation or the like. The construct containing the above DNA fragments is preferably linearized for efficient integration into a chromosome or to an extrachromosomal element. Additionally, inducible promoters are preferably used to induce site-specific recombinase to actively proliferating cells.

(3) Introducing Directly Said Site-Specific Recombinase Protein.

[0075] Site-specific recombinase is prepared by expressing and purifying large amount of the enzyme. The enzyme is introduced into the above transformed cells using commercial protein delivery reagent (i.e. Targeting System Co., Profect; Genlantis Co., BioPORTER Protein Delivery Reagent) and the like. It is preferable to introduce the site-specific recombinase into cells at actively proliferating and progressing the cell cycle, or into cells synchronized in S phase, since the site-specific recombinase should be induced into actively proliferating cells.

[0076] In the stage, wherein the site-specific recombinase acts, one of the replication folks must be located between two first target sequences and the other replication folk must be located between two second target sequences after initiation of the replication (FIG. 2 (b)). However, it is not necessary that all of the prepared cells are affected with the site-specific recombinase in such a specific situation. Since practically DNA replication in a number of cells is in various situations, it is enough for part of cells to be in such a specific situation. The target gene is amplified explosively in the cells in the above situation. Therefore, only a fraction of cells are good enough to be amplified.

[0077] Although amplification is induced as above description, it is preferable to select the cells with amplified DNA by drugs corresponding to target gene to be amplifieds (dihydrofolate reductase (DHFR), glutamine synthetase (GS), asp artate transcarbamylase (CAD), metallothionein (MT), adenosine deaminase (ADA), adenylate deaminase (AMPD1, 2), UMP synthetase, P-glycoprotein (P-gp), asp aragine synthetase (AS), ornithine decarboxylase (OP C) and the like). Those cell lines with high level of expression of a target gene are thus selected, and cultured. Large amount of the protein encorded by the target gene is prepared by purification from the culture supernatant.

[0078] The following examples illustrate the present invention, but are not intended to limit the scope of the present invention.

EXAMPLE 1

[0079] In this example, a construct (FIG. 3) for amplification was composed. Firstly, a DNA fragment structure 1 (structure of telomere side) was constructed, wherein the DNA fragment structure 1 contains a pair of loxP sequences with inverted arrangement, amplification-selection marker gene leu2d, and TRP1 gene, (SEQ ID NO.1, bases 1-34 of structure 1 is loxP sequence, bases 36-1988 is amplification marker gene leu2d, bases 1993-2845 (complementary strand) is TRP1 gene, and bases 5699-5732 is loxP sequence of inversion).

[0080] A DNA fragment was constructed, wherein the DNA fragment structure 1 is linked PCR fragment of bases 263177-264016 (SEQ ID No. 3) of chromosome 6 (Genebank Accession No. NC_001138) to the upstream of the DNA fragment structure 1 and linked PCR fragment of bases 264017-264685 (SEQ ID No. 4) of chromosome 6 (Genebank Accession No. NC_001138) to the downstream of the DNA fragment structure 1. Host yeast cells lines were transformed with the DNA fragment by Frozen-EZ Yeast Transformation II (ZYMO RESEARCH Co.). TRP1 marker gene allows cells to form colonies on agarose medium without tryptophan. The chromosomal structure of the selected cells was analyzed and cell lines with inserted structure flanked by loxP pair were established.

[0081] Then, DNA fragment structure 2 (structure of centromere side) was constructed, wherein the DNA fragment structure 2 contains a pair of loxm2 sequences with inverted arrangement, amplification-selection marker gene leu2d, and LYS5 gene, ((SEQ ID NO.2, bases 1-34 of structure 2 is loxm2 sequence, bases 3936-5888 (complementary strand) is amplification marker gene leu2d, bases 2891-3930 is LYS5 gene, and bases 5890-5923 is loxm2 sequence of inversion)).

[0082] A DNA fragment was constructed, wherein the DNA fragment structure 2 is linked PCR fragment of bases 257941-258821 (SEQ ID No. 5) to the upstream of the DNA fragment structure 2 and linked PCR fragment of bases 258822-259719 (SEQ ID No. 6) to the downstream of the DNA fragment structure 2. The DNA fragment was introduced into cells containing the above DNA structure 1 (a structure flanked by loxP pair). LYS5 marker gene allows cells to form colonies on agarose medium without lysine. The chromosomal structure of the selected cells was analyzed and cell lines with inserted structures flanked by loxP pair and loxm2 pair were established.

[0083] Additionally, amplification-selection marker gene leu2d lacks most of the promoter sequence and the expression level is very law. Therefore, the gene can complement leucine auxotrophy only when amplified.

[0084] It has been observed that Orel protein involved in replication initiation binds to the region between the above two DNA fragment structures (nature, 424: 1078, 2003). Therefore, the DNA region is supposed to be functional as replication origin. Furthermore, the DNA region contains WTTTAYRTTTWB (SEQ ID No.: 7), which is a consensus sequence of replication origin in Saccharomyces cerevisiae (bases 258889-258900).

EXAMPLE 2

[0085] In this example, the construct (FIG. 3) obtained in Example 1 was inserted to chromosome 6 of Saccharomyces cerevisiae, Cre gene was expressed and the double rolling-circle replication (DRCR) was induced.

[0086] The plasmid (FIG. 4, Genebank Accession No. AF298782, gifted from University of Washington, Yeast Resource Center), wherein Cre gene (SEQ ID No.: 8) is linked to the down stream of GAL promoter, was introduced into Saccharomyces cerevisiae cell line obtained in Example 1 by Frozen-EZ Yeast Transformation II (ZYMO RESEARCH). Furthermore, URA3 marker gene allows cells to form colonies on agarose medium without uracil.

[0087] The Ura.sup.+ cells with the plasmids obtained above were cultured for three hours in liquid medium supplemented with galactose to induce Cre expression or glucose to suppress Cre expression as control. These cells were plated on glucose agar plate without leucine and then Leu.sup.+ colonies were counted. The Leu.sup.+ cells were further cultured and chromosomal DNA was prepared using low-melting temperature agarose.

[0088] The chromosomal DNA was separated by pulsed-field gel electrophoresis (PFGE, BIO-RAD, CHEF Mapper XA, Auto Algorithm, range: size from 220 to 500 kb), or the DNA digested with a restriction enzyme, SmaI, was separated by Field-inversion gel electrophoresis (FIEG, BIO-RAD, CHEF Mapper XA, Auto Algorism, range: size from 3 to 50 kb) and were analyzed by Southern blotting.

Result and Interpretation

[0089] The Leu.sup.+ colony counts showed that there was about seven folds increase in colony forming activity in the case of induction of Cre expression in contrast to the control (addition of glucose) the induction of Cre expression gave about seven-fold higher frequency of Leu.sup.+ colonies than the control condition as shown in FIG. 5. The result strongly suggests that the Cre recombination contributes to the amplification.

[0090] Then, FIG. 6 (a) shows the result of structural analysis of chromosomal DNA, which is separated by PFGE, by Southern blotting using leu2d as a probe. As shown in FIG. 6(a), amplified product (i) on chromosome 6, wherein the construct for amplification is inserted, and (ii) multi-copies of mini-chromosome were detected. Additionally, chromosome 3 (*) of host cell lines containing leu2 fragments at 345 kb in size, chromosome 6 containing the construct for amplification originally (e.g. NS) or containing slight amplification at size from 290 to 320 kb were detected.

[0091] Then, the above chromosomal DNA was digested with a restriction enzyme (SmaI) and separated by FIGE. The result of Southern blot for structural analysis using leu2d probe is shown in FIG. 6(b).

[0092] Based on these results, the structure of the amplified product was elucidated as follows.

[0093] SmaI fragments with about 11 kb (10.9 and 11.1 kb) and 17 kb (16.8 kb) in size were detected from clones with strong signal highly amplified products (i) on chromosome (FIG. 6 (a) (i) #32, 48, 52, 53: black lanes). These fragments were derived from the product with inversions through lox pairs in a designed DRCR product and deemed to contain highly repeated sequence containing leu2d with at least more than several tens of copies, as shown in FIG. 7.

[0094] In contrast, mini chromosome (FIG. 6 (ii)) observed in most of clones (grey lanes) generated SmaI amplified fragments at about 6.3 kb in size. It is interpreted that these fragments are generated through reversal of replication from telomere side of the structure by Cre-loxP recombination, and that these fragments present as multi-copies, as shown in FIG. 8.

[0095] In addition to the above fragments, chromosomal products without inversions (FIG. 7(a), #34, 41, 47) and other types of mini chromosome (FIG. 9, #29-31, 49,56) through reversal of replication by similar recombination are observed. Furthermore, a number of clones containing both amplified product on chromosome and mini chromosome are detected (#22, 31, 34, 41, 47, 58). Also, weak signal originating from four fragments in addition to two SmaI fragments (* of FIG. 6 (b)) derived from host cell lines are confirmed in the construct not amplified (NS of FIG. 6 (b), FIG. 10).

[0096] Highly amplified products through the expected molecular mechanism was observed (#32, 48, 52 and 53). Since these products are observed in one tenth of the analyzed clones, these type of amplification occurred at frequency of one tenth of the total colony forming frequency 4.4%, i.e. 0.44%.

Sequence CWU 1

1

815732DNAArtificial SequenceArtificially synthesized replication unit 1ataacttcgt ataatgtatg ctatacgaag ttatggatct agaggttaac taagcgaatt 60tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat 120tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt 180aggtcaggtt gctttctcag gtatagcatg aggtcgctct tattgaccac atctctaccg 240gcatgccgag caaatgcctg caaatcgctc cccatttcac ccaattgtag atatgctaac 300tccagcaatg agttgatgaa tctcggtgtg tattttatgt cctcagagga caacacctgt 360tgtaatcgtt cttccacacg gatcttatat atatttcaag gatataccat tctaatgtct 420gcccctaaga agatcgtcgt tttgccaggt gaccacgttg gtcaagaaat cacagccgaa 480gccattaagg ttcttaaagc tatttctgat gttcgttcca atgtcaagtt cgatttcgaa 540aatcatttaa ttggtggtgc tgctatcgat gctacaggtg tcccacttcc agatgaggcg 600ctggaagcct ccaagaaggt tgatgccgtt ttgttaggtg ctgtgggtgg tcctaaatgg 660ggtaccggta gtgttagacc tgaacaaggt ttactaaaaa tccgtaaaga acttcaattg 720tacgccaact taagaccatg taactttgca tccgactctc ttttagactt atctccaatc 780aagccacaat ttgctaaagg tactgacttc gttgttgtca gagaattagt gggaggtatt 840tactttggta agagaaagga agacgatggt gatggtgtcg cttgggatag tgaacaatac 900accgttccag aagtgcaaag aatcacaaga atggccgctt tcatggccct acaacatgag 960ccaccattgc ctatttggtc cttggataaa gctaatgttt tggcctcttc aagattatgg 1020agaaaaactg tggaggaaac catcaagaac gaattcccta cattgaaggt tcaacatcaa 1080ttgattgatt ctgccgccat gatcctagtt aagaacccaa cccacctaaa tggtattata 1140atcaccagca acatgtttgg tgatatcatc tccgatgaag cctccgttat cccaggttcc 1200ttgggtttgt tgccatctgc gtccttggcc tctttgccag acaagaacac cgcatttggt 1260ttgtacgaac catgccacgg ttctgctcca gatttgccaa agaataaggt caaccctatc 1320gccactatct tgtctgctgc aatgatgttg aaattgtcat tgaacttgcc tgaagaaggt 1380aaggccattg aagatgcagt taaaaaggtt ttggatgcag gtatcagaac tggtgattta 1440ggtggttcca acagtaccac ggaagtcggt gatgctgtcg ccgaagaagt taagaaaatc 1500cttgcttaaa aagattctct ttttttatga tatttgtaca taaactttat aaatgaaatt 1560cataatagaa acgacacgaa attacaaaat ggaatatgtt catagggtag acgaaactat 1620atacgcaatc tacatacatt tatcaagaag gagaaaaagg aggatgtaaa ggaatacagg 1680taagcaaatt gatactaatg gctcaacgtg ataaggaaaa agaattgcac tttaacatta 1740atattgacaa ggaggagggc accacacaaa aagttaggtg taacagaaaa tcatgaaact 1800atgattccta atttatatat tggaggattt tctctaaaaa aaaaaaaata caacaaataa 1860aaaacactca atgacctgac catttgatgg agtttaagtc aataccttct tgaaccattt 1920cccataatgg tgaaagttcc ctcaagaatt ttactctgtc agaaacggcc ttaacgacgt 1980agtcgacgga tcgatctttt atgcttgctt ttcaaaaggc ctgcaggcaa gtgcacaaac 2040aatacttaaa taaatactac tcagtaataa cctatttctt agcatttttg acgaaatttg 2100ctattttgtt agagtctttt acaccatttg tctccacacc tccgcttaca tcaacaccaa 2160taacgccatt taatctaagc gcatcaccaa cattttctgg cgtcagtcca ccagctaaca 2220taaaatgtaa gctttcgggg ctctcttgcc ttccaaccca gtcagaaatc gagttccaat 2280ccaaaagttc acctgtccca cctgcttctg aatcaaacaa gggaataaac gaatgaggtt 2340tctgtgaagc tgcactgagt agtatgttgc agtcttttgg aaatacgagt cttttaataa 2400ctggcaaacc gaggaactct tggtattctt gccacgactc atctccatgc agttggacga 2460tatcaatgcc gtaatcattg accagagcca aaacatcctc cttaggttga ttacgaaaca 2520cgccaaccaa gtatttcgga gtgcctgaac tatttttata tgcttttaca agacttgaaa 2580ttttccttgc aataaccggg tcaattgttc tctttctatt gggcacacat ataataccca 2640gcaagtcagc atcggaatct agagcacatt ctgcggcctc tgtgctctgc aagccgcaaa 2700ctttcaccaa tggaccagaa ctacctgtga aattaataac agacatactc caagctgcct 2760ttgtgtgctt aatcacgtat actcacgtgc tcaatagtca ccaatgccct ccctcttggc 2820cctctccttt tcttttttcg accgatccgt cgaccgatgc ccttgagagc cttcaaccca 2880gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc 2940tttatcatgc aactcgtagg acaggtgccg gcagcgctct tccgcttcct cgctcactga 3000ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 3060acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 3120aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 3180tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 3240aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 3300gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 3360acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 3420accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 3480ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 3540gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 3600aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 3660ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 3720gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 3780cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 3840cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 3900gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 3960tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4020gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4080agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4140tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4200agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 4260gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 4320catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 4380ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 4440atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 4500tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 4560cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 4620cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 4680atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 4740aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 4800ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 4860aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg 4920tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 4980cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 5040ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 5100gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg 5160atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 5220ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt 5280gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 5340taacaaaata ttaacgctta caatttgcca ttcgccattc aggctgcgca actgttggga 5400agggcgatcg gtgcgggcct cttcgctatt acgccagccc aagctaccat gataagtaag 5460taatattaag gtacgggagg tacttggagc ggccgcaata aaatatcttt attttcatta 5520catctgtgtg ttggtttttt gtgtgaatcg atagtactaa catacgctct ccatcaaaac 5580aaaacgaaac aaaacaaact agcaaaatag gctgtcccca gtgcaagtgc aggtgccaga 5640acatttctct atcgataggt accgagctct tacgcgtgct agcccgggct cgagatctat 5700aacttcgtat agcatacatt atacgaagtt at 573225923DNAArtificial SequenceArtificially synthesized replication unit 2ataacttcgt ataagaaacc atatacgaag ttatagatct cgagcccggg ctagcacgcg 60taagagctcg gtacctatcg atagagaaat gttctggcac ctgcacttgc actggggaca 120gcctattttg ctagtttgtt ttgtttcgtt ttgttttgat ggagagcgta tgttagtact 180atcgattcac acaaaaaacc aacacacaga tgtaatgaaa ataaagatat tttattgcgg 240ccgctccaag tacctcccgt accttaatat tacttactta tcatggtagc ttgggctggc 300gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 360aatggcaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 420gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 480ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 540actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 600caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 660ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga 720agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 780ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg 840gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 900tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 960ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 1020ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 1080gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 1140gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 1200acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 1260actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 1320ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 1380cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 1440gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 1500caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 1560aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 1620ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 1680tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 1740ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 1800ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 1860ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1920tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 1980cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 2040taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 2100gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 2160acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 2220ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2280ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2340cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2400aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2460gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2520gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2580gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 2640ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 2700ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgct 2760gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata 2820gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt 2880cgacggatcc tacataaatg tgagcaagcg aaaaaaaaaa attggcatta taaaccatca 2940ttttcgatga aataatcaat caacgtagat aagctgatat tatataattt tggtctgttc 3000gtgttgattt tatcactgat ggactttggc atacagatag tgacaatttc gttattgaac 3060cattgagaat ggaaaatcaa tggaacttca tccagagtta tgcacataga agctccctca 3120gccggaaaaa agctgatagc gccaaaatct attagtgaca agtctgtgtt aaggccagtt 3180ccagtaaatt ttgtatacga ctccttcaag gaccataagt aagtaaatat tgtgcatgga 3240tcagacgctt tcagtaaacc gttaaattct ctttcactaa aaacttcttt aaatagctcc 3300aactcttccc tcccgccata attgcacgga gaagcgatat caattccgac atcctggtat 3360tcatctgtac ttacacattt tacgaggaac atagctacat attgttcacc gatggtcatg 3420ctaaatggaa gaaaacgatt gttgtctaag aatggcttac cgaagctgcc cttgtcaaat 3480ttcagctctt gaaaatttaa gcccgttact atagagcagc caaacaactg cagcagctgg 3540ctgcatagat ttgaacatct atcgtgaaac gattttttat tgaggattct ggcttgagac 3600gccaatggca aagttctcat taatgcctcg aacgtaaact catccgcgag tatatcctct 3660tgaatttcaa caacgaatat acctgcccat ggtcttacac ctgccacctt tgaaacttcg 3720cttactactt cagtcgtttt aaccatccac ggtttttttg ctgagtgatt ctctttctcc 3780tcattctcat tttagtcata gcggttttaa taagcgcccg aaagataatt gtaaaacata 3840tattcaatgc ttaaaaatat aagaaattgc ccatcaattt gaaaactcaa gtaaaacaga 3900gaagttgtaa ggtgaataag gaatgagtga ggatccgtcg actacgtcgt taaggccgtt 3960tctgacagag taaaattctt gagggaactt tcaccattat gggaaatggt tcaagaaggt 4020attgacttaa actccatcaa atggtcaggt cattgagtgt tttttatttg ttgtattttt 4080ttttttttag agaaaatcct ccaatatata aattaggaat catagtttca tgattttctg 4140ttacacctaa ctttttgtgt ggtgccctcc tccttgtcaa tattaatgtt aaagtgcaat 4200tctttttcct tatcacgttg agccattagt atcaatttgc ttacctgtat tcctttacat 4260cctccttttt ctccttcttg ataaatgtat gtagattgcg tatatagttt cgtctaccct 4320atgaacatat tccattttgt aatttcgtgt cgtttctatt atgaatttca tttataaagt 4380ttatgtacaa atatcataaa aaaagagaat ctttttaagc aaggattttc ttaacttctt 4440cggcgacagc atcaccgact tccgtggtac tgttggaacc acctaaatca ccagttctga 4500tacctgcatc caaaaccttt ttaactgcat cttcaatggc cttaccttct tcaggcaagt 4560tcaatgacaa tttcaacatc attgcagcag acaagatagt ggcgataggg ttgaccttat 4620tctttggcaa atctggagca gaaccgtggc atggttcgta caaaccaaat gcggtgttct 4680tgtctggcaa agaggccaag gacgcagatg gcaacaaacc caaggaacct gggataacgg 4740aggcttcatc ggagatgata tcaccaaaca tgttgctggt gattataata ccatttaggt 4800gggttgggtt cttaactagg atcatggcgg cagaatcaat caattgatgt tgaaccttca 4860atgtagggaa ctcgttcttg atggtttcct ccacagtttt tctccataat cttgaagagg 4920ccaaaacatt agctttatcc aaggaccaaa taggcaatgg tggctcatgt tgtagggcca 4980tgaaagcggc cattcttgtg attctttgca cttctggaac ggtgtattgt tcactatccc 5040aagcgacacc atcaccatcg tcttcctttc tcttaccaaa gtaaatacct cccactaatt 5100ctctgacaac aacgaagtca gtacctttag caaattgtgg cttgattgga gataagtcta 5160aaagagagtc ggatgcaaag ttacatggtc ttaagttggc gtacaattga agttctttac 5220ggatttttag taaaccttgt tcaggtctaa cactaccggt accccattta ggaccaccca 5280cagcacctaa caaaacggca tcaaccttct tggaggcttc cagcgcctca tctggaagtg 5340ggacacctgt agcatcgata gcagcaccac caattaaatg attttcgaaa tcgaacttga 5400cattggaacg aacatcagaa atagctttaa gaaccttaat ggcttcggct gtgatttctt 5460gaccaacgtg gtcacctggc aaaacgacga tcttcttagg ggcagacatt agaatggtat 5520atccttgaaa tatatataag atccgtgtgg aagaacgatt acaacaggtg ttgtcctctg 5580aggacataaa atacacaccg agattcatca actcattgct ggagttagca tatctacaat 5640tgggtgaaat ggggagcgat ttgcaggcat ttgctcggca tgccggtaga gatgtggtca 5700ataagagcga cctcatgcta tacctgagaa agcaacctga cctacaggaa agagttactc 5760aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta tacacttatt 5820ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc ttagttaacc 5880tctagatcca taacttcgta tatggtttct tatacgaagt tat 59233840DNASaccharomyces cerevisiae 3aggtggagcg caggtcatag gtatgccggc tcattgtttt ctattttaaa aagtaaaaaa 60tatgctgcta aaggaacacg tgagaaatta cattctccct aggtctgcga taacgcggta 120atattacact gccgccgcct tccatgcctt tggaaagcag acaatgatgc taggcggcgc 180ccagcagtat aaacttttct tgcttataac cagaacctct atcacaaaat tagaaactgc 240gatactatgg gtcagatcga cacataggga gcactattag gcgcaaggcg tatacatagg 300cattgcgtgt tcaaaaattg tcgtatgaga aaagttccaa actttccacc attactcacc 360aacaacttac accagcccgg atttaagatt tagcttccga gaatattgtg actcagccac 420tggtctcttg aatgttgcgt gtagcttgat taagattatg gcataaccgt tttttttact 480tggcaagagt gaacgtcctt ttactccaaa aggctcctga tgaaactgga gagtctcttt 540gttctgaaat ttttaaagtt tagcacacca tattcacgct cgaggtgaac ccaagttttc 600ctgaaaaatg tgccatgaac ctgaaaaaaa gaattattct cgaaaataaa aaaggcaatc 660aagatcggaa agataagcat tttttttcaa tccgtatcta acattcataa agtgataaaa 720aaattgataa cgattttatt gtcgcctctt gttttgagta tattttttta acgttctttt 780tcggcattca aattccgtat aatcaactca attgtaaggc gccgtagcat ccaaataatg 8404669DNASaccharomyces cerevisiae 4ttgaaagtgc caatttgctc atcagtgcta aatattcctt gataaaaata tagaagacaa 60ggacatataa aaagaaagac tgctctagtg ttgggacacc acaatgaaaa aatacttaac 120gtgtttcgaa actgtgaata taaaattcca gcaaaaacca aaatattcac tacaatgatt 180gatcgtaccg agttatcgaa gtttggtatt actacgcaac tgtctgttat tggacgtaat 240ccagatgaac aaagtggctt tgttaatcca cctttgtata aggggtcaac catcattctt 300aaaaaactta gtgatttaga acaaaggaaa ggaagatttt acgggacagc aggttctcca 360actattgaca atttagaaaa tgcctggacg catttaaccg gcggtgctgg gacagtgcta 420tcagcttctg ggcttggttc tatctctttg gcgctattgg ccctttcgaa agctggtgat 480catatcttga tgactgatag tgtctacgtg ccaacacgta tgctatgtga tggtttattg 540gccaagttcg gtgttgaaac ggattattat gacccatcaa tagggaagga tatagaaaaa 600ctagttaagc caaatacaac cgtcattttc ctcgaaagcc cgggttctgg gaccatggaa 660gtacaggat 6695881DNASaccharomyces cerevisiae 5gcaactaaaa cgcccgtgga ttgaggttca gatttgctac tgtcgctttc gaagaagcta 60gatgaaccac gggtaaagta ttctgcatct aatgtgttca ataaatattg agtgacgtta 120tcgtaatgtt acagtactaa caccgctaga aaatgctggt gtgaatgtga atgacgatag 180acggactgat gcacttttcc attgtacgat aacattactt acaagattgg gagaagcatg 240attgaaaatt tgactggaag aaccacttat attaggagtg gcggtattag tagaaaattg 300actaaacgca tccgaaaatt aaatagaatt taaagtttcc ttgggtgcac tgttttgggc 360tgcagtgcta aaatccagaa gtgttggagt caggttactt gtttgcatac agacattact 420gaagttttca gaaggccttt gaatcgagaa cgagataagg aagtgtcctc taaatgcaat 480tttagagctc aaagtgagga tagtggcact gaaacttgat ttagttaatg gcctttagat 540ttgctgctgt ctgaaaagct catcatcgag aagctcacaa aatggagttc tagttgccct 600ttcactatac aatcgatgta aagatggctt ataagtattt gaattgtaag ttttgtgcta 660gctgaggaat caaaaaatgt atttagtgct tccttactgg tcaattgtgt attatttcca 720gatgaaacag aaaatatgta ttttatggat tgactaatca agtcacttgc tgattgtgta 780atagtggtgg ttaaagaaag tacagtaagt ttgcttgaaa tacaggcagg attcctggaa 840tactgcctac tactgcttat tgaatagagt gtaatctcgc c 8816898DNASaccharomyces cerevisiae 6aaacccagta atagcatcgt ttaagaaatg gtgcttactt gtaggtaaaa cttctgaagg 60atactgagta aatgtaaaat tatatgagtt aaggcagaat gactgtaaac ttttgtgacg 120aatctggaag atgcattcgt cgattggcct tcattaagtg aagattggta acctattgca 180tccaaaccag aagtaataca accagaatgt ggagatgagg agccaacagg tgtgtatcca 240gaaggttcag ccacaggttg gtcatcaata ccactggggg agcatgcaat ataatcagat 300ggttgcgagg aatagctagt agagctaaaa ctgaacacat tagttaagat tagactagcc 360atgctcaaag aaacaattag agaggcacct acatgttcgt tatccatttt tgaggaaaaa 420atagaagtga taataataat tttgctcgaa ctactcgtaa agctacttga aaaacggctc 480gagattacgg aagagtcggt agtaaaccga ctctcagtgt cacggaatgg aagcgccttg 540aaactactaa tatcaggtat gcattgaggg gcaaggcaac ctgaatatgc aaagagcata 600gtcttaactt tcgtagtacg taatacttcg gcattaattt ggcctaccgc tttgccacag

660ttgagtggtc actggagtat tagccatgaa aaaatgatcc cttgtatatc caggcccaaa 720gtctaaaatg tacttcctgc caatggttgt cacagctaat attccatttt gaattgactt 780gatttttaga ttattatcat ggaaccaagt tgatgtcttg caatttctga tttttaacga 840tgtactggag gttgacgact aggcaaatct gcgaaacatc ctagtacaat ggcatttg 898712DNASaccharomyces cerevisiaemisc_feature(1)..(1)w can be a or t 7wtttayrttt wb 1281032DNABacteriophage P1 8atgtccaatt tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt 60gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat 120acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac 180cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg 240cgcggtctgg cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt 300cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc 360cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact 420gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat 480ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc 540agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg 600aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg 660gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc 720cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc 780ctggaaggga tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt 840cagagatacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc 900cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt 960gtcatgaact atatccgtac cctggatagt gaaacagggg caatggtgcg cctgctggaa 1020gatggcgatt ag 1032

Claims

1. A double-stranded DNA represented by a-b-c-d or a-c-b-d, wherein one of a and b is a double-stranded DNA fragment comprising a first target sequence of a site-specific recombinase, and the other is a double-stranded DNA fragment comprising an inverted sequence of said first target sequence; and one of c and d is a double-stranded DNA fragment comprising a second target sequence of the site-specific recombinase and the other is a double-stranded DNA fragment comprising an inverted sequence of said second target sequence; a replication origin and at least one target gene to be amplified are inserted anywhere between a and d; and arbitrary DNA sequences may be inserted among above fragments.

2. The double-stranded DNA of claim 1, wherein b and care combined and said double-stranded DNA is represented by a-b-d, wherein a and d are the same sequence with the same direction and the other letters are the same as defined previously.

3. The double-stranded DNA of claim 1, which is represented by a-b-X-c-d or a-c-X-b-d, wherein X represents a replication origin and the other letters are the same as defined previously.

4. The double-stranded DNA of claim 3, which is represented by a-A-b-X-c-B-d or a-A-c-X-b-B-d, wherein at least one of A and B represents the target gene, arbitrary DNA sequences may be inserted among these fragments, and the other letters are the same as defined previously.

5. The double-stranded DNA of claim 1, wherein the first target sequence and the second target sequence of the site-specific recombinase are different.

6. The double-stranded DNA of claim 1, wherein each of said the first and the second target sequences is selected from the group comprising loxP, lox511, lox5171, lox2272, lox2372, loxm2, loxFAS, lox71, lox66 and the mutants thereof in a case where the site-specific recombinase is Cre recombinase or its derivative; each of said the first and the second target sequences is selected from the group comprising FRT, F3, F5, FRT mutant-10, FRT mutant+ 10 and the mutants thereof in a case where the site-specific recombinase is Flp recombinase or its derivative; and each of said the first and the second target sequences is selected from the group comprising attB, attP and the mutants thereof 10 in a case where the site-specific recombinase is phiC31 integrase or its derivative.

7. A vector comprising the double-stranded DNA of claim 1.

8. A transformant, which is introduced with the double-stranded DNA of claim 1.

9. The transformant of claim 8, wherein the host is an animal cell.

10. A set of double-stranded DNA fragments, which is obtained by dividing any one of the double-stranded DNA of claim 1 into at least two, wherein each said fragment contains a double-stranded DNA region with at least 50 bp at both ends for homologous recombination; said double-stranded DNA region for homologous recombination comprises a part of the sequences of said host chromosome or an extrachromosomal element so that the double-stranded DNA can be integrated into the host chromosome or the extrachromosomal element by homologous recombination; and said replication origin may be a replication origin of said host or said exogeneous origin.

11. The set of double-stranded DNA of claim 10 comprising a double-stranded DNA fragment represented by e-a-A-b-f and a double-stranded DNA fragment represented by g-c-B-d-h, wherein one of a and b is a double-stranded DNA fragment comprising a first target sequence of a site-specific recombinase, and the other is a double-stranded DNA fragment comprising an inverted sequence of said first target sequence; and one of c and d is a double-stranded DNA fragment comprising a second target sequence of the site-specific recombinase and the other is a double-stranded DNA fragment comprising an inverted sequence of said second target sequence; each of letters from e to h is a double-stranded DNA fragment of at least 50 bp in size, which are arranged on a chromosome or an extrachromosomal element that is a host for integration of the set of double-stranded DNA in order of e, f, a replication origin of the chromosome element or the extrachromosomal element, g and h; at least one of A and B represents the target gene to be amplified; and said replication origin or a part of it may be included in for g; and an arbitrary DNA sequence may be inserted among these.

12. The set of double-stranded DNA of claim 10, wherein the first target sequence and the second target sequence of the site-specific recombinase are different.

13. The set of double-stranded DNA of claim 10, wherein each of said the first and the second target sequences is selected from the group comprising loxP, lox511, lox5171, lox2272, lox2372, loxm2, loxFAS, lox71, lox66 and the mutants thereof in a case where the site-specific recombinase is Cre recombinase or its derivative; each of said the first and the second target sequences is selected from the group comprising FRT, F3, F5, FRT mutant-10, FRT mutant+10 and the mutants thereof in a case where the site-specific recombinase is Flp recombinase or its derivative; and each of said the first and the second target sequences is selected from the group comprising attB, attP and the mutants thereof in a case where the site-specific recombinase is phiC31 integrase or its derivative.

14. A set of vectors, wherein each vector contains each of two kinds of the double-stranded DNA of claim 10.

15. A transformant, which is introduced with two kinds of the double-stranded DNA of claim 10, wherein said replication origin locates on a host chromosome or an extrachromosome.

16. The transformant of claim 15, wherein the host is an animal cell.

17-19. (canceled)

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