JP2001513991A - Impaired BRCA2 function in cells and non-human transgenic animals - Google Patents

Abstract

  (57) [Summary]   ScRAD51, a member of the RAD52 epistasis group of Saccharomyces cerevisiae, is a major component in the recombinant repair pathway used to repair genetic damage caused by ionizing radiation. MmRad51, a mouse homolog of ScRad51, appears to have a similar function, but the exact mechanism of action is poorly understood. For ScRad51, protein: protein coupling is important for function. Therefore, to better understand recombination repair in mammalian cells, the yeast two-hybrid system was used to isolate proteins related to MmRad51 and to isolate mouse Brca2. In humans, BRCA2 is an important tumor suppressor gene in the pathogenesis of breast cancer. Phenotypic comparison with MmRad51 and Brca2-deficient embryos and cells suggests that protein: protein coupling is important for their function. Like MmRad51, the function of Brca2 is important in repairing gamma irradiation induced damage. In addition, clever mutations that remove only a small portion of Brca2, which is directly or indirectly related to MmRad51, showed a phenotype suggesting partial function. These homozygous mutant cells are still viable while still being hypersensitive to ionizing radiation and undergo precocious replication senescence. Cells and mice were generated using a model for tumor development, a model for analyzing genotoxic agents, and impaired Brca2 function that was found to be useful as a tool for studying precocious replication senescence.

Description

DETAILED DESCRIPTION OF THE INVENTION       [0001]     TECHNICAL FIELD OF THE INVENTION (1.0. Field of the Invention)   The present invention relates to the Brca2 gene (G
EnBank accession number U65594) and cells that have been genetically integrated
For sgenic animals. In particular, the domain encoding Rad51 or the Rad51 complex
Or targeted disruption of the Brca2 gene to remove the interaction region. Reduced Brca2 activity in the vesicle, but the rest of the coding sequence remained intact, Is to be expressed. Then, the genetically modified cells are transformed into modified Brca2 protein Was used for the production of transgenic animals.       [0002] (2.0. Background of the Invention)   Cellular DNA usually exists in a dynamic environment. Repair, recombination, replication and cells Cell functions for cycle regulation are intertwined to maintain genomic stability and
(See Petes et al., 1991, Recombination in yeast (Reco
mbination in yeast), molecular and cell biology of the yeast Saccharomyces (Molecular a
nd Cellular Biology of the Yeast Saccharomyces) (J.R.Boach, J.R.Pringle,
And E.W.Jones), pp.407-521, Cold Spring Harbor Laboratory Press, Co.
ld Spring Harbor, New York; Drapkin et al., 1994, Cell, 77: 9-12; Kuhn et al., 1995.
, Genes Dev. 9: 193-203; Friedberg et al., 1995, DNA repair and mutagenesis (DNA
 repair and mutagenesis), pp. 147-192, ASM Press Washington, D.C .; Li et al.,
1995, Cell 83: 1079-1089)). The products of these processes
Mutations in genes that are important for either cause neurological disorders, immunodeficiency
And a variety of clinical signs, including predisposition to cancer.       [0003]   Understanding the molecular mechanisms of repair and recombination is a defect in these processes
It is helpful to understand the etiology of the disease caused by. Mice have DNA Ideal for studying dynamic properties. Intron-exon range and regulation
Human and mouse, including the location of elements and the spatial transcriptional regulation of homologous genes
Similarities in genomic structure are striking (Lyon and Searle, 1989, experimental mouse remains). Genetic variants and strains of the laboratory mouse
, 2nd edition, Oxford University Press, Oxford). Furthermore, between mouse and human
Because of the anatomical similarities, there is a direct physiological comparison opportunity. p53 tumor support Lesser (Donehower et al., 1992, Nature 356: 215-221), an unsuitable base pair repair protein
(Baker et al., 1995, Cell 82: 309-319; de Wind et al., 1995, Cell 82: 321-330)
And xeroderma pigmentosum complementation group (Sands et al., 1995, Nature 377: 169-173; de Vries et al., 19
95, Nature 377: 169-173; Nakane et al., 1995, Nature 377: 165-168) Target disruption of the gene encoding the protein product has led to significant genetic disorders in humans.
A new similarity has been identified.       [0004]   Several different DNA repair pathways are responsible for correcting various specific DNA lesions.
These pathways include nucleotide excision repair, mismatched base pair repair, and duplex
Destruction (DSB) repair. Nucleotide excision repair and mismatch repair The responsible mechanism is fairly well understood and affects these processes
The mutation has been characterized (this is Friedberg, 1992, Cell 71: 887-889; Cl
eaver, 1994, Cell 76: 1-4). However, it is responsible for DSB restoration. The understanding of the mechanism in question remains low. Some genetic disorders in mammals include:
Characterized by deficits in DSB repair associated with hypersensitivity to ionizing radiation and immunodeficiency I do. These include telangiectasia in humans (AT) (Lehmann and Ca
rr, 1995, Trends in Genet. 11: 375-377) and mice (Roth et al., 1992
, Cell 70: 983-991) and horses (Wiler et al., 1995, Proc. Natl. Acad. Sci. 92:
11485-11489), including autosomal recessive scid (severe combined immunodeficiency).       [0005]   ScRad51 is a member of the RAD52 epistasis group in Saccharomyces cerevisiae
And the yeast DSB repair pathway (by homologous recombination); a pathway called recombination repair (Friedberg, et al., 1995, DNA repair and mutagenesis)
, pp. 523-594, outlined in ASM Press Washington, D.C.). This path is for ionizing radiation
Repair the genetic damage caused by it. MmR, a mouse homolog of ScRad51
ad51 appears to have a similar function (Shinohara et al., 1993, Nature Genet.
 4: 239-243; Lim and Hasty 1996, Mol. Cell. Biol. 16: 7133-7143),
The exact mechanism is not well understood. For ScRad51, Protein: Tan Park relation is important for function. Thus, recombination in mammalian cells
MmRad51 and yeast cells using the yeast two-hybrid system to better understand repair A related protein was isolated and mouse Brca2 was isolated (Sharan et al., 1997, Natu
re, 386: 804-810). For phenotypic comparison of MmRad51 and Brca2-deficient embryos and cells More importantly, that protein: protein linkages are important for their function.
Is suggested. Like MmRad51, Brca2 function is involved in early embryo development, cell proliferation or
Important for viability and repair of gamma-induced damage.       [0006]   People with a mutation in Brca2 are more susceptible to breast cancer (Wooster et al., 1994, Natu
re 265: 2088-2090; Smith et al., Nature Genet. 2: 128-131; Easton et al., 1993, A.J.
Hum. Genet. 52: 678-701). Neoplasia is a non-mutated allele in tumors
Is associated with loss of conjugation, indicating that Brca2 is a tumor suppressor. Suggestive. Brca2 has no significant homology to any other gene. 3,418 amino acid proteins. Mouse Brca2 protein has 3,328 Amino acid, the overall identity of mouse and human Brca2 is 58% (Sharan et al., 1
997, Genomics 40: 234-241).       [0007] (3.0. Summary of the Invention)   Brca2 is a tumor suppressor and mediates Rad51 function. Therefore, Brca2 Lacks or destabilizes or reduces Rad51 function and then becomes mutagenic there is a possibility. Some of these mutations can promote cancer. Mouse and thin
Cells that inhibit the direct or indirect association of Brca2 with mouse Rad51 (MmRad51) Created by strange mutation. The data described here is directly compatible with MmRad51. A clever mutation that removes only a small portion of the associated or indirectly Brca2 It demonstrates that it exhibits a phenotype that suggests function. These brca2 Mutant cells can survive and remain hypersensitive to ionizing radiation,
Suggests that the repair of DNA double-strand breaks is deficient. Sa Furthermore, embryonic fibroblasts undergo precocious replication senescence, as do cells with insufficient Ku autoantigen.
(US patent application Ser. No. 08 / 695,866, filed Aug. 8, 1996),
Involved in the repair of double-strand breaks.       [0008]   It is an object of the present invention to provide a Brc with impaired ability to directly or indirectly associate with MmRad51.
An object of the present invention is to provide an animal cell expressing the modified form of a2.       [0009]   A further object of the present invention is to reduce the ability to directly or indirectly associate with MmRad51. Expressing a modified form of the damaged Brca2, a mammalian, preferably a mouse embryo or mammal Providing an animal, preferably a mouse.       [0010]   These and other objects of the present invention, which are apparent from the detailed description of the invention,
Illustrated with a mouse cell containing two chromosomal alleles of the Brca2 gene. Where before
At least one of the alleles has the ability to be directly or indirectly associated with MmRad51.
Includes mutations that produce impaired Brca2.       [0011]   Another embodiment of the present invention is directed to direct or indirect Mutant mouse embryos producing Brca2 with impaired ability associated with.       [0012] (5.0. Detailed Description of the Invention)   The present invention relates to the production of Brca2 impaired cells and Brca2 impaired non-human animals. The present invention
The intended non-human transgenic animal will generally contain a Brca2 homolog. Any vertebrate, preferably a mammal, is included. Such non-human tiger
Examples of transgenic animals include transgenic pigs and transgenic pigs.
Nick rat, transgenic rabbit, transgenic cow, trans
Transgenic goats and other transgenic animal species known in the art
And especially mammalian species. In addition, cattle, sheep and pig species, rodents
Family animals, such as rats, and rabbits and guinea pigs, and chimpanzees
Non-human primates, such as humans, can be used in the practice of the present invention. A particularly preferred animal is la
Pigs, rabbits and guinea pigs, most preferably mice.       [0013]   The obvious similarities between the repair mechanisms of yeast and mouse DSB have been demonstrated herein. The mouse Brca2 sequence utilized in Can be used as a heterologous probe to identify and isolate E. coli. Typically
, Hybridization conditions are adjusted by the relationship between the probe and the target sequence
. For example, if the cDNA library (or target sequence)
If derived from a different organism than the type of product, hybridization / washing
The matter should be of low stringency. Using a mouse Brca2 probe for the Brca2 homolog
For cloning, for example, hybridization is performed, for example, by Church's relaxation.
Impact liquid (7% SDS, 250 mM NaHPO_Four , 2 μM EDTA, 1% BSA) at 65 ° C. overnight
. Washing is performed at 65 ° C. using 2 × SSC, 0.1% SDS, and then at 65 ° C. using 0.1 × SSC, 0.1% SDS.
Can be performed.       [0014]   Low stringency conditions are well known to those skilled in the art and, as expected, the library and label sequences
Will vary depending on the particular organism from which it is induced. For guidance on such conditions
For example, see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manu.
al, Cold Springs Harbor Press, N.Y .; and Ausaubel et al., 1989, Current Pro.
tocols in Molecular Biology, Green Publishing Associates and Wiley Inter
See science, N.Y.       [0015]   On the other hand, again using a labeled Brca2 nucleotide probe, Screen genomic libraries derived from the organism of interest using the conditions
obtain.       [0016]   In addition, two oligonucleotides designed from the Brca2 sequence utilized herein By performing PCR using otide, the Brca2 gene homolog can be replaced with the nucleic acid of the target organism.
Can be isolated from The template for the reaction, for example, expresses the Brca2 gene allele Human or non-human cells, such as the choroid plexus, which is known or suspected to be
It can be cDNA obtained by reverse transcription of mRNA prepared from a system or tissue.       [0017]   By subcloning and sequencing the PCR product, the amplified sequence
It can be confirmed that it shows the sequence of the a2 gene. Then, using the PCR fragment Full-length cDNA clones can be isolated by various methods. For example, label the amplified fragment,
Screening of cDNA libraries such as bacteriophage cDNA libraries
Can be used for On the other hand, the labeled fragments are
Can be used to isolate genomic clones.       [0018]   PCR techniques can also be used to isolate full-length cDNA sequences. For example, standard operation Depending on the crop, the RNA is transferred to a suitable cell or tissue source (ie, for example, the choroid plexus). Or is known or suspected to express the obR gene, such as brain tissue ). The reverse transcription reaction is the priming of the first strand synthesis.
On the RNA using oligonucleotide primers specific for the 5 'end of the amplified fragment Can be done. Then, using a standard terminal transferase reaction, the resulting
Guanine can be `` tailed '' to RNA / DNA hybrids The bridge is digestible with RNAase H, and then the second strand synthesis is
May be primed with an immer. Therefore, the cDNA sequence upstream of the amplified fragment can be easily isolated.
I can do it. For a review of cloning strategies that can be used, see, for example, Sambrook et al.
, 1989, (as above).       [0019]   The present invention can be practiced by utilizing virtually any animal cell.
However, a preferred embodiment of the present invention comprises two chromosomal alleles of the Brac2 gene
Includes diploid mouse cells, mouse embryos and mice, where alleles of Brca2 At least one of the cells wherein the cell is directly or indirectly associated with MmRad51. Mutations that produce several Brca2 proteins whose function is impaired Including. Such Brca2 impaired cells and mice are particularly useful for the analysis and testing of therapeutic agents. And the effects of mutagenic stimuli such as radiation and chemical mutagens
It is considered to be useful as a disease model for use.       [0020]   Replication or cellular senescence leads to its terminal arrest and probably to tumor formation
A process common to cells that acts as a control means and may reflect the aging of an organism.
(Campisi 1996, Cell 84: 497-500). Brca2 impaired cells, and According to this, animals have been shown to exhibit the characteristics of accelerated aging, and are described here.
Cells and animals are also affected by biological aging and agonists to slow it down
It is considered useful for research.       [0021]   In particular, the proliferation and / or senescence abnormalities associated with Brca2 impaired cells are partially or Methods for screening for completely rescuing compounds, conditions, or compensatory mutations
Is contemplated. Such conditions include, but are not limited to, for example,
Overexpression of transfected gene or mutation of gene
And the like. Such compounds include, for example, peptides, peptides
Analogs, antisense or aptamer oligonucleotides, prostaglandins
And other organic molecules.       [0022]   As described above, in one embodiment of the present invention, two stains of the Brca2 gene Mouse cells containing a body allele, wherein at least one of said alleles
One is such that the cells produce Brca2 with impaired ability to associate with MmRad51.
Includes mutations. In a further embodiment of the invention, a Brca2 impaired cell is incorporated. Non-human animal embryos and non-human transgenic animals.       [0023]   As used herein, “brca2 impairment” refers to two wild-type Brca2 chromosome pairs.
At least one of the progeny genes is MmRad51 or any other associated with MmRad51.
Encodes Brca2 with impaired ability to associate directly or indirectly with the protein To be mutated. Brca2 impaired product It can be easily measured using standard molecular biology techniques. For example, reverse transcriptase
Modified Brca2 message by using elemental polymerase chain reaction (RT-PCR)
RNA levels can be measured. Therefore, the term brca2 impairment is
Homozygous and heterozygous genotypes are included, but homozygous genotypes are preferred.
Good.       [0024]   Mutations in the Brca2 gene are preferably direct or indirect with MmRad51.
Or all of the nucleotides encoding the domain that mediates the local association
Deletion mutations, substitution mutations, frameshift mutations, and / or
Alternatively, insertion mutations are included in the scope of the present invention. Substitution mutations are described in Hasty et al., 199.
1, associated with a stop codon or MmRad51, as described in Nature 350: 243-246.
Introduce other mutations near the region encoding the linked domain, or
Or indirectly truncated Brca2 protein product with impaired ability to associate with MmRad51
Prepared by site-directed mutagenesis to produce directly or indirectly
Can be Similarly, insertional mutations are introduced into the Brca2 gene at convenient Restriction sites, such as exon restriction sites, or exon sequencing and Brca2 gene And restriction mapping (Figure 1), and described in Hasty et al., 1991; Joyner et al., 1989. Using any of the other sites easily identified by the
You can enter. Another way to introduce insertions or other mutations is to use von
Brca2 locus as described in Melchner et al., Genes and Dev 6: 919-927.
Infects retroviruses that are integrated into, thereby mutating brca2 alleles Making a gene. However, the mutants of the invention have been produced.
Brca2 protein is impaired in its ability to directly or indirectly associate with MmRad51
DNA sequence encoding Brca2, such that a defective brca2 allele is produced Is preferably missing.       [0025]   The size of the coding region of the Brca2 gene is about 9984 bp. For the purposes of the present invention The nucleotide encoding the Brca2 gene is Genebank Accession # U65594. They are numbered accordingly. The deletion mutant is responsible for the Brca2 protein in this complex. Proper folding or substrate binding of proteins to MmRad51 or another protein Removal of the DNA fragment from the coding region of the Brca2 gene so that the
Therefore, it can be manufactured. The size of the deletion can vary. On the other hand,
One or two base pairs or any number of base pairs
Thus, a loss of activity may occur. In the latter case, polypeptide synthesis is frameshifted
Blocked by an induced stop codon, a truncated polypeptide can be produced.
For a general overview of mutagenesis and mutations, see "Introduction to Genetic Analysis" ("
An Introduction to Genetic Analysis "), 4th edition, 1989 (D. Suzuki, A. Griffiths
, J. Miller and R. Lewontin), W.H.Freeman & Co., N.Y., New York I want to be illuminated.       [0026]   Changing one base pair in the coding region of the brca2 gene is an amino acid change.
Can alter the proper folding of Brca2 protein if And may also be a mutation that can cause loss of Brca2 activity. The single amino acid change so created is responsible for the affinity of Brca2 for its substrate.
Can also be changed, thereby causing MmRad51 or Can also impair its association with another protein. On the other hand, the appropriateness of Brca2 messenger RNA
Deletions in the non-coding region of the Brca2 gene, which affect proper splicing Or other mutations can be made. With such a mutation, wild-type Brca2 Sudden excursion where all exons or some exons are missing compared to the message
Different Brca2 transcripts can be produced effectively. It also lacks non-coding regulatory regions. The expression of the Brca2 gene can be reduced. In addition, the Brca2 gene
Promoter sequences that reduce transcription can be deleted or altered, resulting in reduced transcription.
Tampering to impair the direct or indirect association between Brca2 and MmRad51
The quality of the material can be dysfunctional.       [0027]   Change the expression of a given gene by changing the codon usage in the gene
It can be changed. Changing this sort can increase or decrease the level of expression.
Conserve the amino acid sequence of the product while minimizing it.       [0028]   Antisense RNA transgenes allow partial or total expression of a particular gene May be used to knock out (Helen, C. and Toulme, J., 1990, Bio
chimica Bioshys. Acta 1049: 99; Pepin et al., 1991 Nature 355: 725; Stout, J.
And Caskey, T., 1990, Somat.Cell Mol. Genet. 16: 369; Munir et al., 1990 Soma.
t Cell Mol. Genet. 16: 383. Each is included herein by reference. ).       [0029]   "Antisense polynucleotide" refers to a reference such as (1) the sequence of the Brca2 gene.
Complementary to all or part of the target sequence, such as chromosomal locus mRNA
A polynucleotide that specifically hybridizes to a complementary target sequence. That's it
Such complementary antisense polynucleotides specifically hybridize to the relevant target sequence.
Substitution, as long as the dilation is maintained as a functional property of the polynucleotide.
It may include additions, deletions or transpositions. Complementary antisense polynucleotides
It can specifically hybridize to various mRNA species,
Prevent or prevent A processing and / or translation of the encoded polypeptide Including antisense capable (Ching et al., 1989, Proc. Natl. Acad. Sci. US
A 86: 10006-10010; Broder et al., Ann.Int.Med. 113: 604-618; Loreau et al., 1990,
FEBS Letters 274: 53-56; Holcenberg et al., WO91 / 11535; WO91 / 09865; WO91 / 04753
WO90 / 13641; and EP 386563. Included herein by reference Is). The antisense sequence is at least about 15 contiguous nucleotides in length, typically
Typically at least 20 to 30 nucleotides, preferably 30 nucleotides or more,
Polynucleotides substantially complementary to one or more target gene sequences in the vesicle Array. In some embodiments, the antisense sequence is a complementary target
May have substitutions, additions, or deletions relative to the sequence, but
As long as the polynucleotide is maintained, the polynucleotide is generally an antisense gene
Acts as an inhibitor.       [0030]   For the purposes of the present invention, the antisense sequence corresponds to the endogenous Brca2 target gene sequence. Complementary. In some cases, the sense sequence corresponding to the brca2 target region Can function to suppress expression, particularly by interfering with transcription. on the other hand,
Antisense polynucleotides generally suppress Brca2 expression at the post-transcriptional level I do.       [0031]   Antisense polynucleotides are used in cells to produce one or more polypeptides. Have been shown to inhibit the production of Transgenic animals.       [0032]   Antisense polynucleotides are available in transgenic pluripotent embryonic stem cells
Can be produced from the inserted heterologous expression cassette, and then described herein.
Brca2-impaired animals.       [0033]   The genetically modified animal cells of the present invention can be prepared using a number of techniques well established in the art.
It can be prepared accordingly. In particular, on November 7, 1995, -Capecchi and Thomas, incorporated herein by reference. U.S. Pat.No. 5,464,764 uses a technique conceptually similar to that taught in U.S. Pat. Can be. In general, Brca2 impaired cells can be manipulated using the following steps Can:   (1) A targeting vector comprising a cloning vector and a DNA fragment
And the DNA fragments are in the same 5 'to 3' In the non-adjacent regions of the two mouse Brca2 genes or genomic loci referenced At least one positive selectable marker gene (positive selection)
Selectable marker);   (2) The targeting vector contains a negative adjacent to one of the homology regions. This gene contains a negative selectable marker gene (negative selectable marker).
Selectable marker recovers desired homologous recombination events that delete portions of the Brca2 gene Increase the likelihood but not need it;   (3) Transfer wild-type Brca2 mouse cells with the targeting vector in step (2). Transfecting;   (4) The obtained marker in the transfected mouse cells of step (3)
Screening or selecting against the car (s); and   (5) Brca2 impaired mouse cells were expressed with the positive marker and
Screening from cells in step (4) that do not express the active selectable marker. When.       [0034]   Exact B that must be present in the targeting vector of step (1)
The rca2 gene or locus sequence is the sequence selected for deletion, and It will depend on the restriction nucleases used in genetic engineering of the mutation.       [0035]   The specific regions of homology required in step (1) are deleted in the targeting vector. Depends on the nature of the loss. Generally, the homology region used in the targeting vector
The size of the zone can be longer or shorter, but at least
Would also be about 400 bp. In general, to guarantee high targeting efficiency, It is preferable to use a homology region of approximately 1.5 kb or more. The target described in detail in FIG. In the setting vector, the 5 'and 3' homology regions on either side of the deletion are at least 1.5 kb. Was.       [0036]   The size of the deletion may also vary, and the homology region used in the targeting vector
It depends on the area. That is, non-adjacent regions of homology are used in the targeting vector.
The region of the wild-type allele located between the homology regions
Region that is deleted by homologous recombination with the vector. Deleted in the present invention
The area is Brca2^lex1Now about 2.5kb and Brca2^lex23.3kb; but positive
The exact size is not important, more or less deleted from the locus
And result in Brca2 deficiency. Deletion mutated Brca2 messenger
At least one exon or exon of the Brca2 gene so as to result in RNA
It is preferable to include a part of       [0037]   The specific positive and negative selectable markers used in the present invention are listed here.
It does not matter. Preferred positive and negative selectable markers are listed in Table 1.
You. The positive selectable marker should be located between the regions of homology,
The selectable marker is located outside the region of homology, if used, FIGS. 1a and 1b.
Should be 5 'or 3' of these regions as shown in Regions of homology
Should be in the same 5 'to 3' direction, but with positive and negative selection
The direction of the car is not important. It is not important to include a negative selectable marker,
, Which can increase the efficiency of targeting.       [0038]   The positive selectable marker is the transforming cell in which gene targeting is performed.
It should be genetically engineered to be functional in the cell. Positive and / or
Alternatively, the negative selectable marker may be a DNA sequence encoding these selectable markers. The phenotype expressed by the sequence is characteristic of the cell expressing the sequence.
Ability to provide positive or negative selection is functional in transformed cells
is there. How to make a positive selectable marker gene functional is important to the present invention
Not. Positive selection is a sub-selection that does not contain the integrated positive selection marker.
Can be achieved by killing the vesicles or exposing them to the appropriate drug of choice
You. Such drugs are listed in Table 1. The positive selectable marker gene is
It may have a promoter driving the present, or may have a positive selectable marker.
One may be driven by a transcription element proximal to the target locus. This latter one
The method requires that these transcription elements be active in the transformed cells.       [0039] [Table 1]      [0040]   Mutations that have been genetically engineered with a targeting vector In between, a DNA sequence, such as an oligonucleotide, in addition to the positive selectable marker A linker, can be included in place of the deleted Brca2 DNA. The oligonucleotide
Drinkers are usually 8-10 nucleotides in length, but longer, e.g., About 50 nucleotides, or short, e.g. 4, 5 or 7 nucleotides Is also good. The preferred length of the oligonucleotide linker is approximately 20-40 nucleobases in length.
Reotide. The DNA sequence of the oligonucleotide linker is not critical.       [0041]   Insert oligonucleotide between homology regions of targeting vector DNA The method used will depend on the type of oligonucleotide linker used. Oh
Paris containing one or more restriction nuclease cleavage sites in the lignonucleotide sequence
A double-stranded linker (New England Biolabs) is inserted by a known method. (Maniatis et al., 1982, Molecular cloning) ), Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.). pM
B9, pBR325, pKH47 (Bethesda Research Laboratories), oligonucleotide The linker can also be modified with a complementary homopolymer using a terminal transferase.
And can be inserted into plasmid DNA deletions (Maniatis et al., Supra. ). Alternatively, for example, complementary to the 3 'recesses and 3' overhanging cohesive ends of the truncated plasmid
By cross-linking of the oligonucleotide containing the terminus, i.e. via annealing
Followed by a gap complementary to the oligonucleotide sequence in a DNA polymerase (eg, For example, by filling with Klenow fragment), oligonucleotides
A linker can be inserted into the deletion in the plasmid. Then T4 DNA rigger
After the ligation reaction, the closed circular DNA molecule can be regenerated. If the deletion region is exo Once the targeting vector is designed to interrupt the oligonucleotide,
By judicious choice of tidlinker length and sequence, frameshift mutations
And / or a stop codon can be made in the mouse Brca2 gene and the mouse Brc
Increase the effect of deletion in the a2 gene.       [0042]   Single-stranded oligonucleotides “loop-out” the desired region of the target gene The use of leotide allows the simultaneous construction of specific deletions using site-directed mutagenesis.
Can be constructed and linker sequences inserted (Krogstad and Champoux, 1990
, J. Virol. 64 (6): 2796-2801, which is incorporated herein by reference. ).       [0043]   Mutations that have been genetically engineered with the targeting vector are added to the positive marker.
Instead, it contains a DNA sequence between the Brca2 gene homology, for example, a splice acceptor sequence.
Can be. Such a sequence facilitates ectopic splicing and creates a mutated message.
Make it.       [0044]   The DNA used as the homology region may be genomic DNA from the mouse-derived Brca2 locus. Or should be derived from a sequence flanking the Brca2 locus. DNA
The strain of mouse to induce is not critical, but preferably, gene targeting is performed.
It should be the same as the mouse strain derived from the cells to be administered. Gene target
By using DNA of the same quality as the cells to be used for the homology region, -The efficiency with which the targeting is achieved can be increased. Mouse homology region
Can be derived from a genomic library of DNA, and Divector, cosmid vector, plasmid vector, p1 phage vector, yeast
Various library vectors, such as mother artificial chromosome vectors, or other vectors
Can be cloned into Used in the targeting vector
Homologous regions are derived directly from genomic DNA using the polymerase chain reaction (PCR).
You can also. This method uses the sequence of the disclosed Brca2 gene (Sharan and And some of the knowledge of Bradley 1997, Genomics 40: 234-241). It becomes possible. Directly targeting vector of homology region thus derived
Can be subcloned into it.       [0045]   Positive selectable marker gene and optional flanking negative selectable marker
Targeting vector comprising two Brca2 homology regions separated by the same
Certain cloning vectors used in the present invention to construct
For example, as long as it contains a gene encoding drug resistance. like this
Examples of cloning vectors are pBR322 and vectors based on pBR322 (Sekiguchi,
 1983 Gene 21: 267), pMB9, pBR325, pKH47 (Bethesda Research Laboratories)
), PBR328, pHC79, phage Charon 28 (Bethesda Research Laboratories, Bo
ehringer Mannheim Biochemicals), pKB11, pKSV-10 (P-L Biochemicals), pM
AR420 (Otsuka, 1981) and oligonucleotide (dg) -processed pBR322 (Bethesda R
esearch Laboratories), pBluescript or a similar plasmid (Stratagene), p
Contains uc19 or a similar plasmid (New England Biolabs).       [0046]   Positive selectable marker gene and optional flanking negative selectable marker
Targeting vector comprising two Brca2 homology regions separated by the same
Indicates λ phage vector, cosmid vector, plasmid vector, p1 phage
Other clones, such as vectors, yeast artificial chromosome vectors, or other vectors
Cloning vector. Other choices are targeting
Vector components are prepared by PCR and synthesized with relative homology to each other. Restriction endonuclease cuts for ligation to ensure proper orientation of the sex region
By simply selecting the position, each component is simply ligated to its appropriate position, and
This is to ensure that the selective marker is located between the homology regions.       [0047]   Unique cloning sites other than those described in FIG. 1 that are useful in the present invention
The cloning vector containing the position is determined by restriction nuclease evaluation.
Can be Can be used to generate fragments containing the mouse Brca2 gene Other restriction nucleases that can be made, and thus other cloni that may be useful in the present invention
Vector is immediately evident from mouse Brca2 gene restriction enzyme cleavage site map Become. In fact, using multiple combinations of restriction endonucleases, Brca2 A Brca2 targeting vector for mutating offspring can be made. These regions of homology include the pBluescript series (Stratagene) and the puc series (Ne
w England Biolabs) or pGEM series (Promega)
It can be cloned into any of the Smids.       [0048]   The particular host used to propagate the targeting vector of the invention is important
Not. An example of such a host is E. coli K12 RRI (Bolivar et al., 1977, Gene 2: 9).
5); E. coli K12 HB101 (ATCC No. 33694); E. coli MM21 (ATCC No. 336780) And E. coli DH1 (ATCC No. 33849). A preferred host of the invention is DH5a
lpha (Life Technologies). Similarly, E. coli or Saccharo Tars that grow on Saccharomyces cerevisiae or both
Getting vector or plasmid vector that grows on B. subtilus Alternative vector / cloning systems can be used (Ure et al.,
 1983, Methods of Enzymology, "Recombinant DNA"
NA "), vol. 101, Part C, Academic Press, N.Y.).       [0049]   The particular mouse cell mutated in the present invention is not critical here, preferably
It is a somatic pluripotent cell. The term precursor is used when the pluripotent cells are prepared according to the invention
It is a precursor of the desired transfected pluripotent cells. Said versatility
Sex cells can be cultured in vivo to produce mutant mice (Evans et al., 1981,
Nature 292: 154-156). Examples of mouse cells that can be used in the present invention are AB1 or Comprises embryonic stem (ES) cells such as AB2.1, preferably a primary isolate of ES cells. ES Primary isolates of cells will generally be described for the EK.CCE cell line or ES cells.
Thus, it can be obtained directly from an embryo. The specific embryonic stem cells used in the present invention are
Not limited to this. Examples of such embryonic stem cells are AB 2.1, hprt^- Cell line, AB1, hpr
t⁺It is a cell line. Use other selectable markers listed in Table 1 for other stem cell lines
Can be.       [0050]   ES cells were obtained from Robertson (Robertson, 1987, In "Teratocarcinomas and embr
yonic stem cells: a practical approach " ), E. Robertson, ed. (Oxford: IRL Press), pp. 71-112).
Preferably on stromal cells such as STO cells and / or primary embryonic fibroblasts Cultured. Stromal (and / or fibroblast) cells are abnormal ES cell clones.
It plays a role in suppressing the growth of the protein.       [0051]   To obtain the Brca2 impaired mice of the present invention, Bradley (1987, In
 "Teratocarcinomas and embryonic stem cells: a practical approach"
Cancer and Embryonic Stem Cells: Practical Research Methods), E. Robertson, Ed. (Oxford: IRL Press), pp. 11
The mutant embryonic stem cells are injected into mouse blastocysts as described in 3-151).       [0052]   The particular mouse fibroblast used in the present invention is not critical here. like this
Examples of naive fibroblasts include C57BL6 mouse, C57BL6 white species, Swiss outbred, CFLP, M
Includes those derived from FI or other. Br made from injected blastocysts
Mice heterozygous for the ca2 mutant allele
For example, using a DNA probe for the above mutation (FIG. 1), Alternatively, it can be screened by PCR.       [0053]   The mutant mice of the present invention are intercrossed to control the mutation in the Brca2 gene. To obtain homozygous embryos and / or hybridize with other mouse strains.
Brca2 mutations can be transmitted to these other strains.       [0054]   The following example provides a more complete description of how to make and use the above invention.
The following is a description of the best mode intended to implement the various aspects of this invention.
stand. These examples are not intended as a way to limit the true scope of the invention, but rather as explanations.
Interpreted for presentation purposes.       [0055]     【Example】     6.0.Example   Embryonic stem cells are essentially derived from the disclosed methods (Teratocarcinomas and embryonics
tem cells: a practical approach, E. J.
 Robertson, eds., IRL Press, Washington, DC, 1987; Zjilstra et al., 1989, Na.
ture 342: 435-438; and Schartzberg et al., 1989, Science 246: 799-803, these
Each of which is incorporated by reference herein).
Was.       [0056]   The DNA cloning method is essentially as described in J. Sambrook et al., In Molec.
ular Cloning: A Laboratory Manual, 2nd edition,
 1989, and its periodic revisions, Cold spring Harbor Laboratory press, Cold Spr
ing Harbor, N.Y., which is hereby incorporated by reference).
Was implemented as follows.       [0057]   Oligonucleotides are available from Applied Bio System
) Oligonucleotide synthesizer according to the instructions provided by the manufacturer.
Synthesized.       [0058]     6.1.Cloning of mouse Brca2 gene   The mouse Brca2 gene was cloned from a mouse 129SvEv strain genomic library.
Was. More specifically, oligonucleotides based on the sequence and mouse cell-derived
Obtain a fragment of the Brca2 gene using reverse transcriptase polymerase chain reaction on RNA.
Was. The fragment of the mouse gene obtained in this manner was converted into a plasmid vector pBluescript SK + (S
tratagene). Release using a subclone of the Brca2 gene
A radiolabeled probe was made. Using the probe, the mouse 129SvEv strain genome lambda fur Phage containing homologous mouse genes by screening dilibrary
It was confirmed. Three positive phages were isolated, expanded and the DNA Restriction enzyme cleavage site mapping on the salt was performed by standard techniques.       [0059]     6.2.Construction of targeting vector   Construction of two targeting vectors to generate Brca2 impaired mice did. brca2 using pMB2TVhprt^lex1Allele (FIG. 1a) was created: the vector The mouse shows a 5.4 kb DNA homologous 5 'to exon 27 of the mouse Brca2 gene, and a mouse. Contains a 1.9 kb DNA homologous 3 'to exon 27 of the mouse Brca2 gene. This Also provides a marker for positive selection (hypoxanthine phosphoribosyl salt).
Transferase, HPRT, minigene cassette) and a marker for negative selection.
(Thymidine kinase, tk, gene).       [0060]   More specifically, mouse Brca2 Regions of homology upstream and downstream of the exons of the gene were used. Upstream homology region
It was isolated by Apa1 and SacI digestion (FIG. 1a), releasing an approximately 5.4 kb DNA fragment. Downstream phase
The homozygous region was isolated by HindIII and SmaI digestion (FIG. 1a) and an approximately 1.9 kb DNA fragment was Released.       [0061]   To prepare pMB2TVhprt, the coding nucleotides 942 from SacI to HindIII
The 2.5 kb genomic fragment containing 0-9984 was removed and replaced with a positive selectable marker I got it. To prepare positive-negative selection targeting vectors , A negative selection tk gene was added outside the 3 'homology region. A unique KpnI site
Used to cut the vector before transfection (FIG. 1a).       [0062]   brca2^lex2Allele preparation: The vector (pMB2TVneo) is the mouse Brca2 gene 4.6 kb DNA homologous 5 'to exon 26 of mouse, and exo of mouse Brca2 gene It contains a 1.9 kb DNA homologous 3 'to protein 27. This vector is also positive
Markers for selection (neomycin phosphotransferase cassette), and
And a marker (tk gene) for negative selection.       [0063]   More specifically, based on the generated restriction site map, mouse Brca
Exon 26 upstream of the two genes (including only a small fraction 5 'of exon 26) and mouse B
The homologous region downstream of exon 27 of the rca2 gene was used. The upstream homology region is Apa1 and
And Cla1 digestion (FIG. 1b), releasing an approximately 4.6 kb DNA fragment. Downstream homology domain
A region was isolated by HindIII and Sma1 digestion (FIG. 1a), releasing an approximately 1.9 kb DNA fragment. .       [0064]   To prepare pMB2TVneo, ClaI to HindIII coding nucleotide 9265-998
The 3.3 kb genomic fragment containing 4 was removed and replaced with a positive selectable marker.
To prepare a positive-negative selection targeting vector, a negative The Tiv selection tk gene was added outside the 3 'homology region. Using a unique KpnI site
The vector was cut before transfection (FIG. 1b).       [0065]     6.3.Transfection of mouse embryonic stem cells   Hprt homologous recombination between the targeting vector and the Brca2 genomic locus Performed on mouse embryonic stem cells that lack activity (see FIG. 1). More specific
Specifically, the positive-negative targeting vector obtained in Section 6.2 above
10 μg, 1 x 10⁷Transfected into 129SvEv mouse strain embryonic stem cells lacking Hprt activity HAT (hypoxanthine, aminopterin, thymidine) ) Select cells grown in selective medium and transfected with the targeting construct.
Brca2^lex1Alleles were created. Negative selection for tk gene Cells that have undergone a homologous recombination event at the Brca2 locus using the drug FIAU The choice was enhanced. Surviving colonies were from Ramirez-Solis, 1992, Anal
Biochem. 201: 331-336) as described by mini-Southern. However, this method uses the targeting vector and the Brca2 locus in the ES cell chromosome. Double reciprocal homologous recombination e
vent) to detect the Br at 3 'of the homology region of the targeting vector.
A fragment of DNA from the ca2 locus was used as a probe. Genomic DNA digested with BglII
And separated by electrophoresis. The desired recombination event is detected using a 3 'probe and the
Probes are compared to the 6.0 kb wild-type allele and 2.9 kb mutant allele and gene target for pMB2TVhprt vector The mutant allele of 9.4 kb was specified for pMB2TVneo after the printing. Many ES cells
The clone is approximately 2.5 kb after gene targeting with the pMB2TVhprt vector. Nome deletion and approximately 3.3 kb genomic deletion after gene targeting with pMB2TVneo
And was identified as a correct replacement event.       [0066]   Brca2^lex1Allele-targeted ES cell clones were subsequently cloned into Brca2^lex2 Alleles were targeted with the vector to create them. Tiger with 10 μg vector After transfection, select the cells in G418 selection medium and construct targeting
Brca2^lex2Alleles were created. medicine Applying negative selection to the tk gene using FIAU Enhanced selection of cells that have undergone a homologous recombination event. Surviving colonies were Ramire-Sori (R
amirez-Solis, 1992, Anal. Biochem. 201: 331-336). Screening using a targeting vector and ES cells.
To detect double reciprocal homologous recombination events between Brca2 loci in chromosomes, A DNA fragment from the Brca2 locus 3 'of the homology region of the
Used as a probe. ES cell genomic DNA for mini-Southern was digested with BglII ( Figures 1a, b). In addition, many of these target clones were mutated in both Brca2 alleles
Let me brca2^lex1/ brca2^lex2A composite heterozygote was made.       [0067]     6.4.Generation of Brca2-impaired mice and embryonic fibroblasts   The next genotype, brca2, obtained in Section 6.3 above^lex1/ +, Brca2^lex2/ +, Brca2^lex1/ brc
a2^lex2An ES cell clone representing Bradley, 1987, In "Teratocar
cinomas and embryonic stem cells: a practical approach
: Practical research method) ", E. Robertson, ed. (Oxford: IRL Press), pp. 113-151).
Injected into C57BL6 white host blastocysts as described. Pseudopregnancy of injected blastocysts
Transplanted into females, and mixed with agouti and albino coat colors (
Agouti from ES cell line and Agouti from wild-type host embryo)
Chimeric offspring were born. The chimeric male mouse was designated as wild-type C57BL6 white female
Agouti offspring were born, and this is the germline transmission of the ES cell components of chimeric mice.
Were successful and the C57BL6 white species / 129SvEv hybrid (called the C57BL6 / 129 hybrid)
Was obtained. At 3 weeks of age, the progeny from the chimeric cross was transformed with the mutant brca2
Alleles were screened as described below.       [0068]   Genomic DNA was isolated from the obtained mice. Then, 10μ of the obtained genomic DNA
g digested with BglII and used for Southern blot analysis using the above 3 'probe for mini Southern
To ES cells mutate alleles through the germline^lex1And brca2 ^lex2 Transmitted to both alleles. Male and female mice are mutant alleles
Heterozygosity was confirmed.       [0069]   Male and female mice heterozygous for the Brca2 mutant were crossed. Chimeric mice also background mutant alleles on the 129SvEv strain. 129SvEv strain mice were bred for the purpose.       [0070]     6.5.The brca2 ^lex1 mutation is not nearly zero   Analyze messenger RNA and brca2^lex2Whether the allele made the modified transcript
Was confirmed. One primer for exon 26 of mouse Brca2 gene and H
RT-PCR (reverse transcriptase-Po) using other primers for exon 3 of PRT minigene Remerase chain reaction). Sequence found in exon 27 of mouse Brca2 Is deleted and exon 26 sequence of mouse brca2 is fused to exon 3 of HPRT minigene
A fusion transcript was detected. Before the stop codon terminates translation, additional
The amino acid is encoded by the HPRT minigene (FIG. 1c).       [0071]     6.6.brca2 ^lex1 / brca2 ^lex2 complex heterozygous ES cells are hypersensitive to ionizing radiation Yes, but not sensitive to UV light   brca2^lex1/ brca2^lex2Combining heterozygous ES cells with two genotoxic factors, gamma irradiation And ability to repair damage caused by ultraviolet light (FIG. 2). gamma illumination
(Fig. 2a): wild type Hprt positive cells (1 clone) Raw Hprt deletion clone (3 clones), brca2^lex1/ + Cells (8 clones), brca2^{le x2} / + Cells (6 clones). No difference was seen between these clones, Were averaged. brca2^lex1/ brca2^lex2Eight clones of cells were observed and averaged. twenty five
Brca2 with 0 RADS, 500 RADS and 750 RADS^lex1/ brca2^lex2Surviving colonies are 2.5, 5, and 10 times less than control. About UV light (Figure 2b): control
brca2^lex1/ + Cells (3 clones) and brca2^lex2/ + Cells (2 clones).
No differences were seen between these clones, and their numbers were averaged. brca2^lex1/ brca2^{l ex2} Five clones of cells were observed and averaged. Control and brca2^lex1/ brca2^lex2cell Showed the same degree of sensitivity to ultraviolet light. Therefore, brca2^lex1/
brca2^lex2Hereditary traits indicate increased sensitivity to factors that induce DNA damage (γ-irradiation).
As shown, there is an increase in sensitivity to factors that induce pyrimidine dimers (ultraviolet light).
Not shown.       [0072]     6.7.brca2 ^lex1 / brca2 ^lex2 complex heterozygous embryonic fibroblasts have premature replicative senescence Receive   Mouse embryoid fibroblasts (MEFs) were analyzed for proliferation and longevity (FIG. 3). Control MEF
Was derived from 129SvEv embryos. brca2^lex1/ brca2^lex2MEFs were used for chimeric 15.5 day embryos (brca2 ^lex1 / brca2^lex2 Derived from Swiss Webster fed embryos injected with 129SvEv cells. Chimeric properties were confirmed by embryos with black eyes, as Swiss Webster is a white species. Was. brca2^lex1/ brca2^lex2Grow MEF for 10 days with G418 and select neo cassette expression And isolated from chimeric embryos. Control and brca2^lex1/ brca2^lex2The growth characteristics of MEF
It was measured. brca2^lex1/ brca2^lex2MEF contaminated with cells induced by Swiss embryos Were grown with or without G418 to ensure that they were not
There was no contamination since no differences in breeding were observed).       [0073]   The growth curve was determined at high density (8 x 10^FourCells / 3.5 cm plate) and brca2 ^lex1 / brca2^lex2Made for MEF. Control MEFs were brca2 at high density^lex1/ brca2^{lex Two} Proliferated slightly faster than MEF (Fig. 3a) and brca2^lex1/ brca2^lex2Is a shadow against growth It was shown that it was affected. MEFs are labeled with BrdU and stained with propidium iodide. The number of cells entering S phase at 48 hours was determined. Brca2 about 10-20% lower than control MEF^lex1/
brca2^lex2MEF enters S phase, higher percentage of brca2^lex1/ brca2^lex2MEF aging (Fig. 3b).       [0074]   Control and brca2 were grown at low density^lex1/ brca2^lex2Created for MEF
did. The difference in proliferation was significantly greater at low densities (FIG. 3c). Colonies (> 3 cells Total colonies (including all colonies) was counted, and when 5000MEF was spread on a 10cm plate, brc
a2^lex1/ brca2^lex2Colonies were 10 times less than control colonies. These colonies
-Determine the percentage of (> 15 cell colonies) for the colony size distribution (CSD) Was. CSD is an accurate indicator of cell longevity. Again brca2^lex1/ brca2^lex2MEF CSD
Was reduced by a factor of 10 compared to the control MEF (FIG. 3d).       [0075]   Control and brca2^lex1/ brca2^lex2The life of the MEF was determined (Fig. 3e). The CSD is brca2^lex1/ b
rca2^lex2Shows that MEFs age more quickly than control MEFs. MEF into 3 plates
Sowed (1 x 10^Five Cells / 3.5 cm plate). Passage them every 3.5 days to I sowed again. As the cell number decreases, the same cell number
Seeding was continued until there were no longer enough cells on one plate. this
At that point, the cells are considered to be senescent. brca2^lex1/ brca2^lex2MEF at 7-8 passages
Although aging, the control MEF was shown to be able to be passaged longer. In addition, one Control MEFs spontaneously immortalized. Like this brca2^lex1/ brca2^lex2MEF is an early onset complex Receive aging.       [0076]     6.8.Screening for Rescue of Proliferation / Aging Defects in Brca2-Depleted Cells G   Embryonic fibroblasts with Brca2 dysfunction rescue proliferation / early replication aging defects Will be used to screen for genetic mutations. Mutation is applied to various technologies
Therefore, the particular technique for making the mutation is not important. Make a mutation
An example of a method for treating cells with a DNA damaging agent, preferably (double-strand breaks Exposure to factors that do not result in double-strand breaks. other
The method is to infect a retroviral virus. Retro virus virus
Integration will introduce mutations.       [0077]   Other methods of rescuing the proliferation are ectopically in woven fibroblasts with impaired Brca2 function. Expressing the transgene. Various expression libraries can be used
The particular type of library is not important.   Other ways to rescue poor growth / early replication senescence phenotype impair Brca2 function To induce the overexpression of endogenous genes in certain fibroblasts. Various expressions
Libraries can be used, and the particular type of library is not important.       [0078]     6.9.B as an animal model for cancer and for testing the mutagenicity of toxic drugs Mice and cells with impaired rca2 function   Mouse and cells with impaired Brca2 function as a model system for carcinogenesis and inheritance Can be used to test the mutagenicity of toxic drugs. Brca2 impaired mice are cancer Onset and type can be observed. Brca2 impaired mice are p53 mutant mice
Mice with a known predisposition to cancer are bred to alter the onset of cancer or
You can observe the cuttle. Brca2-lesioned mice and cells are treated with various toxic Exposure to the agent may result in a spectrum of onset and tumorigenesis or cell survival, proliferation and
Can be tested for mutagenicity by observing and chromosomal damage
.       [0079]   All publications, patents and patent applications mentioned in this specification are incorporated herein by reference.
Incorporated by light. Various modifications and alterations of the described invention will be apparent to those skilled in the art.
is there. Although the invention has been described in connection with specific preferred embodiments, it is not intended to limit the scope of the invention.
It is understood that the present invention should not be unduly limited to such specific embodiments.
Should. Indeed, the methods, techniques and various modifications of cells and animals described above
Positive is intended to be within the scope of the following claims. [Brief description of the drawings]     FIG.   Figure 1: Targeting strategy for the Brca2 locus. A. Targeting Deletion of exon 27 (coding nucleotides 9420-9998) by the vector pMB2TVhprt
Lost. This target allele is brca2^lex1 Called. Upstream of HPRT selection cassette (5 ')
Is flanked by a 5.4 kb Apa1 / Sma1 genomic Brca2 fragment and 1.9 kb HindII downstream (3 ′).
2.5 kb flanking the I / SmaI genomic fragment, thus removing all of exon 27 of Brca2
A deletion is created. Positive selection, HPRT minigene; Negative selection, thymidine
Kinase (tk) cassette; plasmid backbone (pKS, Stratagene), wavy line
. Southern analysis is a BglII digestion. B. By targeting vector pMB2TVneo
Most of exon 26 and all of exon 27 (coding nucleotides 9265-9964)
) Deletion. This allele is brca2^lex2 Called. Neomycin phosphotran
Upstream (5 ') of the spherase (neo) selection cassette is a 4.6 kb Apa1 / Cla1 genome Brca2.
The fragments are adjacent, and the downstream (3 ') is adjacent to the 1.9 kb HindIII / SmaI genomic fragment, Thus, removing most of exon 26 and all of exon 27 of Brca2 3.3
A kb deletion occurs. Positive selection, neo cassette; negative selection, thymidine kinase (tk) Cassette; plasmid backbone (pKS, Stratagene), wavy line. Southern analysis Is BglII digestion.     FIG. 2   Figure 2: Control and brca2^lex1 / Brca2^lex2 Exposure of cells to genotoxic agents. Raw
Fraction (100% x number of colonies after exposure to genotoxic substance / genotoxic substance) Number of colonies that have not been exposed to
After 10 days, colonies were counted and measured. A. Dose response curve for gamma rays.
Controls were wild-type Hprt-positive cells (AB1, 1 clone) and wild-type Hprt-negative cells (AB2.2, 3 Clone), brca2^lex1 / + Cells (8 clones) and brca2^lex2 / + (6 black
). The same viable fraction was obtained by each of the three groups of control clones, The average is taken for this curve. brca2^lex1 / Brca2^lex2 Average of 8 clones of cells
did. B. Dose response curve for ultraviolet light. Three brca2^lex1 / + Cell clones
Two brca2^lex2 The average of the / + clones is shown for controls. 5 brca2^lex1 / Brca
Two^lex2 The average of cell clones is shown.     FIG. 3   Figure 3: brca2^lex1 / Brca2^lex2 Proliferative characteristics of embryonic fibroblasts. Mouse embryo fibroblasts
(MEF) was isolated from wild-type E15.5-day-old 129SvEv embryos and brca2^lex1 / Brca2^lex2 MEF
E15.5-day-old chimeric embryos (129SvEv cells injected into undifferentiated Swiss Webster germ cells
). brca2^lex1 / Brca2^lex2 Select MEFs in 90 mM G418 for 10 days
Were isolated from embryos with eyelid subcutaneous hemorrhage. Brca for all experiments
Two^lex1 / Brca2^lex2 MEFs were maintained with and without G418 selection (G41
The presence or absence of 8 did not affect proliferation). All experiments are 1 passage Start with the vesicle. MEF was purified with M10 (10% fetal calf serum from HyClone, Dulbecc from GibcoBRL)
o's modified Eagle medium, 2 mM L-glutamine, 49.5 U / ml penicillin and 38.8 μg
/ ml of streptomycin). A. Growth curve. 8 × 10^Four MEF 8
Plate on 3.5 cm plates and trypsinize individual wells of cells for 11 days. Counting during the period. B. Percentage of cells in S phase (%). MEF (4 × 10^Five ) On a 6cm plate for 2 days Growth. Continuous exposure of MEF to 10 μM 5-bromo-2'-deoxyuridine (BrdU)
And collected over a 48 hour period. Infiltrate cells, and use fluorescently labeled anti-BrdU antibody and
DNA was stained by exposure to propidium iodide. Fluorescence activated sorter (FACS) analysis Cells (2 × 10^Five Cells) and at each time point BrdU integrated cells (DNA synthesis fingers). Which is a benchmark and therefore an indicator of cell cycle progression)
It was measured. C. Colony formation in low density plating. 5000ME for each clone
Plate F on 10 cm plates (3 plates for each clone) and increase for 14 days Bred. Stain the colonies with crystal violet and count the number of colonies.
Was. Colonies are> 3 cells. D. Colony size distribution (CSD). > 15 cells The percentage of Ronnie is compared to the total number of colonies> 3 cells. E. FIG. Survival spa Measurement. The survival span is measured by the number of passages that the MEF could receive before growth stopped. It was decided by doing. MEFs were plated on three 6 cm plates (1 × 10^Five Fine Vesicle / plate). MEFs were trypsinized every 3.5 days and the total number of cells was counted. Next
Then, re-plate the MEFs on three 6 cm plates and plate on three plates
The operation continued until there were not enough MEFs to do so. Then, 1 × 10^Five 2 MEFs
Only on one plate, and finally on only one pre-auto plate . 1 × 10 cells remaining^Five If less than cells, MEF is considered completely senescent
. One clone of wild-type cells became naturally immortalized.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 C12N 15/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK) , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR) , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN , MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW F term (reference) 2G045 AA35 CB01 FA11 4B024 AA20 CA04 DA02 4B063 QA01 QA08 QQ08 QQ43 QQ58 QR32 QR55 QR62 QS25 QS35 4B065 AA91X AA91Y AB01 BA02 BA03 CA24 CA46

Claims (1)

Claims 1. A diploid animal cell comprising a genetically engineered mutation in at least one allele of the Brca2 gene. 2. The cell of claim 1, wherein the cell is homozygous for a mutant brca2 allele. 3. The cell of claim 1, wherein said mutation is a deletion mutation. 4. A diploid animal cell comprising a genetically modified mutation in the first allele of the Brca2 gene and another genetically modified mutation in the second allele of the Brca2 gene. 5. A non-human transgenic animal comprising a genetically engineered mutation that alters the expression or function of at least one allele of the Brca2 gene. 6. A mutant embryo progeny of the non-human transgenic animal according to claim 5. 7. The animal of claim 5, which is homozygous for said mutation in the Brca2 gene. 9. The animal of claim 5, wherein said mutation is a deletion mutation. 10. A step of: (A) growing the genetically modified Brca2-depleted cells and observing the cells that have not aged after 8 passages; (B) 3T3 or 3T3 in the cells that have not aged after 8 passages. Performing 3T9 or population doubling analysis to determine the span of survival of said cells; (C) observing the morphology of non-senescent cells after 8 passages for characteristics found in senescent versus proliferating cells; A method for screening for mutations that save the precocious replicative senescent phenotype of Brca2 impaired cells, comprising the step of identifying genomic alterations in the cells of step C. 11. The method of claim 10, wherein said method further comprises overexpression of a transfected or endogenous gene. 12. The method of claim 10, wherein said method further comprises ectopic expression of a transfected or endogenous gene. 13. The method, wherein the method comprises one or more brca2 mutations, or Br
Incorporate any mutation that results in loss or impairment of ca2 function into a genetic background that lacks negative regulation of the cell cycle, progresses through the cell cycle, or alters the span of survival of the cell or organism 2. The method of claim 1, further comprising:
The method according to 0. 14. The method of claim 10, wherein the method further comprises exposing the Brca2 impaired cells to a molecule, compound or peptide. 15. A method for screening a genetic property that increases the incidence of cancer in a Brca2 impaired animal, said property comprising: (A) overexpression of one or more transgenes; (C) ectopic expression of one or more transgenes; (D) ectopic expression of one or more endogenous genes; (E) one or more And (F) underexpression of one or more endogenous genes. 16. A method for screening a genetic property that reduces the incidence of cancer in a Brca2 impaired animal, said property comprising: (A) overexpression of one or more transgenes; (C) ectopic expression of one or more transgenes; (D) ectopic expression of one or more endogenous genes; (E) one or more And (F) underexpression of one or more endogenous genes. 17. A method for screening for a compound or molecule that increases the number of cancers in a Brca2 impaired animal, comprising exposing the Brca2 impaired animal to the compound and increasing the number of cancers in a Brca2 impaired animal. The method comprising the step of identifying 18. A method of screening for a compound or molecule that reduces the number of cancers in a Brca2 impaired animal, comprising exposing the Brca2 impaired animal to the compound and reducing the number of cancers in the Brca2 impaired animal. The method comprising the step of identifying