WO2006019153A1 - NONHUMAN ANIMAL WITH INHIBITED GdsmA GENE FUNCTION AND PROMOTED OR INHIBITIED CANCERATION-ASSOCIATED GENE FUNCTION - Google Patents

NONHUMAN ANIMAL WITH INHIBITED GdsmA GENE FUNCTION AND PROMOTED OR INHIBITIED CANCERATION-ASSOCIATED GENE FUNCTION Download PDF

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WO2006019153A1
WO2006019153A1 PCT/JP2005/015126 JP2005015126W WO2006019153A1 WO 2006019153 A1 WO2006019153 A1 WO 2006019153A1 JP 2005015126 W JP2005015126 W JP 2005015126W WO 2006019153 A1 WO2006019153 A1 WO 2006019153A1
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gene
mouse
gsdma
human
cell
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PCT/JP2005/015126
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French (fr)
Japanese (ja)
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Toshihiko Shiroishi
Masaru Tamura
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Inter University Research Institution Corporation, Research Organization Of Information And Systems
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Priority to JP2006531868A priority Critical patent/JP4742272B2/en
Publication of WO2006019153A1 publication Critical patent/WO2006019153A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases

Definitions

  • the present invention relates to a non-human animal and animal cell in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene is promoted or suppressed, and a compound that complements the phenotype of the non-human animal or the animal cell
  • the present invention relates to a screening method.
  • mutant mice are very powerful analysis methods for understanding human functions such as gene function, development, and cancer. Recently the present inventors have found that proliferation 'differentiation, hair formation' of epithelial cells focused on a playback, the analysis is conducted using mutant mice such Rim3, Re den, Re. R e is, at spontaneous mutant mice found by Crew and Auerbach in 1939, shows the phenotype of curled beard and hair (Non-Patent Documents:! ⁇ 2).
  • Re combinant-induced mutation-3 (Rim3) and Re den are spontaneous mutation mice found in 1983 and 1968 in the congeneric strains of 810.81? 228) and 810.129, respectively.
  • Non-Patent Documents 1, 3, and 4 Hyperproliferation, hyperkeratinization and hair loss. From the results of linkage analysis, Rim3 and Re den are considered to be alleles of Re (Non-Patent Document 4). These mutations are located on mouse chromosome 11 and in humans correspond to a closely related region of cancer called 17ql2 amplicon on chromosome 17.
  • Chromosome instability is well studied in human carcinogenesis. For example, deletion or amplification of part of a chromosome and translocation are phenomena that are prominently observed in cancer cells. These genome rearrangements are caused by DNA double strand break down, which may lead to amplification of oncogenes and loss of tumor suppressor genes (non-patented). Reference 5).
  • Non-Patent Document 3 Snell, GD, and Bunker, HP (1968) Mouse News Lett. 39. 28.
  • Non-Patent Document 4 Sato, H., Koide, T "Masuya, H” Wakana, S., Sagai, T ., Umezawa, Tsuji, Ishiguro, S., Tama, M., and Shiroism, T. (1998)
  • a new mutation Rim3 resembling R e (den) is mapped close to retinoic acid receptor alpha (Rara) gene on mouse chromo some 11. Mamm Genome.; 9 (1), 20-25.
  • Non-Patent Document 5 Cottle, A "Pipiras, E., Toledo, F., Buttin, G., and Debatisse, M. (1997). Expression of fragile sites triggers intrachromosomal mammalian gene amplification and sets boundaries to early amplicons. Cell 89, 215-225.
  • Non-Patent Document 6 van de Vijver, M, van de Bersselaar, R., Devilee, P., Cornelisse, C., Peterse, J, and Nusse, R. (1987). Amplification of the neu (c-erbB- 2) oncogene in h uman mammary tumors is relatively frequent ana is often accompanied by amplificati on of the linked c-erbA oncogene. Ol. Cell. Biol. 7, 2019-2023.
  • Non-Patent Document 7 Yokota, J., Yamamoto, T., Miyajima, N., Toyoshima, K., Nomura, N., Sakamoto, H., Yoshida, T., Terada, M, and Sugimura, T. ( 1988). Genetic alteratio ns of the c erbB-2 oncogene occur frequently in tubular adenocarcinoma of the sto mach and are often accompanied by amplification of the v-erbA homologue.Oncoge ne 2, 283-287.
  • Patent Document 8 Saeki, N., Kuwahara, Tsuji, Sasaki, H., Satoh H., and Shiroishi, T. Gas dermin (Gsdm) localizing to mouse chromosome 11 is predominantly expressed in up per gastrointestinal tract but significantly suppressed in human gastric cancer cells.
  • GsdmA3 a causative gene of Recombinant-induced mutation 3
  • GsdmA cluster a gene in the GasderminA cluster
  • PCT / JP0 3/02345 a dominant spontaneous mutant mouse that exhibits a phenotype of hyperkeratinization and hair loss.
  • the causative gene of this mutant mouse is GsdmA3, it is speculated that the function of GsdmA3 is deeply involved in cell growth and differentiation and hair formation in the epithelium.
  • the human homologous gene of the mouse GsdmA cluster is the human GasderminA (hereinafter referred to as GSDMA) gene present on human chromosome 17.
  • GSDMA human GasderminA
  • the mouse GsdmA cluster is composed of three genes, GsdmAl (GasderminA-1 GsdmA2 (GasderminA-2 GsdmA3), whereas in humans, only the GSDMA gene is present on chromosome 17.
  • Mouse chromosome 11 The region of GsdmA cluster in GSDMA and the region of human chromosome 17 GSDMA are very well conserved, especially the region where human chromosome 17 GSDMA is present, called 17ql2 plicon, skin cancer, esophageal cancer. In addition, it is known that genome amplification is observed in gastric cancer, breast cancer, etc., and it is thought to be deeply related to canceration.
  • the present inventors have clarified that the human GSDMA gene has the ability to suppress cancer in gastric cancer and esophageal cancer.
  • the tumor suppressive ability of the mouse GsdmA cluster gene was unknown (PCT / JP03 / 02345).
  • Gli_2 and A Lef-l transgenic mice are transgenic mice expressing the Gli_2 gene under the control of the keratin 14 promoter, and the N-terminal region of the Lef- ⁇ protein under the control of the keratin 14 promoter, respectively.
  • Trans for expressing the deficient gene ( ⁇ Lef-1) It is a dienic mouse.
  • Gli-2, A Lef-1 transgenic mice develop skin cancers in the ears, limbs, tail, and eye margins (Grachtchouk et al., Nat Genet, 24, 216-217, 2 000.; Niemann et al, Development, 129, 95-109, 2002.). These positions are all regions with little body hair. Based on this, the present inventors speculated that GsdmA3 or GsdmAl may suppress the occurrence of cancer in the region where hair is present in these transgenic animals.
  • the present invention has been made based on the above considerations, and an object of the present invention is to produce a non-human animal in which the function of the GsdmA gene exhibiting a cancer phenotype is suppressed. Is to provide the law.
  • the present inventors introduced a knockout allele of Trp53 (p53), which is known as a tumor suppressor gene, into Rim3 having a mutation in GsdmA3, so that GsdmA-3 Rim3 / +; TYp53-/ + individuals (Hereinafter referred to as Rim3 heterozygote ⁇ ⁇ 53 knockout heterozygous individuals).
  • p53 Trp53
  • TYp53-/ + individuals (Hereinafter referred to as Rim3 heterozygote ⁇ ⁇ 53 knockout heterozygous individuals).
  • Rim3 heterozygote ⁇ ⁇ 53 knockout heterozygous individuals As a result, multiple cases of squamous cell carcinoma occurred spontaneously from 7 months after birth, and similar squamous cell carcinomas were observed in more than 80% of individuals at 12 months of age. Furthermore, it was found that the cancer cells were transferred to lymph nodes, and the present invention was completed.
  • [1] A non-human animal in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene is promoted or suppressed.
  • the canceration-related gene is at least one gene selected from the group consisting of Trp53 gene, Rb gene, Ras gene, Cyclin_D gene, Cdk gene, TNF-a gene, and TGF- ⁇ gene.
  • [4] A mouse in which the function of at least one gene selected from the group consisting of GsdmAl gene, GsdmA2 gene and GsdmA3 gene is suppressed, and the function of canceration-related gene is promoted or suppressed.
  • Canceration-related genes are Trp53 gene, Rb gene, Ras gene, Cyclin_D gene, C
  • Canceration-related genes are Trp53 gene, Rb gene, Ras gene, Cyclin_D gene,
  • [12] A mouse cell in which the functions of the GsdmA3 gene and the ⁇ 53 gene are suppressed.
  • a protein functionally equivalent to the protein consisting of the amino acid sequence set forth in any one of SEQ ID Nos: 2, 4, 6, and 8.
  • FIG. 1 is a photograph showing the expression of GSDMA gene in a skin cancer cell line. As a result of investigating the expression of GSDMA gene in normal skin tissue and skin cancer tissue by RT-PCR, the expression of GSDMA gene disappeared in all cancer cell lines.
  • FIG. 2 is a schematic diagram of a method for producing a Rim3 heterozygous ⁇ 53 knockout heteromouse.
  • Rim3 heterozygous individuals GsdmA3 Rim3 / +
  • p53 knockout heterozygous individuals TYp53 — / +
  • FIG. 3 is a graph showing the onset of skin cancer in Rim3 heterozygous ⁇ 53 knockout heterozygous mice (GsdmA3 Rim3 / +; TYp53 ⁇ / +).
  • Rim3 heterozygous ⁇ 53 knockout heterozygous mice skin cancer spontaneously developed from 6 months of age. At 12 months of age, more than 80% (17/21) of Rim3 heterozygous ⁇ 53 knockout hetero mice were observed with multiple skin cancers. In mice of other genotypes, only Rim3 heterozygous (GsdmA3 Rim3 / +; TYp53 + / +) Skin cancer was seen, but the rate was as low as 10% (2/19). The n on the right of the graph indicates the number of individual genotype mice used in the experiment.
  • FIG. 4 is a photograph showing skin cancer in a Rim3 hetero- ⁇ 53 knockout hetero mouse.
  • ⁇ Image of Rim3 heterozygous ⁇ 53 knockout heteromouse that developed skin cancer. The white line in the figure shows the section plane at ⁇ .
  • ⁇ Whole image of skin cancer section.
  • C Enlarged view of C region in B (non-cancerous part).
  • D Enlarged view of D region in B (cancerous part).
  • Rim3 heterozygous ⁇ ⁇ 53 knockout heterozygous skin cancer showed a typical squamous cell carcinoma image.
  • E Rim3 heterozygous ⁇ ⁇ 53 knockout Skin cancer cells in heterozygous mice metastasize to regional lymph nodes, and epithelial structure in lymph nodes
  • FIG. 5 is a photograph showing the specificity of an anti-GsdmA / GSDMA polyclonal antibody.
  • the antigen specificity of the anti-GsdmA / GSDMA polyclonal antibody was verified by Western blotting. As a result, a single band was detected in the normal skin sample in the vicinity of the expected molecular weight of 50 k Dalton of GsdmA / GSDMA, indicating the anti-specificity of the anti-GsdmA / GSDMA polyclonal antibody.
  • A is a molecular weight marker and B is a normal skin sample.
  • FIG. 6 is a photograph showing the disappearance of GsdmA protein in Rim3 heterozygous ⁇ 53 knockout heteromouse skin cancer.
  • A is a non-cancerous part
  • C is a hematoxylin / eosin stained image of the cancerous part.
  • B and D are stained images with GsdmA antibody of adjacent sections of A and C, respectively.
  • non-cancerous B localization of GsdmA (1/3) protein is observed from spinous cells, whereas in typical squamous cell carcinoma cells (* area in Figures C and D) GsdmA (1/3) protein was lost.
  • FIG. 7 is a photograph showing the expression of GSDMA protein in human skin and skin cancer.
  • the GSDMA protein is expressed in normal skin (A) as well as normal hair (B), similar to the mouse GsdmA protein.
  • human squamous cell carcinoma cells (C) and basal cell carcinoma cells (D) the expression of GSDMA protein was lost, as in Rim3 hetero- ⁇ 53 knockout hetero skin cancer cells.
  • the present invention relates to a non-human animal in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene (oncogene and a tumor suppressor gene other than the GsdmA gene) is promoted or suppressed.
  • a non-human animal in which the function of the GsdmA gene is suppressed and the function of the oncogene is promoted, or a non-human animal in which the function of the GsdmA gene is suppressed and the function of the tumor suppressor gene is suppressed is provided.
  • the GsdmA gene in the non-human animal of the present invention is as follows: 1) If the non-human animal is a mouse, it is at least one selected from the group consisting of GsdmAl gene, GsdmA2 gene, GsdmA3 gene, and mutants thereof. Gene, 2) If the non-human animal is a non-human animal other than a mouse, a GsdmAl gene, a GsdmA2 gene, a GsdmA3 gene, or a homolog of the GSDMA gene can be exemplified. An example is at least one gene selected from the group consisting of the plurality of genes.
  • GsdmAl GsdmA2 GsdmA3 or GSDMA include the ability to suppress abnormal cell proliferation or differentiation, the ability to induce apoptosis, the ability to induce epithelial hyperproliferation, hyperkeratosis, and hair loss. Can be mentioned.
  • the biochemical functions of GsdmAl GsdmA2 GsdmA3 or GSDMA include the binding between the gas dermin families, the phosphorylation of GsdmA clusters by growth differentiation control proteins, modification of sugar chains, etc. Examples include localization to the vesicle membrane and secretion outside the cell.
  • the functions of a plurality of canceration-related genes may be promoted or suppressed.
  • the canceration-related genes of the present invention include those known to those skilled in the art, for example, as 3 ⁇ 4 regression gene, Ras (Harvey rat sarcoma virus oncogene (Hrasl): h-ras) (mouse : NM_008284, human: NM_005343) Cyclin-D (cyclin D l (Ccndl)) (mouse: NM_00 7631, human: BC000076), Cdk (cyclin-dependent kinase 2: Cdk2) (mouse: BC005654, human: BC003065), Tumor suppressor genes include TYp53 Rb (retinoblastoma 1 (Rbl): Retinobl astoma) (mouse: NM_009029, human: NM_000321) TNF_ (tumor necrosis factor) (mouse: NM_013693, human: NM_000594) TGF- ⁇ (transforming growth factor
  • the canceration-related gene in the non-human animal of the present invention is as follows. 1) If the non-human animal is a mouse, for example, mouse Trp53 gene, mouse Rb gene, mouse Ras gene, mouse Cyclin-D gene, Mouse Cdk gene, mouse TNF-a gene, mouse TGF- ⁇ gene, And at least one gene selected from the group consisting of these mutants, and 2) if the non-human animal is a non-human animal other than a mouse, for example, a gene such as the mouse Trp53 gene ( Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene, mouse TNF- ⁇ gene, mouse TGF- ⁇ gene, human ⁇ ⁇ 53 gene, human Rb gene, human Ras gene, human Cyclin-D gene , Human Cdk gene, human TNF-a gene, human TGF- ⁇ gene) at least one gene selected from the group consisting of homologs.
  • the mouse Trp53 gene Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene,
  • mouse ⁇ 53 or human ⁇ ⁇ 53 include the ability to suppress cancer (DNA) that is activated in response to various stresses such as DNA damage, telomere shortening, abnormal growth signals, and active oxygen. Repair, cell cycle arrest, induction of apoptosis, etc.).
  • the biochemical functions of mouse ⁇ 53 or human ⁇ 53 include binding to DNA and binding to other cancer-related genes.
  • Biological functions of mouse Rb or human Rb include cell cycle control, apoptosis control, transcriptional regulation, and biochemical functions include binding to DNA and binding to E2F protein.
  • Biological functions of mouse Ras or human Ras include cell cycle control, and biochemical functions include binding to GTP and GTPase activity.
  • Biological functions of mouse Cyclin-D or human Cyclin-D include cell cycle control and
  • Biochemical functions include binding to Cdk4 and Cdk6, and phosphorylation of Rb protein.
  • Biological functions of mouse Cdk or human Cdk include cell cycle control, and biochemical functions include binding to Cyclin-E and phosphorylation of Rb protein.
  • Biological functions of mouse TNF-a or human TNF-a include cell growth control, apoptosis induction, and biochemical functions include binding to a TNF receptor.
  • Biological functions of mouse TGF- ⁇ or human TGF- ⁇ include cell growth control, induction of apoptosis, and biochemical functions include binding to the TGF_j3 receptor.
  • non-human animal in the present invention means le, vertebrates and invertebrates including humans.
  • Non-human animals in the present invention include non-human mammals, birds, insects and the like. More preferably, it is a non-human mammal, and examples thereof include rodents such as mice and rats, sal, pigs, and rabbits.
  • the non-human animal of the present invention is useful as a model animal for cancer.
  • cancer include skin cancer, laryngeal cancer, tongue cancer, esophageal cancer, stomach cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, knee cancer, kidney cancer, lung cancer, melanoma, leukemia and the like. However, it is not limited to these.
  • mice in which the function of the GsdmA3 gene expressed in the skin is suppressed develops skin cancer.
  • mice in which the functions of the GsdmA3 gene and Trp53 gene are suppressed frequently spontaneously develop skin cancer frequently and metastasize to lymph nodes (Example 2).
  • the GsdmAl gene is expressed in the skin, esophagus, and frontal stomach (these are all squamous epithelia). Therefore, it is considered that mice in which the functions of the GsdmAl gene and the Trp53 gene are suppressed will develop cancer in the skin, esophagus, and foregutoma.
  • mice in which the functions of the GsdmA2 gene and TYp53 gene are suppressed are thought to develop gastric cancer.
  • mice in which the functions of multiple genes and TYp53 gene among GsdmAl, GsdmA2 and GsdmA3 are suppressed develop the above-mentioned various cancers.
  • the present invention is an animal cell in which the function of the GsdmA gene is suppressed and the function of the canceration-related gene is promoted or suppressed, that is, the function of the GsdmA gene is suppressed and the function of the cancer gene is promoted. Or an animal cell in which the function of a GsdmA gene is suppressed and the function of a cancer suppressor gene is suppressed. Such cells are useful as cancer model cells.
  • GsdmA in the animal cell of the present invention 1) if the animal cell is a human cell, at least one gene selected from the group consisting of a GS DMA gene and a mutant of the gene, 2) an animal cell Is a mouse cell, GsdmAl gene, GsdmA2 gene, GsdmA3 gene, and at least one gene selected from the group consisting of these mutants; 3) If the animal cell is a cell other than human or mouse, the GsdmAl gene GsdmA2 gene, GsdmA3 gene, or homolog of GSDMA gene can be exemplified, and when there are multiple GsdmA genes such as mice, at least one gene selected from the group consisting of the multiple genes can be exemplified . [0030] In the animal cell of the present invention, the function of one or more canceration-related genes may be promoted or suppressed.
  • the canceration-related genes in the animal cells of the present invention are as follows: 1) If the animal cells are human cells, human TYp53 gene, human Rb gene, human Ras gene, human Cyclin_D gene, human Cdk gene, human TNF-a gene Human TGF- ⁇ gene and at least one gene selected from the group consisting of these mutants; 2) if the animal cell is a mouse cell, for example, mouse Trp53 gene, mouse Rb gene, mouse Ras gene, Mouse Cyclin-D gene, mouse Cdk gene, mouse TNF-a gene, mouse TGF- ⁇ gene, and at least one gene selected from the group consisting of these mutant forces; 3) animal cells other than human or mouse For example, a gene such as mouse ⁇ 53 gene (in addition to mouse Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene, mouse TNF-a gene, mouse TGF-gene, human ⁇ 53 gene, human Rb gene, human Ras gene, human Cyclin-D gene, human Cdk gene, human TNF-a gene,
  • the present invention provides a method for suppressing the function of a GsdmA gene in a non-human animal or animal cell and promoting the function of an oncogene, or suppressing the function of a GsdmA gene, and the function of a cancer suppressor gene.
  • a method of suppressing is provided. Cancer is thought to develop by accumulating mutations in many canceration-related genes. Therefore, non-human animals or animal cells can be cancerized by suppressing the function of the GsdmA gene and promoting or suppressing the function of the canceration-related gene.
  • the base sequences of DNAs encoding GsdmAl, GsdmA2, GsdmA3, GSDMA, mouse Trp53 and human Trp53 are represented by SEQ ID NOs: 1, 3, 5, 7, 9 and 11 respectively.
  • the amino acid sequences of the proteins encoded by are shown in SEQ ID NOs: 2, 4, 6, 8, 10 and 12, respectively.
  • GsdmAl, GsdmA2, GsdmA3, GSDMA, mouse T53, and human cho 53 mutants, variants, and homologs are shown in the following (a) or (b).
  • “functionally equivalent” refers to biological functions equivalent to the target protein strength GsdmAl, GsdmA2, GsdmA3, GSDMA, mouse TYp53, or human ⁇ 53 (biological harm ⁇ biology) Property), biochemical function (biochemical activity).
  • mouse Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene, mouse TNF-a gene, mouse TGF- ⁇ gene, human Rb gene, human Ras gene, human Cyclin Mutants, variants and homologues of the -D gene, human Cdk gene, human TNF-a gene, and human TGF- ⁇ gene are also defined as described above.
  • GsdmAl, GsdmA2, Gsdm A3, GSDMA mouse T ⁇ 53, mouse Rb, mouse Ras, mouse Cyclin_D, mouse Cdk, mouse TNF-a, mouse TGF- ⁇ , human Trp53, human Rb,
  • a DNA sequence that encodes human Ras, human Cyclin_D, human Cdk, human TNF-s, human TGF-j3, or a portion thereof, or a part thereof, is used to singly homologous DNA. Separation is a well-known technique.
  • the present invention includes a DNA that hybridizes with a DNA encoding GsdmAl or the like under stringent conditions and encodes a naturally derived protein that is functionally equivalent to GsdmAl or the like.
  • Hybridization conditions for isolating a DNA encoding a naturally occurring protein functionally equivalent to GsdmAl or the like can be appropriately selected by those skilled in the art.
  • the hybridization conditions include low stringency conditions.
  • Low stringency conditions are often associated with post-hybridization washing, for example 42 ° C. 5xSSC, 0.1% SDS, preferably 50.
  • the conditions are C, 5xSSC, and 0.1% SDS.
  • Highly stringent conditions can be cited as the conditions for the more preferred and noisy hybridization.
  • Highly stringent conditions are, for example, conditions of 65 ° C, O.lxSSC and 0.1% SDS. Under these conditions, it can be expected that DNA with high homology will be obtained efficiently as the temperature is increased. However, multiple factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art will realize the same stringency by appropriately selecting these factors. It is possible.
  • a gene amplification method using a primer synthesized based on the sequence information of DNA encoding GsdmAl or the like for example, a natural equivalent functionally to GsdmAl or the like using a polymerase chain reaction (PCR) method. It is also possible to isolate the DNA encoding the protein of origin.
  • PCR polymerase chain reaction
  • Naturally-derived proteins functionally equivalent to GsdmAl and the like encoded by DNA isolated by these hybridization and gene amplification techniques usually have high homology in amino acid sequence with GsdmAl and the like.
  • the protein of the present invention includes naturally derived proteins that are functionally equivalent to GsdmAl and the like and have high homology with the amino acid sequence of the protein.
  • High homology generally refers to at least 50% identity, preferably 75% identity, more preferably 85% identity, more preferably 95% identity at the amino acid level. In the base level, it usually means at least 50% identity, preferably 75% identity, more preferably 85% identity, more preferably 95% identity.
  • proteins that are composed of amino acid sequences in which one or more amino acids are mutated in the amino acid sequence such as GsdmAl and are functionally equivalent to the protein are also included in the protein of the present invention.
  • amino acid mutations can also occur in nature.
  • the number of amino acids to be varied is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids (for example, within 3 amino acids), and even more preferably within 2 amino acids.
  • amino acid residue to be mutated should be mutated to another amino acid that preserves the properties of the amino acid side chain.
  • the properties of amino acid side chains include hydrophobic amino acids (A, I, M, F, P, W, Y, V), hydrophilic degenerate amino acids (R, D, N, C, E, Q, G , H, K, S, T), amino acids having an aliphatic side chain (G, A, V, L, I, P), amino acids having a hydroxyl group-containing side chain (S, T, ⁇ ), sulfur atom Amino acids with side chains (C, M), amino acids with side chains containing carboxylic acids and amides (D, N, E, Q), amino acids with side chains (R, K, ⁇ ), aromatics Amino acids having side chains (H, F, Y, W) can be mentioned (the parentheses indicate single letter amino acids).
  • the functional suppression of the GsdmA gene and the tumor suppressor gene in the present invention includes both complete suppression and partial suppression.
  • suppression of gene function includes suppression of the function of one or both of a gene pair.
  • Suppression of gene function can be performed by suppressing gene expression and introducing a mutation into the gene.
  • a mutation There is no restriction on the type, number, site, etc. of the mutation. Examples of the type of mutation include addition, deletion (which may be complete deletion or partial deletion), substitution, or insertion mutation.
  • mutations may be mutations that occur in nature or artificially introduced mutations (for example, mutations caused by mutagen treatment, Mutations introduced by genetic modification techniques), or even deviations.
  • non-human animal of the present invention a non-human animal having a mutation in one of the gene pair of GsdmA gene and T ⁇ 53 gene (hereinafter referred to as mutant GsdmA hetero-mutant Trp53 hetero-non-human animal) ), A non-human animal having a mutation in one of the GsdmA gene pairs and a mutation in both of the Trp53 3 gene pairs (hereinafter referred to as mutant GsdmA hetero'mutant Trp53 homo non-human animals), GsdmA A non-human animal having a mutation in both gene pairs and a mutation in one of the TYp53 gene pairs (hereinafter referred to as mutant GsdmA homo'mutant Trp53 hetero-non-human animal), GsdmA gene and TYp53 gene Non-human animals having mutations in both gene pairs (hereinafter referred to as mutant GsdmA homo'mutant ⁇ ⁇ 53 homo non-human animals).
  • Mutant GsdmA hetero-mutant Trp53 hetero non-human animal mutant GsdmA hetero 'mutant Trp 53 homo non-human animal, mutant GsdmA homo' mutant Trp 53 hetero non-human animal, mutant GsdmA homo.
  • Mutant TYp53 Homo non-human animals can be obtained by mating the following (i) to (iv) and mating individuals obtained by this mating.
  • a non-human animal having a naturally occurring mutation and a non-human animal having an artificially introduced mutation, and a non-human animal having a naturally occurring mutation are crossed. Appropriate selection of mating between non-human animals with artificially introduced mutations You can choose.
  • Suppression of the function of a specific gene can be performed by methods known to those skilled in the art.
  • a method using a gene modification technique including a conditional gene modification technique by introducing an enzyme that promotes recombination of a target gene site, for example, Cre in Cre-lo X
  • a method using an antisense DNA examples include a method using DNA encoding a ribozyme, a method using RNAi technology, and the like.
  • the production of a non-human animal in which the function of a gene is suppressed can be performed, for example, as follows. First, a genomic DNA fragment containing the target gene is cloned, and based on this, a vector for homologous recombination for endogenous target gene modification is constructed.
  • the homologous recombination vector has a nucleic acid-ligated IJ in which at least a part of the target gene or its expression control region has been deleted / mutated, and a nucleotide or polynucleotide inserted into the target gene or its expression control region.
  • the nucleic acid sequence IJ includes a nucleic acid sequence in which another gene is inserted into the target gene or its expression control region, but the deletion / mutation site and insertion site described above can be used as long as the target gene activity is lost. It ’s not limited.
  • inserted genes include neomycin resistance gene, thymidine kinase gene, diphtheria toxin gene and the like. Combinations of these are also possible.
  • pKO Scrambler Series Longet al.
  • the constructed vector for homologous recombination is introduced into a non-human animal cell (for example, embryonic stem cell (ES cell)) capable of differentiating into an individual, and homologous recombination with an endogenous target gene is performed.
  • a non-human animal cell for example, embryonic stem cell (ES cell)
  • ES cell embryonic stem cell
  • the cell in which the expression of one side of the gene pair is suppressed it can be obtained by, for example, a method of selecting a cell with neomycin.
  • the vector for homologous recombination can be introduced into cells by methods known to those skilled in the art. Specifically, the ability to exemplify the Elect Mouth Position Method etc. can be demonstrated.
  • ES cells are used as animal cells that can be differentiated into individuals in the present invention
  • chimeric embryos are produced by injecting the cells into blastocysts and pseudopregnant animals. Transplant into the uterus of the baby to obtain a litter. Chimeric motion with tissue derived from genetically modified ES cells More preferably, the external characteristics (eg, hair color) of the created individual are different between the tissue derived from the genetically modified ES cell and the tissue derived from the blastocyst. Select a blastocyst.
  • the determination of whether or not a chimeric animal has a reproductive tissue derived from a genetically modified ES cell is generally obtained by crossing the chimeric non-human animal with an appropriate non-human animal of the same strain.
  • Other methods include, for example, performing a PCR reaction using the DNA extracted from the tail or ear of the chimeric non-human animal as a cage, and the presence or absence of the inserted gene. It is also possible to use a method for detecting selenium or a Southern hybridization method.
  • the pup power obtained by mating the chimeric animal with the same species of the same species is heterozygous. For example, whether or not it is a genetically modified non-human animal can be determined by analyzing DNA extracted from the genetically modified animal. It can be determined by PCR or Southern hybridization. In addition, homozygous genetically modified non-human animals can be produced by crossing heterozygous genetically modified non-human animals. Whether or not the offspring obtained by mating is a non-human animal having a desired genotype also follows the above-mentioned determination method.
  • the production of the genetically modified non-human animal of the present invention is not limited to the above method.
  • an animal can be produced according to a technique for producing a somatic cell cloned animal.
  • animal cells can be produced using somatic cells other than ES cells (for example, skin cells) according to the same method as for ES cells.
  • a non-human animal can be produced from the non-human animal cell by applying a somatic cell clone production technique.
  • the production of a non-human animal in which the function of a gene is suppressed can also be performed by using a DNA encoding an RNA complementary to the transcription product of the gene of the present invention.
  • DNA includes antisense DNA.
  • the antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above.
  • designing an antisense probe lj complementary to the untranslated region near the 5 ′ end of the mRNA of a gene would be effective for inhibiting translation of the gene.
  • the DNA containing the antisense sequence of the sequence of the untranslated region as well as the translated region of the gene is also included in the antisense DNA used in the present invention.
  • the antisense DNA to be used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.
  • the DNA thus prepared can be transformed into a desired cell by a known method.
  • the sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene or a part of the cell to be transformed, but it must be completely complementary as long as the gene expression can be effectively inhibited. Also good.
  • the transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene.
  • the length of the antisense DNA is at least 15 bases, preferably 100 bases or more, more preferably 500 bases or more. is there. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.
  • RNA complementary to the transcription product of the gene of the present invention is dsRNA complementary to the transcription product of the gene of the present invention.
  • RNAi is a phenomenon in which the expression of the introduced foreign gene and target endogenous gene is suppressed when double-stranded RNA (hereinafter referred to as dsRNA) having the same or similar sequence as the target gene sequence is introduced into the cell. .
  • siRNA short interference RNA
  • RISC RNA-induced silencing complex
  • the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. It is also considered that this dsRNA becomes a dicer's base again, generating a new siRNA and amplifying the action.
  • RsRP RNA-dependent RNA polymerase
  • the RNA of the present invention is expressed from an antisense coding DNA encoding an antisense RNA for any region of the target gene mRNA and a sense coding DNA encoding a sense RNA of any region of the target gene mRNA. Can be made. DsRNA can also be prepared from these antisense RNAs and sense RNAs.
  • antisense RNA and the sense RNA are expressed from the same vector and the antisense RNA and the sense RNA are expressed from the same vector, respectively. May be expressed.
  • antisense RNA and sense RNA can be expressed from the same vector by combining an antisense RNA and a promoter capable of expressing a short RNA such as ⁇ upstream of the sense code DNA. It can be constructed by constructing an expression cassette and a sense RNA expression cassette, respectively, and inserting these cassettes into the vector in the same direction or in the opposite direction.
  • RNA-encoding DNA double-stranded DNA
  • antisense RNA coding strand and a sense RNA coding strand are paired
  • antisense RNA and sense RNA are connected from each strand to both sides.
  • a promoter is provided oppositely so that it can be expressed.
  • a terminator is added to the 3 'end of each strand (antisense RNA coding strand, sense RNA coding strand).
  • Each is preferably provided.
  • this terminator a sequence in which four or more A (adenine) bases are continued can be used.
  • the two promoter types are preferably different.
  • a short promoter such as a ⁇ ⁇ system, which can express RNA
  • a short promoter such as a ⁇ ⁇ system, which can express RNA
  • It can be constructed by constructing a linked antisense RNA expression cassette and sense RNA expression cassette, and holding these cassettes in different vectors.
  • siRNA may be used as dsRNA.
  • siRNA means a double-stranded RNA consisting of short strands that are not toxic in the cell, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably It can be 21-30 base pairs.
  • the siRNA to be expressed is transcribed and the length of the final double-stranded RNA portion is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, more preferably 21 to 30 base pairs. can do.
  • the DNA used for RNAi need not be completely identical to the target gene, but has at least 70%, preferably 80%, more preferably 90%, most preferably 95% or more sequence homology. .
  • the double-stranded RNA part of the dsRNA in which RNAs are paired with each other is not limited to a perfect pair, but includes mismatches (corresponding bases are not complementary), bulges (corresponding to one strand) An unpaired portion may be included due to the base that is not used. In the present invention, both bulges and mismatches may be contained in the double-stranded RNA region where RNAs in dsRNA pair with each other.
  • Ribozyme refers to an RNA molecule having catalytic activity.
  • ribozymes that have various activities, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for the purpose of site-specific cleavage of RNA.
  • Some ribozymes have a group I intron type or a force hammerhead type or hairpin type having an active domain of about 40 nucleotides such as M1RNA contained in RNaseP having a size of 400 nucleotides or more.
  • the self-cleaving domain of the hammerhead ribozyme cleaves on the 3 'side of C15 of G13U14C15, but it is important for U14 to base-pair with A at position 9 for activity. It has been shown that the base of can be cleaved by A or U in addition to C.
  • the ribozyme substrate binding site is designed to be complementary to the RNA sequence in the vicinity of the target site, a restriction enzyme-like RNA cleavage ribozyme that recognizes UC, UU, or UA in the target RNA will be created. It is possible to do.
  • Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (J.M.Buzayan Nature 323: 349, 1986). This ribozyme has also been shown to be designed to cause target-specific RNA cleavage.
  • a ribozyme designed to cleave the target is linked to a promoter such as the cauliflower mosaic virus 35S promoter and a transcription termination sequence so that it is transcribed in plant cells.
  • ribozyme activity may be lost if extra sequences are added to the 5 'or 3' end of the transcribed RNA.
  • another trimming ribozyme that acts as a cis for trimming is provided on the 5 ′ side or 3 ′ side of the ribozyme portion.
  • the functions of the GsdmA gene and the tumor suppressor gene are suppressed by introducing the above-mentioned DNA that suppresses gene expression into a desired non-human animal using transgene technology. Human animals can also be produced.
  • Transgenic animals can be obtained by the method described in SA 77: 7380-7384 (1980). Specifically, the above-mentioned DNA that suppresses gene expression is introduced into a totipotent cell of an animal, and this cell is generated into an individual. By selecting individuals from which the transgene has been incorporated into somatic cells and germ cells, the desired transgeneic animal can be produced. Examples of totipotent cells into which genes are introduced include fertilized eggs and early embryos, and cultured cells such as ES cells having multipotency. A person skilled in the art can appropriately modify the above method to produce a transgenic animal in which the expression of a desired gene is modified.
  • the DNA that suppresses gene expression is generally a recombinant gene construct (expression vector) linked to a promoter that can be expressed in the cells of an animal into which the DNA is to be introduced.
  • the recombinant gene construct of the present invention can be constructed by inserting the above-described DNA that suppresses gene expression into a vector that can be cloned using an appropriate host and a promoter upstream thereof and cloning. it can.
  • the promoter that can be used in the present invention is not particularly limited as long as it is a promoter that can be expressed in animal cells.
  • promoters derived from mammalian cells, cytomegalovirus, retrovirus, polio Examples include virus promoters such as mavirus, adenovirus, and simian virus 40 (SV40).
  • virus promoters such as mavirus, adenovirus, and simian virus 40 (SV40).
  • the above construct can be prepared according to the method of Mulligan et al. (Nature (1990) 277, 108).
  • any expression vector known to those skilled in the art may be used as long as it can induce expression of a transgene in a wide range in an animal body. it can.
  • the recombinant gene construct excised from the vector with an appropriate restriction enzyme is sufficiently purified and used to produce a transgenic animal.
  • a transgenic animal is produced by introducing the construct into an unfertilized egg, a fertilized egg, a sperm and a germ cell containing a primordial cell thereof.
  • the cells into which the construct is introduced are usually used at the stage of embryonic development in the development of non-human mammals, more specifically at the stage of single cells or fertilized egg cells, and usually those before the 8-cell stage.
  • the calcium phosphate method for example, the calcium phosphate method, the electopore position method, the repofusion method, the aggregation method, the microinjection method, the particle gun method, the DEAE-dextran method and the like are known. Furthermore, it is also possible to produce a transgenic animal by fusing the thus obtained transformed cell with the above-mentioned embryo cell.
  • a fertilized egg capable of introducing a construct can be collected.
  • the construct is generally introduced by microinjection into the male pronucleus.
  • the cells into which the construct has been introduced are cultured outside the body, and then the cells that appear to have been successfully introduced are transplanted into the surrogate mother's fallopian tube, and a transgenic chimeric animal is born.
  • a surrogate mother is usually a female that has been pseudopregnant by mating with males that have had their vagina cut.
  • Transborn chimeric animals are bred with normal animals for the birth of F1 animals after confirming that the DNAs that suppress the above gene expression have been incorporated.
  • foreign DNA introduced as a construct has multiple copies integrated in series in the same part of the genome.
  • the higher the number of integrated copies the greater the expression of the gene, and a clearer phenotype can be expected.
  • the incorporation of DNA that suppresses the above gene expression in the somatic cell genome can be confirmed by PCR using a primer specific for the construct or Southern blotting using a specific probe. It is out.
  • heterozygotes Among F1 animals born as a result of this mating, those that have foreign genes in the somatic cells (DNA that suppresses the above gene expression) (heterozygotes) can transmit foreign genes to germ cells. Transgenic animal. The ability to obtain homozygotes that are F2 animals can be obtained by selecting F1 animals that have foreign genes in somatic cells and using them as parents.
  • non-human kinetics in which the function of an oncogene is promoted by introducing DNA encoding the oncogene into a desired non-human animal using transgene technology. Things can also be made.
  • the function of the GsdmA gene is obtained by introducing DNA encoding an oncogene into a non-human animal in which the function of the GsdmA gene is suppressed, using transgene technology. It is also possible to produce non-human animals in which the function of oncogenes is promoted.
  • the present invention relates to an animal cell in which the function of the GsdmA gene is suppressed and the function of the canceration-related gene is promoted or suppressed (an animal in which the function of the GsdmA gene is suppressed and the function of the oncogene is promoted).
  • the biological species from which the animal cell of the present invention is derived is a cell derived from various biological species including humans without particular limitation.
  • the animal cell of the present invention is not limited to these, including, for example, a somatic cell, a fertilized egg, an ES cell, and a cell established from the non-human animal of the present invention.
  • a method for establishing the cell line derived from the non-human animal a known method can be used.
  • rodents it is possible to use a method of primary culture of fetal cells (Neochemistry Laboratory, 18 pp. 125-129, Tokyo Chemistry Doujin, and Mouse Embryo Operation Manual, 262 Pp. 264, modern publication).
  • the present invention provides a screening method for a compound that complements the phenotype of the non-human animal of the present invention.
  • test sample in the method of the present invention is not particularly limited, for example, a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, a peptide, or an antibody, and a compound library, gene library, antibody Examples include library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts. .
  • the test sample can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.
  • the first aspect of the screening method of the present invention relates to the screening of compounds that complement the phenotype of the non-human animal of the present invention.
  • a test compound is administered to the non-human animal of the present invention. Examination of non-human animals of the present invention Administration of the compound can be performed, for example, orally or parenterally, but is not limited thereto.
  • the test compound is a protein, for example, a viral vector having a gene encoding the protein can be constructed, and the gene can be introduced into the non-human animal of the present invention using its infectivity. Is possible.
  • Non-human animal phenotypes include cancer (eg, skin cancer, laryngeal cancer, tongue cancer, esophageal cancer, gastric cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, knee cancer, kidney cancer. Onset, progression, metastasis, etc. of lung cancer, melanoma, leukemia etc., especially onset, progression, metastasis, etc. of skin cancer.
  • examples of complementation of the phenotype of non-human animals include suppression of cancer onset, progression, metastasis and the like.
  • the second embodiment of the screening method of the present invention relates to screening for compounds that complement the phenotype of animal cells of the present invention.
  • the test compound is brought into contact with the animal cell of the present invention.
  • contact can be performed, for example, by adding a test sample to a cell culture medium.
  • the test sample is a protein
  • a vector containing DNA encoding the protein can be introduced into the cell.
  • test compound complements the phenotype of the animal cell.
  • a compound that complements the phenotype of the animal cell is selected as compared to the case where the test compound is not administered.
  • Examples of the cell phenotype of the present invention include abnormal growth, abnormal differentiation, abnormal adhesion, and enhanced mobility.
  • complementation of cell phenotype includes suppression of proliferation abnormality, differentiation abnormality, adhesion abnormality, enhancement of mobility and the like.
  • the present invention binds to GsdmAl, GsdmA2, GsdmA3, GSDMA, or mutants, variants, homologs (hereinafter referred to as GsdmAl, GsdmA2, GsdmA3, or GSDMA) of these proteins, or fragments thereof.
  • An antibody is provided.
  • the antibody of the present invention can be used for detection of the protein in cancer screening, cancer research, and the like.
  • the anti There are no particular restrictions on the type of body. For example, both monoclonal antibodies and polyclonal antibodies can be used.
  • the antibody can be prepared by methods known to those skilled in the art.
  • a polyclonal antibody can be obtained as follows.
  • GsdmAl, GsdmA2, GsdmA3, GSDMA, and the like, and recombinant proteins expressed in microorganisms such as Escherichia coli as a fusion protein of these with GST, or a partial fragment thereof, are immunized to small animals such as rabbits to obtain serum.
  • This is prepared, for example, by purifying with ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, GsdmAl, GsdmA2, GsdmA3, GSDMA, etc., or a affinity ram with a synthetic peptide coupled. .
  • a monoclonal antibody for example, GsdmAl, GsdmA2, GsdmA3, or GSDMA or a partial fragment thereof is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to isolate cells.
  • the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is purified by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography.
  • GsdmAl, GsdmA2, GsdmA3, GSDMA or the like, or a purification column coupled with a synthetic peptide can be used for the preparation.
  • the protein fragment is a fragment described in the examples, or a C-terminal region such as GsdmAl, GsdmA2, GsdmA3, or GSDMA.
  • a C-terminal region such as GsdmAl, GsdmA2, GsdmA3, or GSDMA.
  • Examples of such a region include the 343rd to 343th amino acids of GsdmA3. 451st, amino acids 334 to 442 of GsdmAl, amino acids 352 to 460 of GsdmA2, amino acids 333 to 441 of GSDMA, and the like, but not limited thereto.
  • C57BL / 6J and C57BL / 10J were purchased from Jackson Laboratory. Mutant mice '' Rim 3 was independently systematized at the National Institute of Genetics, and p53 (Trp53) knockout mice were provided by Dr. Shinichi Aizawa (currently RIKEN Research Center for Developmental Sciences) . These mouse strains were bred and managed at the National Institute of Genetics' Center for Biological Biology, and animals bred at the Center for Biological Biology were used for experiments. Animal experiments were approved in accordance with the guidelines of the National Institute of Genetics' Animal Experimentation Committee.
  • Rim3 heterozygous ⁇ ⁇ 53 knockout heterozygous individuals (GsdmA3 Rim3 / +; TYp53 — / +) were created by natural mating of Rim3 heterozygous individuals (GsdmA3 Rim3 / +) and p53 knockout heterozygous individuals (TYp53 — / +). It was.
  • the genotypes of children obtained from this parental lineage combination include Rim3 heterozygous individuals (GsdmA3 Rim3 / +; TYp53 + / +), p53 knockout heterozygous individuals (GsdmA3 + / +; TYp53 — / +), Normal individuals (GsdmA3 + / +; TYp53 + / +). All these offspring were raised under the same conditions (light / dark conditions, temperature, humidity, feed, drinking water, flooring, etc.), and the rate of skin cancer was observed over time.
  • HSC1 squamous cell carcinoma of the skin
  • HSC4 squamous cell carcinoma of the tongue
  • HSC5 squamous cell carcinoma of the skin
  • A431 epihelioid carcinoma of the vulva
  • HSC1 is 20% fetal urine serum and DMEM supplemented with penicillin-streptomycin
  • HSC4 is 10% fetal urine serum
  • MEM supplemented with penicillin-streptomycin
  • HSC5 is 10% fetal urine serum
  • A431 10% fetal ⁇ shea serum and penicillin - streptomycin was cultured in DMEM supplemented.
  • RT-PCR analysis Preparation of total RNA from cultured skin cancer cells was performed using ISOGEN (Nippon Gene) according to the procedure recommended by the supplier. Human normal skin total RNA was purchased from Invitrogen. Total RNA obtained from each cultured skin cancer cell was treated with a DNA-degrading enzyme at 37 ° C. for 30 minutes to remove genomic DNA in the sample. The procedure for synthesizing cDNA from total RNA is as follows.
  • RNA of cultured skin cancer cells and human normal skin total RNA 7.5 ⁇ g, 10 ⁇ m dNTP (lOmM dATP, lOmM dCTP, lOmM dGTP, lOmM dTTP) 3 ⁇ 1, Oligo (dT)
  • ript III Reverse Transcriptase Invitrogen was reacted with 3 ⁇ l and reacted at 55 ° C. for 50 minutes. After completion of the reaction, incubate at 70 ° C for 15 minutes to remove the enzyme activity of reverse transcriptase, and use this final reaction product for each cultured skin cancer cell and PCR sample of human normal skin total RNA. It was.
  • the GSDMA expression in each cultured skin cancer cell and human normal skin sample was confirmed by PCR.
  • the primer used is a set of (GSDMAFATGACCATGTTTGAAAAT GTC (SEQ ID NO: 13)) and (GSDMAR: TTAGGAGGCCTTGGTAAGCTG (SEQ ID NO: 14)).
  • the reaction conditions were DNA denaturation reaction at 94 ° C for 30 seconds, annealing reaction at 57 ° C for 30 seconds, extension reaction at 72 ° C for 1 minute 30 seconds, reaction cycle of 35 cycles, and the machine used was The company's DNA amplification device. After completion of the reaction, PCR samples were electrophoresed on a 1% agarose gel to evaluate GSDMA expression in each sample.
  • the anti-GsdmA / GSDMA antibody has a region corresponding to the 37th to 12th amino acids (CLVLRKR KSTLF (SEQ ID NO: 15)) of the GsdmAl protein and the 248th to 14th amino acids (QMIS EEPEEEKLIG (SEQ ID NO: 16)) of the GsdmA3 protein.
  • Peptides were synthesized and prepared by immunizing chickens.
  • Antibody titer was measured by ELISA. When the obtained antibodies were verified for their antigen specificity by Western blotting, these antibodies It was confirmed that GsdmAl, GsdmA2, GsdmA3 and human GSDMA were all specifically recognized.
  • Anti-GsdmA / GSDMA (diluted by 50 times) was used as the primary antibody, and peroxidase-labeled donkey anti-nitrate IgY polyclonal antibody (diluted by 500 times, Jacson ImmunoResearch) was used as the secondary antibody.
  • peroxidase-labeled donkey anti-nitrate IgY polyclonal antibody (diluted by 500 times, Jacson ImmunoResearch) was used as the secondary antibody.
  • 3 '3-daminobencidine tetrahydrochloride (Wako Pure Chemical Industries) was used. The specimen after antibody staining was observed with an upright light microscope (OLYMPUS BX51) covered with a rubber glass.
  • GSDMA Human homologous gene of mouse GsdmA cluster: GSDMA has been shown to have its gene expression suppressed in gastric cancer and esophageal cancer, and to have tumor suppressive ability. However, it has been clarified in the skin, and it has been revealed whether it has the same expression pattern and function.
  • GSDMA gene expression using normal skin and four skin cancer cell lines: HSC1 (squamous squamous cell carcinoma), HSC4 (squamous carcinoma of the tongue), HSC5 (squamous cell carcinoma of the skin), and A431 (epidermoid carcinoma)
  • HSC1 squamous squamous cell carcinoma
  • HSC4 squamous carcinoma of the tongue
  • HSC5 squamous cell carcinoma of the skin
  • A431 epidermoid carcinoma
  • Rim3 heterozygote ⁇ ⁇ 53 knockout heterozygote spontaneously develops skin cancer
  • GsdmAl and GsdmA3 are expressed in the skin among the GsdmA cluster genes (present on mouse chromosome 11).
  • the causative gene of epithelial morphogenesis mutant mouse Rim3 is GsdmA3.
  • the function of GsdmA3 gene is expected to be deeply involved in cell growth and differentiation.
  • GsdmA3 may have cancer suppressive ability in mouse skin.
  • T p53
  • the present inventors introduced a knockout allele of p53 (T) 53), which is known as a tumor suppressor gene, to the epithelial morphogenic mutant mouse Rim3, which is a causative gene of GsdmA3.
  • Rim3 heterozygous ⁇ 53 knockout heterozygous individuals (GsdmA3 Rim3 / +; TYp53 — / +) were prepared, and skin cancer incidence in the animal individuals was verified.
  • Figure 2 shows a schematic diagram of the crossing used in the experiment.
  • Rim3 heterozygous individuals (GsdmA3 Rim3 / +) and p53 knockout heterozygous individuals (TYp53 — / +) can be crossed to obtain Rim3 heterozygous, ⁇ 53 knockout heterozygous individuals, Rim3 heterozygous individuals, p53 knockout heterozygous individuals, and normal individuals. Obtained in street proportions. All these genotype individuals were bred under the same conditions and without contact with carcinogens, and observed over time. If skin cancer develops in these animals, it can be said that the cause is a spontaneous disease caused only by the genotype. As a result of time-lapse observation, the development of skin cancer was not observed until 6 months after birth in all genotypes.
  • Rim3 heterozygous ⁇ 53 knockout heterozygous animals were observed only in Rim3 heterozygous ⁇ 53 knockout heterozygous animals for the first time at 7 months of age. In double heterozygous individuals, the incidence increases with age of caro, with 38% (8/21) at 8 months, 62% (13/21) at 10 months, and 81% (17/21) at 12 months. ) Individuals developed skin cancer (Fig. 3). Interestingly, multiple skin cancers occurred in each individual, and the affected sites were concentrated in the head and neck as in human skin cancer (Table 1). Rim3 heterozygous individuals other than ⁇ 53 knockout heterozygous individuals: In Rim3 heterozygous individuals, p53 knockout heterozygous individuals, and normal individuals, only Rim3 heterozygous individuals developed skin cancer at 11 months of age. The incidence was very low, about 10% even at 12 months of age.
  • Table 1 shows the incidence of spontaneous skin cancer in cross-breed mice obtained from crosses of Rim3 / + and TYp53-/ + mice.
  • Example 3 Histochemical observation of Rim3 hetero- ⁇ 53 knockout heteromouse skin cancer
  • tissue sections were prepared.
  • the ⁇ in Fig. 4 shows an overall image of skin cancer that developed in a Rim3 heterozygous ⁇ 53 knockout heterozygous individual
  • the ⁇ in Fig. 4 is a slice image of the individual's cancer.
  • the white line in Fig. 4 ⁇ coincides with the cut surface in Fig. 4 ⁇ .
  • Skin cancer in the Rim3 heterozygous ⁇ 53 knockout heterozygote was keratoacantoma-like, with many highly keratinized regions (Fig. 4 ⁇ ).
  • the present inventors prepared a GsdmA / GSDMA antibody as a starting point for examining whether or not the expression of GSDMA in the cancer site in humans and animal individuals (in vivo) has disappeared.
  • the present inventors succeeded in producing an anti-GsdmA / GSDMA polyclonal antibody by producing a synthetic peptide against GsdmA / GSDMA and immunizing a chicken.
  • the specificity of the anti-GsdmA / GSDMA polyclonal antibody by Western blot is shown in FIG.
  • the GsdmA / GSDMA polyclonal antibody prepared this time surely recognizes GsdmA / GSDMA (mouse GsdmAl to A3 and human GSDMA protein).
  • GSDMA protein was expressed from the spinous cell layer, which is not expressed in the basement membrane cell layer, to the granular layer and the transparent layer, as in normal mouse skin (FIG. 7A).
  • the expression of GSDMA protein in human hair follicles was similar to that in mice (FIG. 7B).
  • squamous cell carcinoma expression of GSDMA protein basal cell carcinoma both were found to be deleted extinction (Fig. 7C, 7D) of zero or more results, not only cultured cancer cell lines It became clear that GsdmA / GSDMA protein disappears with canceration in human and mouse individuals.
  • the present inventors have introduced a new skin cancer model that induces spontaneous skin cancer by introducing p53 (TYp53) knockout aryl into Rim3, an epithelial dysplasia mutant mouse having a mutation in GsdmA3.
  • p53 TYp53
  • An animal was developed.
  • Rim3 heterozygous ⁇ 53 knockout heterozygous individuals develop squamous cell carcinoma around 7 months of age, and the proportion exceeds 80% at 12 months of age.
  • the site of skin cancer is concentrated in the head and neck where human skin cancer is common, and multiple cancers are observed per individual. Surprisingly, the cancer cells were confirmed to metastasize to the regional lymph nodes.
  • Oncogene which is a canceration-related gene, and tumor suppressor g ene
  • Oncogene is originally derived from normal genes, and many of them play extremely important roles such as cell growth / differentiation regulation. If the canceration-related gene is mutated for any reason, abnormal gene hyperfunction (oncogene) or functional decline (tumor suppressor gene) occurs, leading to abnormal cell proliferation signal abnormalities or cell cycle abnormalities. As a result, gene repair or the like becomes impossible, and mutations accumulate in more than one gene, leading to cancerous cells. In fact, in many cases, mutations accumulate in multiple cancer-related genes in cancer.
  • the Rim3 heterozygote ⁇ ⁇ 53 knockout heterozygote is considered to reflect this mechanism of cancer development. Mutation of GsdmA3 or p53 gene alone does not cause skin cancer. This means that in the presence of mutations in the GsdmA3 gene (Rim3 homo / hetero individuals), the occurrence of cancer is suppressed by the p53 gene, and conversely, if there is a mutation in the p53 gene (p53 knockout homo / hetero). It is speculated that the GsdmA3 gene suppresses cancer development in individuals. Skin cancer develops only when mutations exist in both genes and age increases.
  • both GsdmA3 gene and p53 gene are normal types. That is, it is half the wild type but has a normal function. Perhaps half of the normal function during the six months until skin cancer develops, gene repair, etc. cannot be performed normally, and mutations accumulate in many canceration-related genes, leading to skin cancer. Conceivable.
  • Rim3 heterozygous ⁇ 53 knockout heterozygous individual both GsdmA3 gene and T ⁇ 53 gene are normal types, so the phenotype is considered to be moderate. Therefore, Rim3 homo ⁇ ⁇ 53 knockout heterozygous individuals (GsdmA3 Rim3 / Rim3 ; T ⁇ 537+), Rim3 head ⁇ ⁇ 53 knockout homozygous individuals (GsdmA3 Rim3 / +; Trp53—n) Skin cancer develops earlier, and Rim3 homo ⁇ ⁇ 53 knockout homozygous individuals (GsdmA3 Rim 3 / Rim3 ; Trp537—) are considered to develop skin cancer earlier.
  • GsdmAl One candidate for a mutation-accumulating canceration-related gene in skin cancer of Rim3 heterozygous ⁇ 53 knockout heterozygous is GsdmAl.
  • Rim3 heterozygous ⁇ No mutation exists in the GsdmAl gene in ⁇ 53 knockout heterozygous individuals.
  • the results of immunostaining with anti-GsdmA / GSDMA antibody revealed that both GsdmAl and GsdmA3 proteins originally expressed in the skin disappeared in skin cancer of Rim3 hetero- ⁇ 53 knockout hetero individuals. Power. Not only GsdmA3 but also the disappearance of GsdmAl without mutation is very interesting.
  • GsdmAl and GsdmA3 are considered to be functionally redundant with very high homology. At the stage where mutations exist only in Gsdm A3 (Rim3 homozygous or heterozygous individuals), it seems that GsdmAl supplements its function and suppresses canceration. For canceration, the disappearance of both genes GsdmAl and Gsdm A3 may be necessary. In addition, we are interested in the ability to suppress the expression of both GsdmAl and GsdmA3, and the factors involved in the transcriptional regulation of both genes are considered to be candidates for mutation-accumulating canceration-related genes in skin cancer.
  • the present inventors produced a skin cancer spontaneous model animal by introducing a p53 knockout aryl into Rim3 having a mutation in GsdmA3 expressed specifically in the skin.
  • GsdmA3 has GsdmAl and GsdmA2 as paralogs.
  • GsdmAl is specifically expressed in the squamous epithelium in the skin, esophagus and foregut region, and GsdmA2 is expressed specifically in the gastric gland epithelium.
  • Squamous cell carcinoma model mice that develop skin cancer, esophageal cancer, etc.
  • p53 knockout homozygous mice are 3 months old and 6 months old, and p53 knockout heterozygous mice spontaneously develop lymphoma at 10 months of age, but do not develop skin cancer (Donehower et al., Nature. 356). , 215-221 ⁇ 1992 ⁇ ).
  • the present inventors have also found that the spontaneous incidence of skin cancer in Rim3 (GsdmA3 Rim3 / + and Gsdm A3 Rim3 / Rim3 ) is extremely low.
  • the Rim3 heterozygous ⁇ 53 knockout heteromaus (GsdmA3 Rim3 / +; TYp53 — / +) provided by the inventors of the present invention is a spontaneous onset of squamous cell carcinoma of the skin more frequently than 7 months after birth.
  • the development of similar squamous cell carcinoma in more than 80% of individuals at 12 months of age was observed. More surprisingly, it was confirmed that squamous cell carcinoma metastasized to regional lymph nodes.
  • Rim3 heterozygous ⁇ 53 knockout heterozygous mouse model of squamous cell carcinoma of the present invention is useful to “spontaneously develop squamous cell carcinoma in a high frequency and frequently occurring” and to metastasize to lymph nodes. It is also useful for elucidating the mechanism of 'squamous cell carcinoma development' and for developing drug discovery and diagnostics.

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Abstract

By transferring Trp53(p53) knockout allele into Rim3 having a mutation in GsdmA3, Rim3 hetero p53 knockout hetero individuals (GsdmA3Rim3/+;Trp53-/+) are constructed. As a result, these individuals spontaneously suffer from multiple skin squamous carcinoma from 7 months after birth. The onset of similar skin squamous carcinoma is observed in more than 80% of the individuals 12 months after birth. It is further observed that these cancer cells spread to lymph nodes.

Description

明 細 書  Specification
GsdmA遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進または 抑制された非ヒト動物  Non-human animals in which GsdmA gene function is suppressed and canceration-related gene function is promoted or suppressed
技術分野  Technical field
[0001] 本発明は、 GsdmA遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進また は抑制された非ヒト動物および動物細胞、並びに該非ヒト動物または該動物細胞の 表現型を相補する化合物のスクリーニング方法に関する。  [0001] The present invention relates to a non-human animal and animal cell in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene is promoted or suppressed, and a compound that complements the phenotype of the non-human animal or the animal cell The present invention relates to a screening method.
背景技術  Background art
[0002] ミュータントマウスを用いた解析は、遺伝子機能、発生、癌を始めとしたヒト疾患など の理解に於いて非常に強力な解析方法である。近年本発明者らは、上皮細胞の増 殖 '分化、毛形成'再生などに焦点を当て、 Rim3, Reden, Reなどの突然変異マウスを 用いて解析を行っている。 Reは、 1939年に Crewと Auerbachにより見いだされた自然 発症突然変異マウスで、カールした髭と体毛の表現型を示す (非特許文献:!〜 2)。 Re combinant-induced mutation-3 (Rim3)と Redenは、それぞれ810.81? 228)と810.129の コンジヱニック系統において 1983年、 1968年に見いだされた自然発症突然変異マウ スで、その表現型は、上皮の過増殖、過角化および脱毛である (非特許文献 1、 3、 4) 。連鎖解析の結果より、 Rim3および Redenは、 Reの alleleであると考えられている (非特 許文献 4)。これらの突然変異は、マウス第 11番染色体に位置しており、ヒトでは 17番 染色体の 17ql2 ampliconと呼ばれる癌に密接に関連する領域に相当している。 [0002] Analysis using mutant mice is a very powerful analysis method for understanding human functions such as gene function, development, and cancer. Recently the present inventors have found that proliferation 'differentiation, hair formation' of epithelial cells focused on a playback, the analysis is conducted using mutant mice such Rim3, Re den, Re. R e is, at spontaneous mutant mice found by Crew and Auerbach in 1939, shows the phenotype of curled beard and hair (Non-Patent Documents:! ~ 2). Re combinant-induced mutation-3 (Rim3) and Re den are spontaneous mutation mice found in 1983 and 1968 in the congeneric strains of 810.81? 228) and 810.129, respectively. Hyperproliferation, hyperkeratinization and hair loss (Non-Patent Documents 1, 3, and 4). From the results of linkage analysis, Rim3 and Re den are considered to be alleles of Re (Non-Patent Document 4). These mutations are located on mouse chromosome 11 and in humans correspond to a closely related region of cancer called 17ql2 amplicon on chromosome 17.
[0003] 染色体の不安定性は、ヒトの癌化において良く研究されている。例えば染色体の一 部の欠損や増幅、及び転座は、癌細胞において顕著に観察される現象である。これ らのゲノムの再構成は、 DNAの二重鎖切断 (DNA double strand break down)により引 き起こされ、その結果、癌遺伝子の増幅や癌抑制遺伝子の欠損につながると考えら れる (非特許文献 5)。  [0003] Chromosome instability is well studied in human carcinogenesis. For example, deletion or amplification of part of a chromosome and translocation are phenomena that are prominently observed in cancer cells. These genome rearrangements are caused by DNA double strand break down, which may lead to amplification of oncogenes and loss of tumor suppressor genes (non-patented). Reference 5).
[0004] ヒト 17番染色体長腕、 17ql2 ampliconの増幅は、乳癌、胃癌、卵巣癌、膀胱癌、及 び子宮癌において良く観察される。原癌遺伝子である erbB2は、この領域に位置して おり、癌化に伴いゲノムあたりのコピー数が増幅されて、乳癌の約 20%及び胃癌にお レ、て過剰発現していることが報告されている (非特許文献 6、 7)。本発明者らは、 17ql 2 ampliconの構造的な解析を行った過程において、この領域の内 CAB1/MLN64, C AB2， ERBB2及び GRB7が位置する領域が癌化において一般に、もしくは主として増 幅していることを見いだした。また、マウスにおいてヒトの 17ql2 ampliconに相同な領 域にガスダーミン（後にマウスガスダーミン A-I (GasderminA-1)と名称変更）が存在し てレ、ることを報告した (非特許文献 8)。 [0004] Amplification of human chromosome 17 long arm, 17ql2 amplicon, is frequently observed in breast cancer, stomach cancer, ovarian cancer, bladder cancer, and uterine cancer. ErbB2, the proto-oncogene, is located in this region, and the number of copies per genome is amplified with canceration, resulting in about 20% of breast cancer and gastric cancer. Overexpression has been reported (Non-Patent Documents 6 and 7). In the process of structural analysis of 17ql2 amplicon, the present inventors generally or mainly amplify the region where CAB1 / MLN64, CAB2, ERBB2, and GRB7 are located in this region. I found out. In addition, it was reported that gasdermin (named as mouse gasdermin AI (GasderminA-1)) was present in a region homologous to human 17ql2 amplicon in mice (Non-patent Document 8).
尚、出願の発明に関連する先行技術文献情報を以下に示す。  Prior art document information related to the invention of the application is shown below.
特 3午文献 1： Lyon M.F., Rostan S. and Brown S.D.M. enetic Variants and Strains of the Laboratory Mouse. 3rd edition (Oxford Univ. Press, Oxford, 1996) 非特許文献 2 : Crew, F.A.E. and Auerbach, C. (1939) Rex: a dominant autosomal mo nogenic coat texture character in the mouse. J. Genet. 38， 341-344.  Special 3 pm Reference 1: Lyon MF, Rostan S. and Brown SDM enetic Variants and Strains of the Laboratory Mouse. 3rd edition (Oxford Univ. Press, Oxford, 1996) Non-patent literature 2: Crew, FAE and Auerbach, C. ( 1939) Rex: a dominant autosomal mo nogenic coat texture character in the mouse. J. Genet. 38, 341-344.
非特許文献 3 : Snell， G.D., and Bunker, H. P. (1968) Mouse News Lett. 39. 28. 非特許文献 4 : Sato， H.， Koide, T" Masuya, H" Wakana，S.， Sagai, T.， Umezawa，Α· ， Ishiguro, S.， Tama, M. , and Shiroism, T. (1998) A new mutation Rim3 resembling R e(den) is mapped close to retinoic acid receptor alpha (Rara) gene on mouse chromo some 11. Mamm Genome. ;9 (1), 20-25. Non-Patent Document 3: Snell, GD, and Bunker, HP (1968) Mouse News Lett. 39. 28. Non-Patent Document 4: Sato, H., Koide, T "Masuya, H" Wakana, S., Sagai, T ., Umezawa, Tsuji, Ishiguro, S., Tama, M., and Shiroism, T. (1998) A new mutation Rim3 resembling R e (den) is mapped close to retinoic acid receptor alpha (Rara) gene on mouse chromo some 11. Mamm Genome.; 9 (1), 20-25.
非特許文献 5 : Coquelle， A" Pipiras, E.， Toledo, F.， Buttin, G.， and Debatisse, M. ( 1997). Expression of fragile sites triggers intrachromosomal mammalian gene amplific ation and sets boundaries to early amplicons. Cell 89， 215-225. Non-Patent Document 5: Coquelle, A "Pipiras, E., Toledo, F., Buttin, G., and Debatisse, M. (1997). Expression of fragile sites triggers intrachromosomal mammalian gene amplification and sets boundaries to early amplicons. Cell 89, 215-225.
非特許文献 6 : van de Vijver, M， van de Bersselaar, R.， Devilee, P.， Cornelisse, C.， Peterse, J， and Nusse, R. (1987). Amplification of the neu (c-erbB-2) oncogene in h uman mammary tumors is relatively frequent ana is often accompanied by amplificati on of the linked c - erbA oncogene. ol. Cell. Biol. 7， 2019-2023. Non-Patent Document 6: van de Vijver, M, van de Bersselaar, R., Devilee, P., Cornelisse, C., Peterse, J, and Nusse, R. (1987). Amplification of the neu (c-erbB- 2) oncogene in h uman mammary tumors is relatively frequent ana is often accompanied by amplificati on of the linked c-erbA oncogene. Ol. Cell. Biol. 7, 2019-2023.
非特許文献 7 : Yokota, J.， Yamamoto, T.， Miyajima, N.， Toyoshima, K.， Nomura, N.， Sakamoto, H.， Yoshida, T.， Terada, M， and Sugimura, T. (1988). Genetic alteratio ns of the c一 erbB - 2 oncogene occur frequently in tubular adenocarcinoma of the sto mach and are often accompanied by amplification of the v - erbA homologue. Oncoge ne 2, 283-287. ^^特許文献 8 : Saeki, N. , Kuwahara, Υ·， Sasaki, H., Satoh H. , and Shiroishi, T. Gas dermin (Gsdm) localizing to mouse chromosome 11 is predominantly expressed in up per gastrointestinal tract but significantly suppressed in human gastric cancer cells. Mammalian Genome. , 11(9): 718—724, 2000. Non-Patent Document 7: Yokota, J., Yamamoto, T., Miyajima, N., Toyoshima, K., Nomura, N., Sakamoto, H., Yoshida, T., Terada, M, and Sugimura, T. ( 1988). Genetic alteratio ns of the c erbB-2 oncogene occur frequently in tubular adenocarcinoma of the sto mach and are often accompanied by amplification of the v-erbA homologue.Oncoge ne 2, 283-287. ^^ Patent Document 8: Saeki, N., Kuwahara, Tsuji, Sasaki, H., Satoh H., and Shiroishi, T. Gas dermin (Gsdm) localizing to mouse chromosome 11 is predominantly expressed in up per gastrointestinal tract but significantly suppressed in human gastric cancer cells. Mammalian Genome., 11 (9): 718—724, 2000.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0006] 本発明者らは、国立遺伝学研究所 ·哺乳動物遺伝研究室において見出された Rec ombinant-induced mutation 3 (Rim3)の原因遺伝子が、マウス第 11番染色体にクラス ターとして存在する GasderminAクラスター（以下 GsdmAクラスターと称す）内の 1遺伝 子である GasderminA_3 (以下 GsdmA3と称す)であることを明ら力としていた（PCT/JP0 3/02345) Rim3は、上皮細胞増殖異常、分化異常、過角化、並びに脱毛の表現型 を示す優性の自然発症突然変異マウスである。この突然変異マウスの原因遺伝子が GsdmA3であることから考えると、 GsdmA3の機能は、上皮における細胞増殖'分化、 毛形成に深く関わっていることが推測される。一方、マウス GsdmAクラスターのヒト相 同遺伝子は、ヒト第 17番染色体に存在するヒト GasderminA (以下 GSDMAと称す）遺伝 子である。マウス GsdmAクラスタ一は、 GsdmAl (GasderminA-l GsdmA2 (Gasdermi nA-2 GsdmA3の 3遺伝子力 形成されているのに対して、ヒトでは GSDMA遺伝子 のみが第 17番染色体に存在する。マウス第 11番染色体の GsdmAクラスター存在領域 とヒト第 17番染色体 GSDMA存在領域は、非常に良く保存されている。特にヒト第 17番 染色体の GSDMAが存在するこの領域は、 17ql2 pliconと呼ばれ、皮膚癌、食道癌 、胃癌、乳癌などでゲノムの増幅が観察されることが知られており、癌化に深く関係し ていることが考えられている。  [0006] The present inventors have found that a causative gene of Recombinant-induced mutation 3 (Rim3) found in the National Institute of Genetics / Mammalian Genetics Laboratory is present on mouse chromosome 11 as a cluster. It was revealed that it was GasderminA_3 (hereinafter referred to as GsdmA3), a gene in the GasderminA cluster (hereinafter referred to as GsdmA cluster) (PCT / JP0 3/02345). Is a dominant spontaneous mutant mouse that exhibits a phenotype of hyperkeratinization and hair loss. Considering that the causative gene of this mutant mouse is GsdmA3, it is speculated that the function of GsdmA3 is deeply involved in cell growth and differentiation and hair formation in the epithelium. On the other hand, the human homologous gene of the mouse GsdmA cluster is the human GasderminA (hereinafter referred to as GSDMA) gene present on human chromosome 17. The mouse GsdmA cluster is composed of three genes, GsdmAl (GasderminA-1 GsdmA2 (GasderminA-2 GsdmA3), whereas in humans, only the GSDMA gene is present on chromosome 17. Mouse chromosome 11 The region of GsdmA cluster in GSDMA and the region of human chromosome 17 GSDMA are very well conserved, especially the region where human chromosome 17 GSDMA is present, called 17ql2 plicon, skin cancer, esophageal cancer In addition, it is known that genome amplification is observed in gastric cancer, breast cancer, etc., and it is thought to be deeply related to canceration.
[0007] 本発明者らは、胃癌、食道癌において、ヒト GSDMA遺伝子が癌抑制能を持つことを 明らかにした。一方、マウス GsdmAクラスター遺伝子の癌抑制能については不明であ つた（PCT/JP03/02345)。  [0007] The present inventors have clarified that the human GSDMA gene has the ability to suppress cancer in gastric cancer and esophageal cancer. On the other hand, the tumor suppressive ability of the mouse GsdmA cluster gene was unknown (PCT / JP03 / 02345).
[0008] Gli_2、 A Lef-lトランスジヱニックマウスは、それぞれ、ケラチン 14プロモーター支配 下に Gli_2遺伝子を発現するトランスジヱニックマウス、ケラチン 14プロモーター支配 下で Lef-Ιタンパク質の N末端領域を欠損させた遺伝子 ( Δ Lef- 1)を発現するトランス ジエニックマウスである。この 2種類の皮膚癌発症モデル動物において非常に興味深 い点は、その発癌部位である。 Gli-2、 A Lef- 1トランスジエニックマウスは、耳、手足、 尾、 目の縁などに皮膚癌を発症する (Grachtchouk et al.， Nat Genet, 24, 216-217， 2 000. ; Niemann et al , Development, 129, 95-109, 2002.)。この位置は、どれも体毛の 少ない領域である。このことから本発明者らは、これらトランスジエニック動物では体毛 の存在する領域において、 GsdmA3もしくは GsdmAlが癌の発生を抑えているのでは ないかと推測した。 [0008] Gli_2 and A Lef-l transgenic mice are transgenic mice expressing the Gli_2 gene under the control of the keratin 14 promoter, and the N-terminal region of the Lef-Ι protein under the control of the keratin 14 promoter, respectively. Trans for expressing the deficient gene (Δ Lef-1) It is a dienic mouse. A very interesting point in these two types of skin cancer model animals is their carcinogenic sites. Gli-2, A Lef-1 transgenic mice develop skin cancers in the ears, limbs, tail, and eye margins (Grachtchouk et al., Nat Genet, 24, 216-217, 2 000.; Niemann et al, Development, 129, 95-109, 2002.). These positions are all regions with little body hair. Based on this, the present inventors speculated that GsdmA3 or GsdmAl may suppress the occurrence of cancer in the region where hair is present in these transgenic animals.
[0009] 本発明は、以上の考察に基づきなされたものであり、その目的は、癌の表現型を示 す GsdmA遺伝子の機能が抑制された非ヒト動物を作出し、該非ヒト動物とその利用法 を提供することにある。  [0009] The present invention has been made based on the above considerations, and an object of the present invention is to produce a non-human animal in which the function of the GsdmA gene exhibiting a cancer phenotype is suppressed. Is to provide the law.
課題を解決するための手段  Means for solving the problem
[0010] 本発明者らは、 GsdmA3に変異を有する Rim3に、癌抑制遺伝子として知られている Trp53 (p53)のノックアウトァリルを導入し、 GsdmA-3Rim3/+;TYp53—/+個体（以下 Rim3へ テロ · ρ53ノックアウトヘテロ個体と称す）の作製を行った。その結果、生後 7ヶ月より自 然発症で多発的に皮膚扁平上皮癌を引き起こし、生後 12ヶ月では 8割以上の個体に おいて同様な扁平上皮癌の発症が観察された。更には、その癌細胞がリンパ節に転 移することが見出され、本発明の完成に至った。 [0010] The present inventors introduced a knockout allele of Trp53 (p53), which is known as a tumor suppressor gene, into Rim3 having a mutation in GsdmA3, so that GsdmA-3 Rim3 / +; TYp53-/ + individuals ( (Hereinafter referred to as Rim3 heterozygote · ρ53 knockout heterozygous individuals). As a result, multiple cases of squamous cell carcinoma occurred spontaneously from 7 months after birth, and similar squamous cell carcinomas were observed in more than 80% of individuals at 12 months of age. Furthermore, it was found that the cancer cells were transferred to lymph nodes, and the present invention was completed.
[0011] すなわち本発明は、以下の〔1〕〜〔： 16〕を提供するものである。  [0011] That is, the present invention provides the following [1] to [: 16].
〔1〕 GsdmA遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進または抑制さ れた非ヒト動物。  [1] A non-human animal in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene is promoted or suppressed.
〔2〕癌化関連遺伝子が、 Trp53遺伝子、 Rb遺伝子、 Ras遺伝子、 Cyclin_D遺伝子、 C dk遺伝子、 TNF- a遺伝子、および TGF- β遺伝子からなる群より選択される少なくと も 1つの遺伝子である、〔1〕に記載の非ヒト動物。  (2) The canceration-related gene is at least one gene selected from the group consisting of Trp53 gene, Rb gene, Ras gene, Cyclin_D gene, Cdk gene, TNF-a gene, and TGF-β gene The non-human animal according to [1].
〔3〕癌のモデル動物である、 〔1〕または〔2〕に記載の非ヒト動物。  [3] The non-human animal according to [1] or [2], which is a model animal for cancer.
〔4〕 GsdmAl遺伝子、 GsdmA2遺伝子および GsdmA3遺伝子からなる群より選択される 少なくとも 1つの遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進または抑 制されたマウス。  [4] A mouse in which the function of at least one gene selected from the group consisting of GsdmAl gene, GsdmA2 gene and GsdmA3 gene is suppressed, and the function of canceration-related gene is promoted or suppressed.
〔5〕癌化関連遺伝子が、 Trp53遺伝子、 Rb遺伝子、 Ras遺伝子、 Cyclin_D遺伝子、 C dk遺伝子、 TNF- a遺伝子、および TGF- β遺伝子からなる群より選択される少なくと も 1つの遺伝子である、〔4〕に記載のマウス。 [5] Canceration-related genes are Trp53 gene, Rb gene, Ras gene, Cyclin_D gene, C The mouse according to [4], which is at least one gene selected from the group consisting of a dk gene, a TNF-a gene, and a TGF-β gene.
〔6〕GsdmA3遺伝子および Trp53遺伝子の機能が抑制されたマウス。  [6] A mouse in which the functions of the GsdmA3 gene and the Trp53 gene are suppressed.
[7]癌のモデル動物である、 〔4〕〜〔6〕のレ、ずれかに記載のマウス。 [7] The mouse according to [4] to [6], which is a model animal for cancer.
〔8〕癌が皮膚癌である、 〔7〕に記載のマウス。 [8] The mouse according to [7], wherein the cancer is skin cancer.
〔9〕 GsdmA遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進または抑制さ れた動物細胞。  [9] An animal cell in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene is promoted or suppressed.
〔10〕 GsdmAl遺伝子、 GsdmA2遺伝子および GsdmA3遺伝子からなる群より選択され る少なくとも 1つの遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進または 抑制されたマウス細胞。  [10] A mouse cell in which the function of at least one gene selected from the group consisting of the GsdmAl gene, GsdmA2 gene and GsdmA3 gene is suppressed, and the function of a canceration-related gene is promoted or suppressed.
〔1 1〕癌化関連遺伝子が、 Trp53遺伝子、 Rb遺伝子、 Ras遺伝子、 Cyclin_D遺伝子、 [1 1] Canceration-related genes are Trp53 gene, Rb gene, Ras gene, Cyclin_D gene,
Cdk遺伝子、 TNF- a遺伝子、および TGF- β遺伝子からなる群より選択される少なく とも 1つの遺伝子である、 〔9〕または〔10〕に記載の細胞。 The cell according to [9] or [10], which is at least one gene selected from the group consisting of a Cdk gene, a TNF-a gene, and a TGF-β gene.
〔12〕GsdmA3遺伝子および ΤΥρ53遺伝子の機能が抑制されたマウス細胞。  [12] A mouse cell in which the functions of the GsdmA3 gene and the ΤΥρ53 gene are suppressed.
〔13〕〔1〕もしくは〔2〕に記載の非ヒト動物、または〔4〕〜〔6〕のいずれかに記載のマウ スから樹立された細胞。  [13] A cell established from the non-human animal according to [1] or [2] or the mouse according to any of [4] to [6].
〔14〕以下の (a)〜 (c)の工程を含む、〔1〕もしくは〔2〕に記載の非ヒト動物、または〔4 〕〜〔6〕のいずれかに記載のマウスの表現型を相補する化合物のスクリーニング方法  [14] The phenotype of the non-human animal according to [1] or [2] or the mouse according to any of [4] to [6], comprising the following steps (a) to (c): Method for screening complementary compounds
(a)〔1〕もしくは〔2〕に記載の非ヒト動物、または〔4〕〜〔6〕のいずれかに記載のマウス に投与する工程 (a) A step of administering to the non-human animal according to [1] or [2] or the mouse according to any of [4] to [6]
(b)該非ヒト動物の表現型を測定する工程  (b) measuring the phenotype of the non-human animal
(c)被検化合物を投与してレ、なレ、場合と比較して、表現型を相補する化合物を選択 する工程  (c) A step of administering a test compound and selecting a compound that complements the phenotype compared to the case.
[ 15]以下の（a)〜（c)の工程を含む、〔9〕〜〔13〕のいずれかに記載の動物細胞の 表現型を相補する化合物のスクリーニング方法。  [15] A screening method for a compound that complements the phenotype of an animal cell according to any one of [9] to [13], comprising the following steps (a) to (c):
(a)〔9〕〜〔： 13〕のいずれかに記載の動物細胞に被検化合物を接触させる工程  (a) contacting the test compound with the animal cell according to any one of [9] to [: 13]
(b)該細胞の表現型を測定する工程 (c)被験試料を接触させないときに比較して、表現型を相補する化合物を選択する 工程 (b) measuring the phenotype of the cell (c) selecting a compound that complements the phenotype compared to when the test sample is not contacted
〔16〕以下の（a)〜（e)のいずれかに記載のタンパク質に結合する抗体。  [16] An antibody that binds to the protein according to any one of (a) to (e) below.
(a)配列番号： 1、 3、 5、 7のいずれかに記載の塩基配列からなる DNAよりコードされ るタンパク質  (a) a protein encoded by DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, and 7
(b)配列番号： 2、 4、 6、 8のいずれかに記載のアミノ酸配列からなるタンパク質  (b) a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, and 8
(c)配列番号： 2、 4、 6、 8のいずれかに記載のアミノ酸配列において 1若しくは複数 のアミノ酸が置換、欠失、挿入、および/または付加したアミノ酸配列からなる天然由 来のタンパク質であって、配列番号： 2、 4、 6、 8のいずれかに記載のアミノ酸配列か らなるタンパク質と機能的に同等なタンパク質  (c) a naturally occurring protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8 A protein functionally equivalent to the protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, and 8.
(d)配列番号： 1、 3、 5、 7のいずれかに記載の塩基配列からなる DNAとストリンジェン トな条件下でハイブリダィズする天然由来の DNAよりコードされるタンパク質であって (d) a protein encoded by a naturally occurring DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7;
、配列番号： 2、 4、 6、 8のいずれかに記載のアミノ酸配列からなるタンパク質と機能 的に同等なタンパク質 A protein functionally equivalent to the protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, and 8.
(e) (a)〜（d)のレ、ずれかに記載のタンパク質の部分タンパク質  (e) A partial protein of the protein according to (a) to (d)
図面の簡単な説明 Brief Description of Drawings
[図 1]皮膚癌細胞株における GSDMA遺伝子の発現を示す写真である。正常皮膚組 織、並びに皮膚癌組織における GSDMA遺伝子の発現を RT-PCR法により調べた結 果、癌細胞株では GSDMA遺伝子の発現が全てで消失してレ、た。 FIG. 1 is a photograph showing the expression of GSDMA gene in a skin cancer cell line. As a result of investigating the expression of GSDMA gene in normal skin tissue and skin cancer tissue by RT-PCR, the expression of GSDMA gene disappeared in all cancer cell lines.
[図 2]Rim3ヘテロ · ρ53ノックアウトヘテロマウスの作出方法の模式図である。 Rim3へテ 口個体（GsdmA3Rim3/+)と p53ノックアウトヘテロ個体 (TYp53—/+)を掛け合わせることに より、 Rim3ヘテロ ·ρ53ノックアウトヘテロ、 Rim3ヘテロ、 p53ノックアウトヘテロ、正常マ ウス個体の作出を行った。 FIG. 2 is a schematic diagram of a method for producing a Rim3 heterozygous ρ53 knockout heteromouse. Rim3 heterozygous individuals (GsdmA3 Rim3 / +) and p53 knockout heterozygous individuals (TYp53 — / +) can be used to create Rim3 heterozygous ρ53 knockout heterozygous, Rim3 heterozygous, p53 knockout heterozygous, and normal mouse individuals. went.
[図 3]Rim3ヘテロ · ρ53ノックアウトヘテロマウス（GsdmA3Rim3/+;TYp53—/+)における皮 膚癌の発症を示すグラフである。 Rim3ヘテロ ·ρ53ノックアウトヘテロマウスにおいて、 生後 6ヶ月齢より皮膚癌を自然発症した。生後 12ヶ月齢になると Rim3ヘテロ · ρ53ノック アウトへテロマウスでは 80%以上（17/21)の個体にぉレ、て複数の皮膚癌が観察された 。他の遺伝型のマウスにおいては、 Rim3ヘテロ（GsdmA3Rim3/+;TYp53+/+)のみにお いて皮膚癌が見られたが、その割合は 10% (2/19)と低い値である。グラフの右の nは、 実験に用いた各遺伝子型マウスの個体数を示す。 FIG. 3 is a graph showing the onset of skin cancer in Rim3 heterozygous ρ53 knockout heterozygous mice (GsdmA3 Rim3 / +; TYp53 − / +). In Rim3 heterozygous ρ53 knockout heterozygous mice, skin cancer spontaneously developed from 6 months of age. At 12 months of age, more than 80% (17/21) of Rim3 heterozygous ρ53 knockout hetero mice were observed with multiple skin cancers. In mice of other genotypes, only Rim3 heterozygous (GsdmA3 Rim3 / +; TYp53 + / +) Skin cancer was seen, but the rate was as low as 10% (2/19). The n on the right of the graph indicates the number of individual genotype mice used in the experiment.
[図 4]Rim3ヘテロ · ρ53ノックアウトヘテロマウスにおける皮膚癌を示す写真である。 Α : 皮膚癌を発症した Rim3ヘテロ · ρ53ノックアウトヘテロマウスの像。図中の白線は Βに おける切片面を示す。 Β :皮膚癌切片の全体像。 C : B中における C領域の拡大図（非 癌部）。 D : B中における D領域の拡大図（癌部）。 Rim3ヘテロ ·ρ53ノックアウトヘテロ マウスの皮膚癌は典型的な扁平上皮癌像を示した。 E : Rim3ヘテロ ·ρ53ノックアウト ヘテロマウスにおける皮膚癌細胞は、所属リンパ節へ転移し、リンパ節内で上皮構造 FIG. 4 is a photograph showing skin cancer in a Rim3 hetero-ρ53 knockout hetero mouse. Α: Image of Rim3 heterozygous ρ53 knockout heteromouse that developed skin cancer. The white line in the figure shows the section plane at Β. Β: Whole image of skin cancer section. C: Enlarged view of C region in B (non-cancerous part). D: Enlarged view of D region in B (cancerous part). Rim3 heterozygous · ρ53 knockout heterozygous skin cancer showed a typical squamous cell carcinoma image. E: Rim3 heterozygous · ρ53 knockout Skin cancer cells in heterozygous mice metastasize to regional lymph nodes, and epithelial structure in lymph nodes
、並びに過角化を引き起こした。 As well as hyperkeratosis.
[図 5]抗 GsdmA/GSDMAポリクローナル抗体の特異性を示す写真である。ウェスタン ブロット法により、抗 GsdmA/GSDMAポリクローナル抗体の抗原特異性の検証を行つ た。その結果、正常皮膚サンプルにおいて GsdmA/GSDMAの予想される分子量 50k ダルトン付近に単一のバンドが検出され、抗 GsdmA/GSDMAポリクローナル抗体の抗 原特異性が示された。 Aは分子量マーカー、 Bは正常皮膚サンプルを示す。  FIG. 5 is a photograph showing the specificity of an anti-GsdmA / GSDMA polyclonal antibody. The antigen specificity of the anti-GsdmA / GSDMA polyclonal antibody was verified by Western blotting. As a result, a single band was detected in the normal skin sample in the vicinity of the expected molecular weight of 50 k Dalton of GsdmA / GSDMA, indicating the anti-specificity of the anti-GsdmA / GSDMA polyclonal antibody. A is a molecular weight marker and B is a normal skin sample.
[図 6]Rim3ヘテロ · ρ53ノックアウトヘテロマウス皮膚癌における GsdmAタンパク質の消 失を示す写真である。 Aは非癌部、 Cは癌部のへマトキシリン/ェォシン染色像。 B、 Dは、それぞれ A、 Cの隣接切片の GsdmA抗体による染色像である。非癌部の Bにお いては、有棘細胞から GsdmA (1/3)タンパク質の局在が観察されるのに対して、典型 的な扁平上皮癌細胞（図 C、 Dにおける *領域)では GsdmA (1/3)タンパク質が消失 していた。 FIG. 6 is a photograph showing the disappearance of GsdmA protein in Rim3 heterozygous ρ53 knockout heteromouse skin cancer. A is a non-cancerous part, C is a hematoxylin / eosin stained image of the cancerous part. B and D are stained images with GsdmA antibody of adjacent sections of A and C, respectively. In non-cancerous B, localization of GsdmA (1/3) protein is observed from spinous cells, whereas in typical squamous cell carcinoma cells (* area in Figures C and D) GsdmA (1/3) protein was lost.
[図 7]ヒト皮膚並びに皮膚癌における GSDMAタンパク質の発現を示す写真である。 G SDMAタンパク質は、マウス GsdmAタンパク質と同様に正常皮膚（A)並びに正常毛 胞（B)において発現している。また、ヒト扁平上皮癌細胞（C)、基底細胞癌細胞（D) においては、 Rim3ヘテロ · ρ53ノックアウトヘテロ皮膚癌細胞同様、 GSDMAタンパク質 の発現が消失していた。  FIG. 7 is a photograph showing the expression of GSDMA protein in human skin and skin cancer. The GSDMA protein is expressed in normal skin (A) as well as normal hair (B), similar to the mouse GsdmA protein. In human squamous cell carcinoma cells (C) and basal cell carcinoma cells (D), the expression of GSDMA protein was lost, as in Rim3 hetero-ρ53 knockout hetero skin cancer cells.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
本発明は、 GsdmA遺伝子の機能が抑制され、癌化関連遺伝子（癌遺伝子と GsdmA 遺伝子以外の癌抑制遺伝子）の機能が促進または抑制されている非ヒト動物、すな わち、 GsdmA遺伝子の機能が抑制され、癌遺伝子の機能が促進されている非ヒト動 物、または GsdmA遺伝子の機能が抑制され、癌抑制遺伝子の機能が抑制されている 非ヒト動物を提供する。 The present invention relates to a non-human animal in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene (oncogene and a tumor suppressor gene other than the GsdmA gene) is promoted or suppressed. In other words, a non-human animal in which the function of the GsdmA gene is suppressed and the function of the oncogene is promoted, or a non-human animal in which the function of the GsdmA gene is suppressed and the function of the tumor suppressor gene is suppressed is provided. .
[0014] 本発明の非ヒト動物における GsdmA遺伝子としては、 1 )非ヒト動物がマウスであれ ば、 GsdmAl遺伝子、 GsdmA2遺伝子、 GsdmA3遺伝子、およびこれらの変異体から なる群より選択される少なくとも 1つの遺伝子、 2)非ヒト動物がマウス以外の非ヒト動物 であれば、 GsdmAl遺伝子、 GsdmA2遺伝子、 GsdmA3遺伝子、または GSDMA遺伝 子のホモログが例示でき、マウスのように複数の GsdmA遺伝子が存在する場合は、そ の複数の遺伝子からなる群より選択される少なくとも 1つの遺伝子が例示できる。  [0014] The GsdmA gene in the non-human animal of the present invention is as follows: 1) If the non-human animal is a mouse, it is at least one selected from the group consisting of GsdmAl gene, GsdmA2 gene, GsdmA3 gene, and mutants thereof. Gene, 2) If the non-human animal is a non-human animal other than a mouse, a GsdmAl gene, a GsdmA2 gene, a GsdmA3 gene, or a homolog of the GSDMA gene can be exemplified. An example is at least one gene selected from the group consisting of the plurality of genes.
[0015] GsdmAl GsdmA2 GsdmA3、または GSDMAの生物学的機能としては、細胞の増 殖異常または分化異常を抑制する機能、アポトーシス誘導能、上皮の過増殖、過角 ィ匕および脱毛の抑制機能等が挙げられる。また、 GsdmAl GsdmA2 GsdmA3、また は GSDMAの生化学的機能としては、ガスダーミンファミリ一間の結合、増殖分化制御 タンパクによる GsdmAクラスターのリン酸化'糖鎖等の修飾、核へのタンパク輸送、細 胞膜への局在、細胞外への分泌等が挙げられる。  [0015] The biological functions of GsdmAl GsdmA2 GsdmA3 or GSDMA include the ability to suppress abnormal cell proliferation or differentiation, the ability to induce apoptosis, the ability to induce epithelial hyperproliferation, hyperkeratosis, and hair loss. Can be mentioned. The biochemical functions of GsdmAl GsdmA2 GsdmA3 or GSDMA include the binding between the gas dermin families, the phosphorylation of GsdmA clusters by growth differentiation control proteins, modification of sugar chains, etc. Examples include localization to the vesicle membrane and secretion outside the cell.
[0016] 本発明の非ヒト動物においては、ほたは複数の癌化関連遺伝子の機能が促進ま たは抑制されてもよい。  [0016] In the non-human animal of the present invention, the functions of a plurality of canceration-related genes may be promoted or suppressed.
本発明の癌化関連遺伝子には、当業者に周知の癌化関連遺伝子が含まれ、例え は、 ¾退伝子としては、 Ras (Harvey rat sarcoma virus oncogene (Hrasl) : h— ras) ( マウス： NM_008284、ヒト： NM_005343) Cyclin-D (cyclin D l (Ccndl)) (マウス： NM_00 7631、ヒト： BC000076)、 Cdk(cyclin-dependent kinase 2 : Cdk2) (マウス： BC005654、ヒ ト： BC003065)、癌抑制遺伝子としては、 TYp53 Rb (retinoblastoma 1 (Rbl)： Retinobl astoma) (マウス： NM_009029、ヒト： NM_000321) TNF_ (tumor necrosis factor) (マ ウス： NM_013693、ヒト： NM_000594) TGF- β (transforming growth factor, beta 1) ( マウス： BC013738、ヒト： NM_000660)、が挙げられ、好ましくは T卬 53が挙げられる。  The canceration-related genes of the present invention include those known to those skilled in the art, for example, as ¾ regression gene, Ras (Harvey rat sarcoma virus oncogene (Hrasl): h-ras) (mouse : NM_008284, human: NM_005343) Cyclin-D (cyclin D l (Ccndl)) (mouse: NM_00 7631, human: BC000076), Cdk (cyclin-dependent kinase 2: Cdk2) (mouse: BC005654, human: BC003065), Tumor suppressor genes include TYp53 Rb (retinoblastoma 1 (Rbl): Retinobl astoma) (mouse: NM_009029, human: NM_000321) TNF_ (tumor necrosis factor) (mouse: NM_013693, human: NM_000594) TGF-β (transforming growth factor , beta 1) (mouse: BC013738, human: NM_000660), preferably T 卬 53.
[0017] また、本発明の非ヒト動物における癌化関連遺伝子としては、 1 )非ヒト動物がマウス であれば、例えば、マウス Trp53遺伝子、マウス Rb遺伝子、マウス Ras遺伝子、マウス Cyclin-D遺伝子、マウス Cdk遺伝子、マウス TNF- a遺伝子、マウス TGF- β遺伝子、 およびこれらの変異体からなる群より選択される少なくとも 1つの遺伝子、 2)非ヒト動 物がマウス以外の非ヒト動物であれば、例えば、マウス Trp53遺伝子などの遺伝子（そ の他には、マウス Rb遺伝子、マウス Ras遺伝子、マウス Cyclin-D遺伝子、マウス Cdk遺 伝子、マウス TNF- α遺伝子、マウス TGF- β遺伝子、ヒト Τ卬 53遺伝子、ヒト Rb遺伝子 、ヒト Ras遺伝子、ヒト Cyclin-D遺伝子、ヒト Cdk遺伝子、ヒト TNF- a遺伝子、ヒト TGF- β遺伝子）のホモログからなる群より選択される少なくとも 1つの遺伝子が例示できる。 [0017] Further, the canceration-related gene in the non-human animal of the present invention is as follows. 1) If the non-human animal is a mouse, for example, mouse Trp53 gene, mouse Rb gene, mouse Ras gene, mouse Cyclin-D gene, Mouse Cdk gene, mouse TNF-a gene, mouse TGF-β gene, And at least one gene selected from the group consisting of these mutants, and 2) if the non-human animal is a non-human animal other than a mouse, for example, a gene such as the mouse Trp53 gene ( Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene, mouse TNF-α gene, mouse TGF-β gene, human Τ 卬 53 gene, human Rb gene, human Ras gene, human Cyclin-D gene , Human Cdk gene, human TNF-a gene, human TGF-β gene) at least one gene selected from the group consisting of homologs.
[0018] マウス ΤΥρ53、またはヒト Τ卬 53の生物学的機能としては、 DNAダメージ、テロメァの 短縮、異常な増殖シグナル、活性酸素など、様々なストレスに反応して活性化する癌 抑制能（DNA修復、細胞周期の停止やアポトーシスの誘導等）が挙げられる。また、 マウス ΤΥρ53、またはヒト ΤΥρ53の生化学的機能としては、 DNAとの結合、他の癌関連 遺伝子との結合等が挙げられる。  [0018] The biological functions of mouse ΤΥρ53 or human Τ 卬 53 include the ability to suppress cancer (DNA) that is activated in response to various stresses such as DNA damage, telomere shortening, abnormal growth signals, and active oxygen. Repair, cell cycle arrest, induction of apoptosis, etc.). The biochemical functions of mouse ΤΥρ53 or human ΤΥρ53 include binding to DNA and binding to other cancer-related genes.
[0019] マウス Rb、またはヒト Rbの生物学的機能としては、細胞周期制御、アポトーシス制御 、転写調節、また、生化学的機能としては、 DNAとの結合、 E2Fタンパク質との結合が 挙げられる。  [0019] Biological functions of mouse Rb or human Rb include cell cycle control, apoptosis control, transcriptional regulation, and biochemical functions include binding to DNA and binding to E2F protein.
[0020] マウス Ras、またはヒト Rasの生物学的機能としては、細胞周期制御、また、生化学的 機能としては、 GTPとの結合、 GTPase活性が挙げられる。  [0020] Biological functions of mouse Ras or human Ras include cell cycle control, and biochemical functions include binding to GTP and GTPase activity.
[0021] マウス Cyclin-D、またはヒト Cyclin-Dの生物学的機能としては、細胞周期制御、また[0021] Biological functions of mouse Cyclin-D or human Cyclin-D include cell cycle control and
、生化学的機能としては、 Cdk4、 Cdk6との結合、 Rbタンパク質のリン酸化が挙げられ る。 Biochemical functions include binding to Cdk4 and Cdk6, and phosphorylation of Rb protein.
[0022] マウス Cdk、またはヒト Cdkの生物学的機能としては、細胞周期制御、また、生化学 的機能としては、 Cyclin-Eとの結合、 Rbタンパク質のリン酸化が挙げられる。  [0022] Biological functions of mouse Cdk or human Cdk include cell cycle control, and biochemical functions include binding to Cyclin-E and phosphorylation of Rb protein.
[0023] マウス TNF- a、またはヒト TNF- aの生物学的機能としては、細胞増殖制御、アポト 一シス誘導、また、生化学的機能としては、 TNFレセプターとの結合が挙げられる。  [0023] Biological functions of mouse TNF-a or human TNF-a include cell growth control, apoptosis induction, and biochemical functions include binding to a TNF receptor.
[0024] マウス TGF- β、またはヒト TGF- βの生物学的機能としては、細胞増殖制御、アポト 一シス誘導、また、生化学的機能としては、 TGF_ j3レセプターとの結合が挙げられる  [0024] Biological functions of mouse TGF-β or human TGF-β include cell growth control, induction of apoptosis, and biochemical functions include binding to the TGF_j3 receptor.
[0025] 本発明における「非ヒト動物」とは、ヒトを含まなレ、脊椎動物や無脊椎動物を意味す る。本発明における非ヒト動物としては、非ヒト哺乳動物、鳥類、昆虫類等が挙げられ るが、より好適には非ヒト哺乳動物であり、例えば、マウスやラットなどのげつ歯類、サ ル、ブタ、ゥシなどが挙げられる。 The “non-human animal” in the present invention means le, vertebrates and invertebrates including humans. Non-human animals in the present invention include non-human mammals, birds, insects and the like. More preferably, it is a non-human mammal, and examples thereof include rodents such as mice and rats, sal, pigs, and rabbits.
[0026] 本発明の非ヒト動物は、癌のモデル動物として有用である。癌としては、皮膚癌、喉 頭癌、舌癌、食道癌、胃癌、乳癌、大腸癌、卵巣癌、膀胱癌、子宮癌、膝臓癌、腎臓 癌、肺癌、メラノーマ、白血病等が例示できるが、これらに限定されるものではない。  [0026] The non-human animal of the present invention is useful as a model animal for cancer. Examples of cancer include skin cancer, laryngeal cancer, tongue cancer, esophageal cancer, stomach cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, knee cancer, kidney cancer, lung cancer, melanoma, leukemia and the like. However, it is not limited to these.
[0027] 例えば、皮膚で発現する GsdmA3遺伝子の機能が抑制されているマウスは、皮膚癌 を発症する。具体的には、 GsdmA3遺伝子および Trp53遺伝子の機能が抑制されて いるマウスは、高頻度で多発的に皮膚癌を自然発症し、かつリンパ節への転移を起 こす（実施例 2)。また、 GsdmAl遺伝子は、皮膚、食道、前胃（これらは全て扁平上皮 )で発現する。よって、 GsdmAl遺伝子および Trp53遺伝子の機能が抑制されている マウスは、皮膚、食道、前胃において癌を発症すると考えられる。さらに、 GsdmA2遺 伝子は、胃の腺上皮で発現する。よって、 GsdmA2遺伝子および TYp53遺伝子の機能 が抑制されているマウスは、胃癌を発症すると考えられる。また、 GsdmAl、 GsdmA2、 GsdmA3のうち複数の遺伝子および TYp53遺伝子の機能が抑制されているマウスは、 上記種々の癌を発症すると考えられる。  [0027] For example, a mouse in which the function of the GsdmA3 gene expressed in the skin is suppressed develops skin cancer. Specifically, mice in which the functions of the GsdmA3 gene and Trp53 gene are suppressed frequently spontaneously develop skin cancer frequently and metastasize to lymph nodes (Example 2). In addition, the GsdmAl gene is expressed in the skin, esophagus, and frontal stomach (these are all squamous epithelia). Therefore, it is considered that mice in which the functions of the GsdmAl gene and the Trp53 gene are suppressed will develop cancer in the skin, esophagus, and foregutoma. In addition, the GsdmA2 gene is expressed in the glandular epithelium of the stomach. Therefore, mice in which the functions of the GsdmA2 gene and TYp53 gene are suppressed are thought to develop gastric cancer. In addition, it is considered that mice in which the functions of multiple genes and TYp53 gene among GsdmAl, GsdmA2 and GsdmA3 are suppressed develop the above-mentioned various cancers.
[0028] また、本発明は GsdmA遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進 または抑制されている動物細胞、すなわち、 GsdmA遺伝子の機能が抑制され、癌遺 伝子の機能が促進されている動物細胞、または GsdmA遺伝子の機能が抑制され、癌 抑制遺伝子の機能が抑制されている動物細胞を提供する。このような細胞は、癌の モデル細胞として有用である。  [0028] Furthermore, the present invention is an animal cell in which the function of the GsdmA gene is suppressed and the function of the canceration-related gene is promoted or suppressed, that is, the function of the GsdmA gene is suppressed and the function of the cancer gene is promoted. Or an animal cell in which the function of a GsdmA gene is suppressed and the function of a cancer suppressor gene is suppressed. Such cells are useful as cancer model cells.
[0029] 本発明の動物細胞における GsdmAとしては、 1)動物細胞がヒト細胞であれば、 GS DMA遺伝子および該遺伝子の変異体からなる群より選択される少なくとも 1つの遺伝 子、 2)動物細胞がマウス細胞であれば、 GsdmAl遺伝子、 GsdmA2遺伝子、 GsdmA3 遺伝子、およびこれらの変異体からなる群より選択される少なくとも 1つの遺伝子、 3) 動物細胞がヒトまたはマウス以外の細胞であれば、 GsdmAl遺伝子、 GsdmA2遺伝子 、 GsdmA3遺伝子、または GSDMA遺伝子のホモログが例示でき、マウスのように複数 の GsdmA遺伝子が存在する場合は、その複数の遺伝子からなる群より選択される少 なくとも 1つの遺伝子が例示できる。 [0030] 本発明の動物細胞においては、 1または複数の癌化関連遺伝子の機能が促進また は抑制されてもよい。 [0029] As GsdmA in the animal cell of the present invention, 1) if the animal cell is a human cell, at least one gene selected from the group consisting of a GS DMA gene and a mutant of the gene, 2) an animal cell Is a mouse cell, GsdmAl gene, GsdmA2 gene, GsdmA3 gene, and at least one gene selected from the group consisting of these mutants; 3) If the animal cell is a cell other than human or mouse, the GsdmAl gene GsdmA2 gene, GsdmA3 gene, or homolog of GSDMA gene can be exemplified, and when there are multiple GsdmA genes such as mice, at least one gene selected from the group consisting of the multiple genes can be exemplified . [0030] In the animal cell of the present invention, the function of one or more canceration-related genes may be promoted or suppressed.
本発明の動物細胞における癌化関連遺伝子としては、 1 )動物細胞がヒト細胞であ れば、ヒト TYp53遺伝子、ヒト Rb遺伝子、ヒト Ras遺伝子、ヒト Cyclin_D遺伝子、ヒト Cdk 遺伝子、ヒト TNF- a遺伝子、ヒト TGF- β遺伝子、およびこれらの変異体力 なる群よ り選択される少なくとも 1つの遺伝子、 2)動物細胞がマウス細胞であれば、例えば、マ ウス Trp53遺伝子、マウス Rb遺伝子、マウス Ras遺伝子、マウス Cyclin- D遺伝子、マウ ス Cdk遺伝子、マウス TNF- a遺伝子、マウス TGF- β遺伝子、およびこれらの変異体 力もなる群より選択される少なくとも 1つの遺伝子、 3)動物細胞がヒトまたはマウス以 外の細胞であれば、例えば、マウス ΤΥρ53遺伝子などの遺伝子（その他には、マウス R b遺伝子、マウス Ras遺伝子、マウス Cyclin-D遺伝子、マウス Cdk遺伝子、マウス TNF- a遺伝子、マウス TGF- 遺伝子、ヒト ΤΥρ53遺伝子、ヒト Rb遺伝子、ヒト Ras遺伝子、ヒ ト Cyclin-D遺伝子、ヒト Cdk遺伝子、ヒト TNF- a遺伝子、ヒト TGF- β遺伝子）のホモ口 グからなる群より選択される少なくとも 1つの遺伝子が例示できる。  The canceration-related genes in the animal cells of the present invention are as follows: 1) If the animal cells are human cells, human TYp53 gene, human Rb gene, human Ras gene, human Cyclin_D gene, human Cdk gene, human TNF-a gene Human TGF-β gene and at least one gene selected from the group consisting of these mutants; 2) if the animal cell is a mouse cell, for example, mouse Trp53 gene, mouse Rb gene, mouse Ras gene, Mouse Cyclin-D gene, mouse Cdk gene, mouse TNF-a gene, mouse TGF-β gene, and at least one gene selected from the group consisting of these mutant forces; 3) animal cells other than human or mouse For example, a gene such as mouse ΤΥρ53 gene (in addition to mouse Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene, mouse TNF-a gene, mouse TGF-gene, human ΤΥρ53 gene, human Rb gene, human Ras gene, human Cyclin-D gene, human Cdk gene, human TNF-a gene, human TGF-β gene) Examples include at least one gene selected from the group consisting of
[0031] また、本発明は非ヒト動物または動物細胞の GsdmA遺伝子の機能を抑制し、かつ 癌遺伝子の機能を促進する方法、または GsdmA遺伝子の機能を抑制し、かつ癌抑 制遺伝子の機能を抑制する方法を提供する。癌は、多数の癌化関連遺伝子に変異 が蓄積することにより発症すると考えられている。したがって GsdmA遺伝子の機能を 抑制及び癌化関連遺伝子の機能を促進または抑制することにより、非ヒト動物または 動物細胞を癌化できる。  [0031] Further, the present invention provides a method for suppressing the function of a GsdmA gene in a non-human animal or animal cell and promoting the function of an oncogene, or suppressing the function of a GsdmA gene, and the function of a cancer suppressor gene. A method of suppressing is provided. Cancer is thought to develop by accumulating mutations in many canceration-related genes. Therefore, non-human animals or animal cells can be cancerized by suppressing the function of the GsdmA gene and promoting or suppressing the function of the canceration-related gene.
[0032] 本発明において、 GsdmAl、 GsdmA2、 GsdmA3、 GSDMA,マウス Trp53およびヒト Tr p53をコードする DNAの塩基配列を、それぞれ配列番号： 1、 3、 5、 7、 9および 1 1に、 該 DNAによりコードされるタンパク質のアミノ酸配列を、それぞれ配列番号： 2、 4、 6、 8、 10および 12に示す。  [0032] In the present invention, the base sequences of DNAs encoding GsdmAl, GsdmA2, GsdmA3, GSDMA, mouse Trp53 and human Trp53 are represented by SEQ ID NOs: 1, 3, 5, 7, 9 and 11 respectively. The amino acid sequences of the proteins encoded by are shown in SEQ ID NOs: 2, 4, 6, 8, 10 and 12, respectively.
[0033] また、 GsdmAl、 GsdmA2、 GsdmA3、 GSDMA,マウス T卬 53、ヒト丁卬53の変異体、バ リアント、ホモログを、以下の（a)または (b)に示す。  [0033] Further, GsdmAl, GsdmA2, GsdmA3, GSDMA, mouse T53, and human cho 53 mutants, variants, and homologs are shown in the following (a) or (b).
(a)配列番号： 2、 4、 6、 8、 10、 12のいずれかに記載のアミノ酸配列において 1若し くは複数のアミノ酸が置換、欠失、挿入、および/または付加したアミノ酸配列からな るタンパク質であって、配列番号：2、 4、 6、 8、 10、 12のいずれかに記載のアミノ酸 配列からなるタンパク質と機能的に同等なタンパク質 (a) From the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12 wherein one or more amino acids are substituted, deleted, inserted and / or added. Na A protein functionally equivalent to a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12
(b)配列番号： 1、 3、 5、 7、 9、 1 1のいずれかに記載の塩基配列を含む DNAとストリン ジヱントな条件下でハイブリダィズする DNAよりコードされるタンパク質であって、配列 番号： 2、 4、 6、 8、 10、 12のいずれかに記載のアミノ酸配列力 なるタンパク質と機 能的に同等なタンパク質  (b) SEQ ID NO: 1, 3, 5, 7, 9, 11 A protein encoded by DNA that hybridizes under stringent conditions with DNA comprising the base sequence described in any one of SEQ ID NO: : A protein functionally equivalent to the protein having the amino acid sequence ability described in any of 2, 4, 6, 8, 10, 12
[0034] ここで「機能的に同等」とは、対象となるタンパク質力 GsdmAl、 GsdmA2、 GsdmA3 、 GSDMA、マウス TYp53、またはヒト ΤΥρ53と同等の生物学的機能（生物学的役害 ι 生 物学的性質)、生化学的機能 (生化学的活性)を有することを指す。  [0034] Here, "functionally equivalent" refers to biological functions equivalent to the target protein strength GsdmAl, GsdmA2, GsdmA3, GSDMA, mouse TYp53, or human ΤΥρ53 (biological harm ι biology) Property), biochemical function (biochemical activity).
[0035] 本明細書においては、マウス Rb遺伝子、マウス Ras遺伝子、マウス Cyclin-D遺伝子 、マウス Cdk遺伝子、マウス TNF- a遺伝子、マウス TGF- β遺伝子、ヒト Rb遺伝子、ヒ ト Ras遺伝子、ヒト Cyclin-D遺伝子、ヒト Cdk遺伝子、ヒト TNF- a遺伝子、ヒト TGF- β 遺伝子の変異体、バリアント、ホモログも上記と同様に定義づけられる。  [0035] In the present specification, mouse Rb gene, mouse Ras gene, mouse Cyclin-D gene, mouse Cdk gene, mouse TNF-a gene, mouse TGF-β gene, human Rb gene, human Ras gene, human Cyclin Mutants, variants and homologues of the -D gene, human Cdk gene, human TNF-a gene, and human TGF-β gene are also defined as described above.
[0036] あるタンパク質と機能的に同等な天然由来のタンパク質をコードする DNAを調製す る当業者によく知られた他の方法としては、ハイブリダィゼーシヨン技術（SambrookJ et ai. , Molecular Cloning 2nd ed.， 9.47-9.58， Cold spring Harbor Lab. press, 1989) を利用する方法が挙げられる。即ち、当業者においては、 GsdmAl、 GsdmA2、 Gsdm A3、 GSDMA、マウス T卬 53、マウス Rb、マウス Ras、マウス Cyclin_D、マウス Cdk、マウ ス TNF— a、マウス TGF- β、ヒト Trp53、ヒト Rb、ヒト Ras、ヒト Cyclin_D、ヒト Cdk、ヒト TNF -ひ、ヒト TGF- j3をコードする DNA配歹 1J (以下 GsdmAl等と称す）もしくはその一部を 利用して、これと相同性の高い DNAを単離することは、周知の技術である。  [0036] Other methods well known to those skilled in the art for preparing a DNA encoding a naturally occurring protein that is functionally equivalent to a protein include the hybridization technique (SambrookJ et ai., Molecular Cloning). 2nd ed., 9.47-9.58, Cold spring Harbor Lab. Press, 1989). That is, those skilled in the art will recognize that GsdmAl, GsdmA2, Gsdm A3, GSDMA, mouse T 卬 53, mouse Rb, mouse Ras, mouse Cyclin_D, mouse Cdk, mouse TNF-a, mouse TGF-β, human Trp53, human Rb, A DNA sequence that encodes human Ras, human Cyclin_D, human Cdk, human TNF-s, human TGF-j3, or a portion thereof, or a part thereof, is used to singly homologous DNA. Separation is a well-known technique.
[0037] 本発明には、 GsdmAl等をコードする DNAとストリンジヱントな条件下でハイブリダィ ズし、 GsdmAl等と機能的に同等な天然由来のタンパク質をコードする DNAが含まれ る。  [0037] The present invention includes a DNA that hybridizes with a DNA encoding GsdmAl or the like under stringent conditions and encodes a naturally derived protein that is functionally equivalent to GsdmAl or the like.
[0038] GsdmAl等と機能的に同等な天然由来のタンパク質をコードする DNAを単離するた めのハイブリダィゼーシヨンの条件は、当業者であれば適宜選択することができる。ハ イブリダィゼーシヨンの条件としては、例えば、低ストリンジェントな条件が挙げられる。 低ストリンジェントな条件とは、ハイブリダィゼーシヨン後の洗浄にぉレ、て、例えば 42°C 、 5xSSC、 0.1%SDSの条件であり、好ましくは 50。C、 5xSSC、 0.1%SDSの条件である。 より好ましレ、ノヽイブリダィゼーシヨンの条件としては、高ストリンジヱントな条件が挙げら れる。高ストリンジェントな条件とは、例えば 65°C、 O.lxSSC及び 0.1%SDSの条件であ る。これらの条件において、温度を上げる程に高い相同性を有する DNAが効率的に 得られると期待できる。但し、ハイブリダィゼーシヨンのストリンジエンシーに影響する 要素としては温度や塩濃度など複数の要素が考えられ、当業者であればこれら要素 を適宜選択することで同様のストリンジエンシーを実現することが可能である。 [0038] Hybridization conditions for isolating a DNA encoding a naturally occurring protein functionally equivalent to GsdmAl or the like can be appropriately selected by those skilled in the art. Examples of the hybridization conditions include low stringency conditions. Low stringency conditions are often associated with post-hybridization washing, for example 42 ° C. 5xSSC, 0.1% SDS, preferably 50. The conditions are C, 5xSSC, and 0.1% SDS. Highly stringent conditions can be cited as the conditions for the more preferred and noisy hybridization. Highly stringent conditions are, for example, conditions of 65 ° C, O.lxSSC and 0.1% SDS. Under these conditions, it can be expected that DNA with high homology will be obtained efficiently as the temperature is increased. However, multiple factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art will realize the same stringency by appropriately selecting these factors. It is possible.
[0039] また、 GsdmAl等をコードする DNAの配列情報を基に合成したプライマーを用いる 遺伝子増幅法、例えば、ポリメラーゼ連鎖反応（PCR)法を利用して、 GsdmAl等と機 能的に同等な天然由来のタンパク質をコードする DNAを単離することも可能である。  [0039] Furthermore, a gene amplification method using a primer synthesized based on the sequence information of DNA encoding GsdmAl or the like, for example, a natural equivalent functionally to GsdmAl or the like using a polymerase chain reaction (PCR) method. It is also possible to isolate the DNA encoding the protein of origin.
[0040] これらハイブリダィゼーシヨン技術や遺伝子増幅技術により単離される DNAがコード する、 GsdmAl等と機能的に同等な天然由来のタンパク質は、通常、 GsdmAl等とアミ ノ酸配列において高い相同性を有する。本発明のタンパク質には、 GsdmAl等と機能 的に同等であり、かつ該タンパク質のアミノ酸配列と高い相同性を有する天然由来の タンパク質も含まれる。高い相同性とは、アミノ酸レベルにおいて、通常、少なくとも 50 %以上の同一性、好ましくは 75%以上の同一性、さらに好ましくは 85%以上の同一 性、さらに好ましくは 95%以上の同一性を指し、塩基レベルにおいて、通常、少なくと も 50%以上の同一性、好ましくは 75%以上の同一性、さらに好ましくは 85%以上の同 一性、さらに好ましくは 95%以上の同一性を指す。  [0040] Naturally-derived proteins functionally equivalent to GsdmAl and the like encoded by DNA isolated by these hybridization and gene amplification techniques usually have high homology in amino acid sequence with GsdmAl and the like. Have The protein of the present invention includes naturally derived proteins that are functionally equivalent to GsdmAl and the like and have high homology with the amino acid sequence of the protein. High homology generally refers to at least 50% identity, preferably 75% identity, more preferably 85% identity, more preferably 95% identity at the amino acid level. In the base level, it usually means at least 50% identity, preferably 75% identity, more preferably 85% identity, more preferably 95% identity.
[0041] アミノ酸配列や塩基配列の同一性は、 Karlin and Altschulによるアルゴリズム BLAS T(Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993)によって決定することができる。こ のアルゴリズムに基づいて、 BLASTNや BLASTXと呼ばれるプログラムが開発されて いる (Altschul et al. J. Mol. Biol.215:403-410, 1990)。 BLASTに基づいて BLASTNに よって塩基配列を解析する場合には、パラメータ一はたとえば score = 100、 wordlengt h = 12とする。また、 BLASTに基づいて BLASTXによってアミノ酸配列を解析する場合 には、パラメータ一はたとえば score = 50、 wordlength = 3とする。 BLASTと Gapped BL ASTプログラムを用いる場合には、各プログラムのデフォルトパラメーターを用いる。こ れらの解析方法の具体的な手法は公知である (http：〃 www.ncbi.nlm.nih.gov·)。 [0042] また、 GsdmAl等のアミノ酸配列において 1もしくは複数のアミノ酸が変異したァミノ 酸配列からなり、該タンパク質と機能的に同等な天然由来のタンパク質もまた本発明 のタンパク質に含まれる。このようなアミノ酸の変異は自然界においても生じうる。変 異するアミノ酸数は、通常、 30アミノ酸以内であり、好ましくは 15アミノ酸以内であり、よ り好ましくは 5アミノ酸以内（例えば、 3アミノ酸以内）であり、さらに好ましくは 2アミノ酸 以内である。 [0041] The identity of amino acid sequences and nucleotide sequences can be determined by the algorithm BLAS T (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993) by Karlin and Altschul. Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When analyzing a base sequence by BLASTN based on BLAST, the parameter 1 is, for example, score = 100 and wordlengt h = 12. In addition, when the amino acid sequence is analyzed by BLASTX based on BLAST, the parameter 1 is, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BL AST programs, use the default parameters of each program. Specific methods of these analysis methods are known (http: 〃 www.ncbi.nlm.nih.gov ·). [0042] In addition, naturally occurring proteins that are composed of amino acid sequences in which one or more amino acids are mutated in the amino acid sequence such as GsdmAl and are functionally equivalent to the protein are also included in the protein of the present invention. Such amino acid mutations can also occur in nature. The number of amino acids to be varied is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids (for example, within 3 amino acids), and even more preferably within 2 amino acids.
[0043] 変異するアミノ酸残基にぉレ、ては、アミノ酸側鎖の性質が保存されてレ、る別のアミノ 酸に変異されることが望ましい。例えばアミノ酸側鎖の性質としては、疎水性アミノ酸（ A、 I、し、 M、 F、 P、 W、 Y、 V)、親水十生アミノ酸（R、 D、 N、 C、 E、 Q、 G、 H、 K、 S、 T)、月旨 肪族側鎖を有するアミノ酸（G、 A、 V、 L、 I、 P)、水酸基含有側鎖を有するアミノ酸 (S、 T、 Υ)、硫黄原子含有側鎖を有するアミノ酸（C、 M)、カルボン酸及びアミド含有側鎖 を有するアミノ酸 (D、 N、 E、 Q)、塩基含有側鎖を有するアミノ酸 (R、 K、 Η)、芳香族 含有側鎖を有するアミノ酸 (H、 F、 Y、 W)を挙げることができる（括弧内はいずれもアミ ノ酸の一文字標記を表す)。  [0043] It is desirable that the amino acid residue to be mutated should be mutated to another amino acid that preserves the properties of the amino acid side chain. For example, the properties of amino acid side chains include hydrophobic amino acids (A, I, M, F, P, W, Y, V), hydrophilic degenerate amino acids (R, D, N, C, E, Q, G , H, K, S, T), amino acids having an aliphatic side chain (G, A, V, L, I, P), amino acids having a hydroxyl group-containing side chain (S, T, Υ), sulfur atom Amino acids with side chains (C, M), amino acids with side chains containing carboxylic acids and amides (D, N, E, Q), amino acids with side chains (R, K, Η), aromatics Amino acids having side chains (H, F, Y, W) can be mentioned (the parentheses indicate single letter amino acids).
[0044] あるアミノ酸配列に対する 1又は複数個のアミノ酸残基の欠失、付加及び/又は他 のアミノ酸による置換により修飾されたアミノ酸配列を有するタンパク質がその生物学 的活性を維持することはすでに知られている（Mark, D. F. et al., Pro Natl. Acad. S ci. USA (1984) 81， 5662-5666、 Zoller, M. J. & Smith, M. Nucleic Acids Research (1 982) 10， 6487-6500、 Wang, A. et al., Science 224, 1431—1433、 Dalbadie-McFarland , G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413)。  [0044] It is already known that a protein having an amino acid sequence modified by deletion, addition and / or substitution by another amino acid residue to one amino acid sequence maintains its biological activity. (Mark, DF et al., Pro Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, MJ & Smith, M. Nucleic Acids Research (1 982) 10, 6487-6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).
[0045] 本発明における GsdmA遺伝子および癌抑制遺伝子の機能抑制には、完全な抑制 および部分的な抑制の双方が含まれる。また、遺伝子の機能抑制には、遺伝子対の 一方または双方の機能の抑制も含まれる。  [0045] The functional suppression of the GsdmA gene and the tumor suppressor gene in the present invention includes both complete suppression and partial suppression. In addition, suppression of gene function includes suppression of the function of one or both of a gene pair.
[0046] 遺伝子の機能の抑制は、遺伝子の発現抑制、および遺伝子への変異導入によつ て行うことができる。該変異は、その種類、その数、その部位などに制限はない。上記 変異の種類としては、例えば、付加、欠失 (完全欠失または部分欠失のいずれであつ てもよい）、置換または揷入変異などが挙げられる。また、変異は、 自然界において 生じた変異または人為的に導入した変異 (例えば、変異原処理によって生じた変異、 遺伝子改変技術によって導入した変異）のレ、ずれであってもよレ、。 [0046] Suppression of gene function can be performed by suppressing gene expression and introducing a mutation into the gene. There is no restriction on the type, number, site, etc. of the mutation. Examples of the type of mutation include addition, deletion (which may be complete deletion or partial deletion), substitution, or insertion mutation. In addition, mutations may be mutations that occur in nature or artificially introduced mutations (for example, mutations caused by mutagen treatment, Mutations introduced by genetic modification techniques), or even deviations.
[0047] 本発明の非ヒト動物の好ましい態様としては、 GsdmA遺伝子および T卬 53遺伝子の 遺伝子対の一方に変異を有する非ヒト動物（以下変異型 GsdmAヘテロ ·変異型 Trp53 ヘテロ非ヒト動物と称す）、 GsdmA遺伝子の遺伝子対の一方に変異を有し、かつ Trp5 3遺伝子の遺伝子対の双方に変異を有する非ヒト動物（以下変異型 GsdmAヘテロ'変 異型 Trp53ホモ非ヒト動物と称す）、 GsdmA遺伝子の遺伝子対の双方に変異を有し、 かつ TYp53遺伝子の遺伝子対の一方に変異を有する非ヒト動物（以下変異型 GsdmA ホモ'変異型 Trp53ヘテロ非ヒト動物と称す）、 GsdmA遺伝子および TYp53遺伝子の遺 伝子対の双方に変異を有する非ヒト動物（以下変異型 GsdmAホモ'変異型 Τ卬 53ホモ 非ヒト動物と称す）が挙げられる。 [0047] As a preferred embodiment of the non-human animal of the present invention, a non-human animal having a mutation in one of the gene pair of GsdmA gene and T 卬 53 gene (hereinafter referred to as mutant GsdmA hetero-mutant Trp53 hetero-non-human animal) ), A non-human animal having a mutation in one of the GsdmA gene pairs and a mutation in both of the Trp53 3 gene pairs (hereinafter referred to as mutant GsdmA hetero'mutant Trp53 homo non-human animals), GsdmA A non-human animal having a mutation in both gene pairs and a mutation in one of the TYp53 gene pairs (hereinafter referred to as mutant GsdmA homo'mutant Trp53 hetero-non-human animal), GsdmA gene and TYp53 gene Non-human animals having mutations in both gene pairs (hereinafter referred to as mutant GsdmA homo'mutant Τ 卬 53 homo non-human animals).
[0048] また、変異型 GsdmAヘテロ ·変異型 TYp53ヘテロ非ヒト動物、変異型 GsdmAヘテロ · 変異型 TYp53ホモ非ヒト動物、変異型 GsdmAホモ'変異型 ΤΥρ53ヘテロ非ヒト動物、変 異型 GsdmAホモ ·変異型 ΤΥρ53ホモ非ヒト動物としては、例えば、それぞれ、 Rim3へテ 口 ·ΤΥρ53ノックアウトヘテロマウス、 Rim3ヘテロ · ΤΥρ53ノックアウトホモマウス（GsdmA- 3Rin +;TYp537_)、 Rim3ホモ · ΤΥρ53ノックアウトヘテロマウス（GsdmA-3Rim3/ Rim3;TYp53 7+)、 Rim3ホモ ·ΤΥρ53ノックアウトホモマウス（GsdmA_3Rin Rim3; Trp537")が挙げら れる力 これらに限定されるものではない。 [0048] Also, mutant GsdmA heterozygous mutant TYp53 heteronon-human animal, mutant GsdmA heterozygous mutant TYp53 homononhuman animal, mutant GsdmA homo'mutant ΤΥρ53 heterononhuman animal, mutant GsdmA homozygous mutant Type 型 ρ53 homozygous non-human animals include, for example, Rim3 mice, ΤΥρ53 knockout heterozygous mice, Rim3 heterozygous ΤΥρ53 knockout homozygous mice (GsdmA-3 Rin +; TYp537_), Rim3 homozygous ΤΥρ53 knockout heterozygous mice (GsdmA- 3 Rim3 / Rim3 ; TYp53 7+), Rim3 homozygous ΤΥρ53 knockout homo mouse (GsdmA_3 Rin Rim3 ; Trp537 ")
[0049] 変異型 GsdmAヘテロ ·変異型 Trp53ヘテロ非ヒト動物、変異型 GsdmAヘテロ '変異 型 Trp53ホモ非ヒト動物、変異型 GsdmAホモ'変異型 Trp53ヘテロ非ヒト動物、変異型 GsdmAホモ.変異型 TYp53ホモ非ヒト動物は、以下の（i)〜（iv)の交配、および、この 交配によって得られた個体同士を交配することにより得ることができる。 [0049] Mutant GsdmA hetero-mutant Trp53 hetero non-human animal, mutant GsdmA hetero 'mutant Trp 53 homo non-human animal, mutant GsdmA homo' mutant Trp 53 hetero non-human animal, mutant GsdmA homo. Mutant TYp53 Homo non-human animals can be obtained by mating the following (i) to (iv) and mating individuals obtained by this mating.
(i)変異型 GsdmAヘテロ非ヒト動物と変異型 Trp53ヘテロ非ヒト動物  (i) Mutant GsdmA hetero-non-human animal and mutant Trp53 hetero-non-human animal
(ii)変異型 GsdmAヘテロ非ヒト動物と変異型 Trp53ホモ非ヒト動物  (ii) Mutant GsdmA hetero-non-human animals and mutant Trp53 homo-non-human animals
(iii)変異型 GsdmAホモ非ヒト動物と変異型 Trp53ヘテロ非ヒト動物  (iii) Mutant GsdmA homo non-human animal and mutant Trp53 hetero non-human animal
(iv)変異型 GsdmAホモ非ヒト動物と変異型 Trp53ホモ非ヒト動物  (iv) Mutant GsdmA homo non-human animal and mutant Trp53 homo non-human animal
また、本発明においては、自然界において生じた変異を有する非ヒト動物と人為的 に導入した変異を有する非ヒト動物の交配、自然界におレ、て生じた変異を有する非ヒ ト動物同士の交配、人為的に導入した変異を有する非ヒト動物同士の交配を適宜選 択しうる。 Further, in the present invention, a non-human animal having a naturally occurring mutation and a non-human animal having an artificially introduced mutation, and a non-human animal having a naturally occurring mutation are crossed. Appropriate selection of mating between non-human animals with artificially introduced mutations You can choose.
[0050] 特定の遺伝子の機能抑制は、当業者に公知の方法によって行うことができる。例え ば、遺伝子改変技術 (標的遺伝子部位の組み換えを促進する酵素、例えば、 Cre-lo Xにおける Cre、の導入による条件的遺伝子改変技術も含む）を用いた方法、アンチ センス DNAを用いた方法、リボザィムをコードする DNAを用いた方法、または、 RNAi 技術を用いた方法等が挙げられる。  [0050] Suppression of the function of a specific gene can be performed by methods known to those skilled in the art. For example, a method using a gene modification technique (including a conditional gene modification technique by introducing an enzyme that promotes recombination of a target gene site, for example, Cre in Cre-lo X), a method using an antisense DNA, Examples include a method using DNA encoding a ribozyme, a method using RNAi technology, and the like.
[0051] 遺伝子の機能が抑制された非ヒト動物の作製は、例えば次にようにして行うことがで きる。まず、標的遺伝子を含むゲノム DNA断片をクローニングして、これを基に、内在 性の標的遺伝子改変のための相同組み換え用ベクターを構築する。該相同組み換 え用ベクターには、標的遺伝子またはその発現制御領域の少なくとも 1部を欠失/変 異させた核酸配歹 IJ、標的遺伝子またはその発現制御領域にヌクレオチドやポリヌクレ ォチドが挿入された核酸配歹 IJ、標的遺伝子またはその発現制御領域に他の遺伝子 が挿入された核酸配列を含むが、標的遺伝子の活性が失われる核酸配列であれば 、前記の欠失/変異部位および挿入部位は限定されなレ、。  [0051] The production of a non-human animal in which the function of a gene is suppressed can be performed, for example, as follows. First, a genomic DNA fragment containing the target gene is cloned, and based on this, a vector for homologous recombination for endogenous target gene modification is constructed. The homologous recombination vector has a nucleic acid-ligated IJ in which at least a part of the target gene or its expression control region has been deleted / mutated, and a nucleotide or polynucleotide inserted into the target gene or its expression control region. The nucleic acid sequence IJ includes a nucleic acid sequence in which another gene is inserted into the target gene or its expression control region, but the deletion / mutation site and insertion site described above can be used as long as the target gene activity is lost. It ’s not limited.
[0052] 挿入される遺伝子としては、例えば、ネオマイシン耐性遺伝子、チミジンキナーゼ遺 伝子、ジフテリア毒素遺伝子等が挙げられる。また、これらの組み合わせも考えられる 。該相同組み換え用ベクターの基本骨格は、特に、制限はなぐ例えば pKO Scrambl er Series (レキシコン）等を用いることができる。  [0052] Examples of inserted genes include neomycin resistance gene, thymidine kinase gene, diphtheria toxin gene and the like. Combinations of these are also possible. As the basic skeleton of the vector for homologous recombination, for example, pKO Scrambler Series (Lexicon) or the like without limitation can be used.
[0053] 構築した相同組み換え用ベクターを、個体へ分化させることが可能な非ヒト動物細 胞（例えば、胚性幹細胞（ES細胞））に導入し、内因性の標的遺伝子との相同組み換 えを行うことで、遺伝子改変非ヒト動物細胞を作製する。遺伝子対の片側の発現が抑 制されている該細胞を作製するには、例えば、ネオマイシンで細胞を選択する方法 で得ることが可能である。相同組み換え用ベクターの細胞への導入は、当業者に公 知の方法によって行うことができる。具体的には、エレクト口ポレーシヨン法等を例示 すること力 Sできる。  [0053] The constructed vector for homologous recombination is introduced into a non-human animal cell (for example, embryonic stem cell (ES cell)) capable of differentiating into an individual, and homologous recombination with an endogenous target gene is performed. To produce genetically modified non-human animal cells. In order to produce the cell in which the expression of one side of the gene pair is suppressed, it can be obtained by, for example, a method of selecting a cell with neomycin. The vector for homologous recombination can be introduced into cells by methods known to those skilled in the art. Specifically, the ability to exemplify the Elect Mouth Position Method etc. can be demonstrated.
[0054] 本発明において個体へ分化させることが可能な動物細胞として ES細胞を用いた場 合は、例えば、該細胞を胚盤胞に注入することによりキメラ胚を作製し、偽妊娠させた 動物の子宮に移植して産仔を得る。遺伝子改変 ES細胞由来の組織を有するキメラ動 物を選別できるようにするために、より好適には、作製された個体の外部的特徴 (例 えば、毛色）が、遺伝子改変 ES細胞由来の組織と胚盤胞由来の組織とで異なるよう に、胚盤胞を選択する。 [0054] When ES cells are used as animal cells that can be differentiated into individuals in the present invention, for example, chimeric embryos are produced by injecting the cells into blastocysts and pseudopregnant animals. Transplant into the uterus of the baby to obtain a litter. Chimeric motion with tissue derived from genetically modified ES cells More preferably, the external characteristics (eg, hair color) of the created individual are different between the tissue derived from the genetically modified ES cell and the tissue derived from the blastocyst. Select a blastocyst.
[0055] また、遺伝子改変 ES細胞由来の生殖組織を有するキメラ動物であるか否かの判定 は、一般的には、該キメラ非ヒト動物と適当な系統の同種非ヒト動物との交配により得 られた仔の毛色で行うが、その他の方法として、例えば、該キメラ非ヒト動物の尻尾も しくは耳から抽出した DNAを铸型とした PCR反応を行レ、、揷入された遺伝子の有無を 検出する方法やサザンハイブリダィゼーシヨン法を用いることも可能である。  [0055] In addition, the determination of whether or not a chimeric animal has a reproductive tissue derived from a genetically modified ES cell is generally obtained by crossing the chimeric non-human animal with an appropriate non-human animal of the same strain. Other methods include, for example, performing a PCR reaction using the DNA extracted from the tail or ear of the chimeric non-human animal as a cage, and the presence or absence of the inserted gene. It is also possible to use a method for detecting selenium or a Southern hybridization method.
[0056] 該キメラ動物と適当な系統の同種動物との交配により得られた仔力 ヘテロ接合型 遺伝子改変非ヒト動物であるか否かは、例えば、該遺伝子改変動物から抽出した DN Aを铸型とする PCRやサザンハイブリダィゼーシヨンで判定することができる。また、へ テロ接合型遺伝子改変非ヒト動物同士の交配により、ホモ接合型遺伝子改変非ヒト 動物を作製することができる。交配により得られた仔が、所望の遺伝子型を有する非 ヒト動物であるか否かも上記の判定法に従う。  [0056] The pup power obtained by mating the chimeric animal with the same species of the same species is heterozygous. For example, whether or not it is a genetically modified non-human animal can be determined by analyzing DNA extracted from the genetically modified animal. It can be determined by PCR or Southern hybridization. In addition, homozygous genetically modified non-human animals can be produced by crossing heterozygous genetically modified non-human animals. Whether or not the offspring obtained by mating is a non-human animal having a desired genotype also follows the above-mentioned determination method.
[0057] また、本発明の遺伝子改変非ヒト動物の作製は、上記の方法に制限されない。例え ば、体細胞クローン動物の作製技術に従って動物を作製することもできる。具体的に は、 ES細胞以外の体細胞（例えば、皮膚細胞等）を用いて、 ES細胞の場合と同様な 方法に従って動物細胞を作製することができる。さらに、該非ヒト動物細胞から、体細 胞クローン作製技術を応用し、非ヒト動物を作製することができる。  [0057] The production of the genetically modified non-human animal of the present invention is not limited to the above method. For example, an animal can be produced according to a technique for producing a somatic cell cloned animal. Specifically, animal cells can be produced using somatic cells other than ES cells (for example, skin cells) according to the same method as for ES cells. Furthermore, a non-human animal can be produced from the non-human animal cell by applying a somatic cell clone production technique.
[0058] 遺伝子の機能が抑制された非ヒト動物の作製は、本発明の遺伝子の転写産物と相 補的な RNAをコードする DNAを用いることでも行うことができる。このような DNAとして は、アンチセンス DNAが挙げられる。  [0058] The production of a non-human animal in which the function of a gene is suppressed can also be performed by using a DNA encoding an RNA complementary to the transcription product of the gene of the present invention. Such DNA includes antisense DNA.
[0059] アンチセンス核酸が標的遺伝子の発現を抑制する作用としては、以下のような複数 の要因が存在する。すなわち、三重鎖形成による転写開始阻害、 RNAポリメラーゼに よって局部的に開状ループ構造がつくられた部位とのハイブリッド形成による転写抑 制、合成の進みつつある RNAとのハイブリッド形成による転写阻害、イントロンとェキ ソンとの接合点でのハイブリッド形成によるスプライシング抑制、スプライソソーム形成 部位とのハイブリッド形成によるスプライシング抑制、 mRNAとのハイブリッド形成によ る核から細胞質への移行抑制、キヤッビング部位やポリ (A)付加部位とのハイブリッド 形成によるスプライシング抑制、翻訳開始因子結合部位とのハイブリッド形成による 翻訳開始抑制、開始コドン近傍のリボソーム結合部位とのハイブリッド形成による翻 訳抑制、 mRNAの翻訳領域やポリソーム結合部位とのハイブリッド形成によるペプチド 鎖の伸長阻止、および核酸とタンパク質との相互作用部位とのハイブリッド形成によ る遺伝子発現抑制などである。これらは、転写、スプライシング、または翻訳の過程を 阻害して、標的遺伝子の発現を抑制する。 [0059] There are several factors as described below for the action of an antisense nucleic acid to suppress the expression of a target gene. That is, transcription initiation inhibition by triplex formation, transcription inhibition by hybridization with a site where an open loop structure has been locally created by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, intron By suppressing splicing by hybridization at the junction of lysine and exon, by suppressing splicing by hybridization with the spliceosome formation site, and by hybridization with mRNA Suppression of translocation from the nucleus to the cytoplasm, suppression of splicing by hybridization with a capping site or poly (A) addition site, suppression of translation initiation by hybridization with a translation initiation factor binding site, hybridization with a ribosome binding site near the initiation codon Examples include suppression of translation by formation, inhibition of peptide chain elongation by hybridization with mRNA translation region and polysome binding site, and suppression of gene expression by hybridization of nucleic acid and protein interaction sites. They inhibit the expression of target genes by inhibiting transcription, splicing, or translation processes.
[0060] 本発明で用いられるアンチセンス配列は、上記のいずれの作用で標的遺伝子の発 現を抑制してもよい。一つの態様としては、遺伝子の mRNAの 5'端近傍の非翻訳領域 に相補的なアンチセンス配歹 ljを設計すれば、遺伝子の翻訳阻害に効果的であろう。 しかし、コード領域もしくは 3'側の非翻訳領域に相補的な配歹 I」も使用し得る。このよう に、遺伝子の翻訳領域だけでなく非翻訳領域の配列のアンチセンス配列を含む DN Aも、本発明で利用されるアンチセンス DNAに含まれる。使用されるアンチセンス DN Aは、適当なプロモーターの下流に連結され、好ましくは 3'側に転写終結シグナルを 含む配列が連結される。このようにして調製された DNAは、公知の方法で、所望の細 胞へ形質転換できる。アンチセンス DNAの配列は、形質転換する細胞が持つ内在性 遺伝子またはその一部と相補的な配列であることが好ましいが、遺伝子の発現を有 効に阻害できる限り、完全に相補的でなくてもよい。転写された RNAは、標的とする 遺伝子の転写産物に対して好ましくは 90%以上、最も好ましくは 95%以上の相補性 を有する。アンチセンス配列を用いて、効果的に標的遺伝子の発現を阻害するには 、アンチセンス DNAの長さは、少なくとも 15塩基以上であり、好ましくは 100塩基以上 であり、さらに好ましくは 500塩基以上である。通常、用いられるアンチセンス DNAの 長さは 5kbよりも短ぐ好ましくは 2.5kbよりも短い。  [0060] The antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above. In one embodiment, designing an antisense probe lj complementary to the untranslated region near the 5 ′ end of the mRNA of a gene would be effective for inhibiting translation of the gene. However, it is also possible to use a coordination I "complementary to the coding region or the 3 'untranslated region. Thus, the DNA containing the antisense sequence of the sequence of the untranslated region as well as the translated region of the gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The DNA thus prepared can be transformed into a desired cell by a known method. The sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene or a part of the cell to be transformed, but it must be completely complementary as long as the gene expression can be effectively inhibited. Also good. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene. In order to effectively inhibit the expression of a target gene using an antisense sequence, the length of the antisense DNA is at least 15 bases, preferably 100 bases or more, more preferably 500 bases or more. is there. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.
[0061] 本発明の遺伝子の転写産物と相補的な RNAの他の一つの態様は、本発明の遺伝 子の転写産物と相補的な dsRNAである。 RNAiは、標的遺伝子配列と同一もしくは類 似した配列を有する二重鎖 RNA (以下 dsRNA)を細胞内に導入すると、導入した外来 遺伝子および標的内在性遺伝子の発現がいずれも抑制される現象である。細胞に 約 40〜数百塩基対の dsRNAが導入されると、ヘリカーゼドメインを持つダイサー (Dice r)と呼ばれる RNaselll様のヌクレアーゼカ ATP存在下で、 dsRNAを 3'末端から約 21〜 23塩基対ずつ切り出し、 siRNA (short interference RNA)を生じる。この siRNAに特異 的なタンパク質が結合して、ヌクレアーゼ複合体（RISC： RNA-induced silencing comp lex)が形成される。この複合体は siRNAと同じ配列を認識して結合し、 RNaselll様の酵 素活性によって siRNAの中央部で標的遺伝子の mRNAを切断する。また、この経路と は別に siRNAのアンチセンス鎖が mRNAに結合して RNA依存性 RNAポリメラーゼ (RsR P)のプライマーとして作用し、 dsRNAが合成される。この dsRNAが再びダイサ一の基 質となって、新たな siRNAを生じて作用を増幅する経路も考えられている。 [0061] Another embodiment of RNA complementary to the transcription product of the gene of the present invention is dsRNA complementary to the transcription product of the gene of the present invention. RNAi is a phenomenon in which the expression of the introduced foreign gene and target endogenous gene is suppressed when double-stranded RNA (hereinafter referred to as dsRNA) having the same or similar sequence as the target gene sequence is introduced into the cell. . When dsRNA of about 40 to several hundred base pairs is introduced into a cell, Dicer with a helicase domain (Dice In the presence of an RNaselll-like nuclease ATP called r), dsRNA is cleaved from the 3 ′ end by about 21 to 23 base pairs to produce siRNA (short interference RNA). A protein specific to this siRNA binds to form a nuclease complex (RISC: RNA-induced silencing complex). This complex recognizes and binds to the same sequence as siRNA, and cleaves the mRNA of the target gene at the center of the siRNA by RNaselll-like enzyme activity. Apart from this pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. It is also considered that this dsRNA becomes a dicer's base again, generating a new siRNA and amplifying the action.
[0062] 本発明の RNAは、標的遺伝子 mRNAのいずれかの領域に対するアンチセンス RNA をコードしたアンチセンスコード DNAと、標的遺伝子 mRNAのいずれかの領域のセン ス RNAをコードしたセンスコード DNAより発現させることができる。また、これらのアン チセンス RNAおよびセンス RNAより dsRNAを作成することもできる。  [0062] The RNA of the present invention is expressed from an antisense coding DNA encoding an antisense RNA for any region of the target gene mRNA and a sense coding DNA encoding a sense RNA of any region of the target gene mRNA. Can be made. DsRNA can also be prepared from these antisense RNAs and sense RNAs.
[0063] 本発明の dsRNAの発現システムを、ベクター等に保持させる場合の構成としては、 同一のベクターからアンチセンス RNA、センス RNAを発現させる場合と、異なるベクタ 一からそれぞれアンチセンス RNA、センス RNAを発現させる場合がある。例えば、同 一のベクターからアンチセンス RNA、センス RNAを発現させる構成としては、アンチセ ンスコード DNAおよびセンスコード DNAの上流にそれぞれ ροΙΙΠ系のような短い RNA を発現し得るプロモーターを連結させたアンチセンス RNA発現カセット、センス RNA 発現カセットをそれぞれ構築し、これらカセットを同方向にあるいは逆方向にベクター に揷入することにより構成することができる。また、異なる鎖上に対向するようにアンチ センスコード DNAとセンスコード DNAと逆向きに配置した発現システムを構成すること もできる。この構成では、アンチセンス RNAコード鎖とセンス RNAコード鎖とが対となつ た一つの二本鎖 DNA (siRNAコード DNA)が備えられ、その両側にそれぞれの鎖から アンチセンス RNA、センス RNAとを発現し得るようにプロモーターを対向して備えられ る。この場合には、センス RNA、アンチセンス RNAの下流に余分な配列が付加される ことを避けるために、それぞれの鎖（アンチセンス RNAコード鎖、センス RNAコード鎖） の 3'末端にターミネータ一をそれぞれ備えることが好ましい。このターミネータ一は、 A (アデニン)塩基を 4つ以上連続させた配列などを用いることができる。また、このパ リンドロームスタイルの発現システムでは、二つのプロモーターの種類を異ならせるこ とが好ましい。 [0063] When the dsRNA expression system of the present invention is held in a vector or the like, the antisense RNA and the sense RNA are expressed from the same vector and the antisense RNA and the sense RNA are expressed from the same vector, respectively. May be expressed. For example, antisense RNA and sense RNA can be expressed from the same vector by combining an antisense RNA and a promoter capable of expressing a short RNA such as ροΙΙΠ upstream of the sense code DNA. It can be constructed by constructing an expression cassette and a sense RNA expression cassette, respectively, and inserting these cassettes into the vector in the same direction or in the opposite direction. It is also possible to construct an expression system in which the antisense coding DNA and the sense coding DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA are connected from each strand to both sides. A promoter is provided oppositely so that it can be expressed. In this case, in order to avoid adding an extra sequence downstream of the sense RNA and antisense RNA, a terminator is added to the 3 'end of each strand (antisense RNA coding strand, sense RNA coding strand). Each is preferably provided. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. This par In a Linde style expression system, the two promoter types are preferably different.
[0064] また、異なるベクターからアンチセンス RNA、センス RNAを発現させる構成としては、 例えば、アンチセンスコード DNAおよびセンスコード DNAの上流にそれぞれ ροΙΠΙ系 のような短レ、 RNAを発現し得るプロモーターを連結させたアンチセンス RNA発現カセ ット、センス RNA発現カセットをそれぞれ構築し、これらカセットを異なるベクターに保 持させることにより構成することができる。  [0064] In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, a short promoter such as a ρο 短 system, which can express RNA, is provided upstream of the antisense code DNA and the sense code DNA, respectively. It can be constructed by constructing a linked antisense RNA expression cassette and sense RNA expression cassette, and holding these cassettes in different vectors.
[0065] 本発明の RNAiにおいては、 dsRNAとして siRNAが使用されたものであってもよレ、。「 siRNA」は、細胞内で毒性を示さない範囲の短鎖からなる二重鎖 RNAを意味し、例え ば、 15〜49塩基対と、好適には 15〜35塩基対と、さらに好適には 21〜30塩基対とす ることができる。あるいは、発現される siRNAが転写され最終的な二重鎖 RNA部分の 長さが、例えば、 15〜49塩基対、好適には 15〜35塩基対、さらに好適には 21〜30塩 基対とすることができる。  [0065] In the RNAi of the present invention, siRNA may be used as dsRNA. “SiRNA” means a double-stranded RNA consisting of short strands that are not toxic in the cell, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably It can be 21-30 base pairs. Alternatively, the siRNA to be expressed is transcribed and the length of the final double-stranded RNA portion is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, more preferably 21 to 30 base pairs. can do.
RNAiに用いる DNAは、標的遺伝子と完全に同一である必要はないが、少なくとも 70 %以上、好ましくは 80%以上、さらに好ましくは 90%以上、最も好ましくは 95%以上の 配列の相同性を有する。  The DNA used for RNAi need not be completely identical to the target gene, but has at least 70%, preferably 80%, more preferably 90%, most preferably 95% or more sequence homology. .
[0066] dsRNAにおける RNA同士が対合した二重鎖 RNAの部分は、完全に対合しているも のに限らず、ミスマッチ（対応する塩基が相補的でない）、バルジ (一方の鎖に対応す る塩基がなレ、）などにより不対合部分が含まれていてもよい。本発明においては、 dsR NAにおける RNA同士が対合する二重鎖 RNA領域中に、バルジおよびミスマッチの両 方が含まれていてもよい。  [0066] The double-stranded RNA part of the dsRNA in which RNAs are paired with each other is not limited to a perfect pair, but includes mismatches (corresponding bases are not complementary), bulges (corresponding to one strand) An unpaired portion may be included due to the base that is not used. In the present invention, both bulges and mismatches may be contained in the double-stranded RNA region where RNAs in dsRNA pair with each other.
[0067] 本発明の遺伝子の発現の抑制は、また、リボザィムをコードする DNAを利用して行 うことも可能である。リボザィムとは触媒活性を有する RNA分子のことをいう。リボザィ ムには種々の活性を有するものがある力 中でも RNAを切断する酵素としてのリボザ ィムの研究により、 RNAの部位特異的な切断を目的とするリボザィムの設計が可能と なった。リボザィムには、グループ Iイントロン型や、 RNasePに含まれる M1RNAのよう に 400ヌクレオチド以上の大きさのものもある力 ハンマーヘッド型やヘアピン型と呼 ばれる 40ヌクレオチド程度の活性ドメインを有するものもある。 [0068] 例えば、ハンマーヘッド型リボザィムの自己切断ドメインは、 G13U14C15の C15の 3' 側を切断するが、活性には U14が 9位の Aと塩基対を形成することが重要とされ、 15位 の塩基は Cの他に Aまたは Uでも切断されることが示されている。リボザィムの基質結 合部を標的部位近傍の RNA配列と相補的になるように設計すれば、標的 RNA中の U C、 UUまたは UAとレ、う配列を認識する制限酵素的な RNA切断リボザィムを作出する ことが可能である。例えば、阻害標的となる本発明の酵素のコード領域中には標的と なりうる部位が複数存在する。 [0067] Inhibition of the expression of the gene of the present invention can also be carried out using a DNA encoding a ribozyme. Ribozyme refers to an RNA molecule having catalytic activity. Among ribozymes that have various activities, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for the purpose of site-specific cleavage of RNA. Some ribozymes have a group I intron type or a force hammerhead type or hairpin type having an active domain of about 40 nucleotides such as M1RNA contained in RNaseP having a size of 400 nucleotides or more. [0068] For example, the self-cleaving domain of the hammerhead ribozyme cleaves on the 3 'side of C15 of G13U14C15, but it is important for U14 to base-pair with A at position 9 for activity. It has been shown that the base of can be cleaved by A or U in addition to C. If the ribozyme substrate binding site is designed to be complementary to the RNA sequence in the vicinity of the target site, a restriction enzyme-like RNA cleavage ribozyme that recognizes UC, UU, or UA in the target RNA will be created. It is possible to do. For example, there are a plurality of sites that can be targets in the coding region of the enzyme of the present invention that is an inhibition target.
[0069] また、ヘアピン型リボザィムも、本発明の目的のために有用である。ヘアピン型リボ ザィムは、例えばタバコリングスポットウィルスのサテライト RNAのマイナス鎖に見出さ れる (J.M.Buzayan Nature 323:349, 1986)。このリボザィムも、標的特異的な RNA切断 を起こすように設計できることが示されてレ、る。  [0069] Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (J.M.Buzayan Nature 323: 349, 1986). This ribozyme has also been shown to be designed to cause target-specific RNA cleavage.
[0070] 標的を切断できるよう設計されたリボザィムは、植物細胞中で転写されるようにカリ フラワーモザイクウィルスの 35Sプロモーターなどのプロモーターおよび転写終結配 列に連結される。しかし、その際、転写された RNAの 5'末端や 3'末端に余分な配列が 付加されていると、リボザィムの活性が失われてしまうことがある。このようなとき、転写 されたリボザィムを含む RNAからリボザィム部分だけを正確に切り出すために、リボザ ィム部分の 5'側や 3'側に、トリミングを行うためのシスに働く別のトリミングリボザィムを 配置させることも可能である (K.Taira et al. (1990) Protein Eng. 3:733、 A.M.Dzianotta nd J.J.Bujarski (1989) Proc.Natl.Acad.Sci.USA. 86:4823、 C.A.Grosshansand R.T.C ech (1991) Nucleic Acids Res. 19:3875、 K.Taira et al. (1991) Nucleic Acids Res. 19 :5125)。また、このような構成単位をタンデムに並べ、標的遺伝子内の複数の部位を 切断できるようにして、より効果を高めることもできる (N.Yuyama et al. Biochem.Biophy s.Res.Commun. l86: 1271, 1992)0このようなリボザィムを用いて本発明で標的となる遺 伝子の転写産物を特異的に切断し、該遺伝子の発現を抑制することができる。 [0070] A ribozyme designed to cleave the target is linked to a promoter such as the cauliflower mosaic virus 35S promoter and a transcription termination sequence so that it is transcribed in plant cells. However, ribozyme activity may be lost if extra sequences are added to the 5 'or 3' end of the transcribed RNA. In such a case, in order to accurately cut out only the ribozyme portion from the RNA containing the transcribed ribozyme, another trimming ribozyme that acts as a cis for trimming is provided on the 5 ′ side or 3 ′ side of the ribozyme portion. (K. Taira et al. (1990) Protein Eng. 3: 733, AMDzianotta nd JJ Bujarski (1989) Proc. Natl. Acad. Sci. USA. 86: 4823, CAGrosshansand RTC ech (1991) Nucleic Acids Res. 19: 3875, K. Taira et al. (1991) Nucleic Acids Res. 19: 5125). In addition, such structural units can be arranged in tandem so that multiple sites in the target gene can be cleaved, thereby further enhancing the effect (N. Yuyama et al. Biochem. Biophys. Res. Commun. L86 : 1271, 1992) 0 Using such a ribozyme, the gene transcript targeted by the present invention can be specifically cleaved to suppress the expression of the gene.
[0071] 本発明においては、トランスジエニック技術を用いて、遺伝子発現を抑制する上記 D NAを所望の非ヒト動物に導入することで、 GsdmA遺伝子および癌抑制遺伝子の機能 が抑制されている非ヒト動物を作製することもできる。  [0071] In the present invention, the functions of the GsdmA gene and the tumor suppressor gene are suppressed by introducing the above-mentioned DNA that suppresses gene expression into a desired non-human animal using transgene technology. Human animals can also be produced.
[0072] トランスジエニック動物の作製方法は公知である。例えば、 Pro Natl. Acad. Sci. U SA 77:7380-7384(1980)に記載の方法により、トランスジヱニック動物を得ることができ る。具体的には、遺伝子発現を抑制する上記 DNAを動物の全能細胞に導入し、この 細胞を個体へと発生させる。得られた個体のうち、体細胞および生殖細胞中に導入 遺伝子が組み込まれた個体を選別することによって、 目的とするトランスジヱニック動 物を作製することができる。遺伝子を導入する全能細胞としては、受精卵や初期胚の ほか、多分化能を有する ES細胞のような培養細胞などが挙げられる。当業者におい ては、上記の方法を適宜改変して、所望の遺伝子の発現が改変されたトランスジェニ ック動物を作製することが可能である。 [0072] Methods for producing transgenic animals are known. For example, Pro Natl. Acad. Sci. U Transgenic animals can be obtained by the method described in SA 77: 7380-7384 (1980). Specifically, the above-mentioned DNA that suppresses gene expression is introduced into a totipotent cell of an animal, and this cell is generated into an individual. By selecting individuals from which the transgene has been incorporated into somatic cells and germ cells, the desired transgeneic animal can be produced. Examples of totipotent cells into which genes are introduced include fertilized eggs and early embryos, and cultured cells such as ES cells having multipotency. A person skilled in the art can appropriately modify the above method to produce a transgenic animal in which the expression of a desired gene is modified.
[0073] 遺伝子発現を抑制する DNAは、該 DNAを導入すべき動物の細胞において発現可 能なプロモーターに連結した組み換え遺伝子コンストラクト（発現ベクター）とするの が一般的である。本発明の組み換え遺伝子コンストラクトは、適当な宿主を利用して クローニング可能なベクターに、前記上記の遺伝子発現を抑制する DNAと、その上 流にプロモーターとを挿入し、クローニングすることによって構築することができる。 [0073] The DNA that suppresses gene expression is generally a recombinant gene construct (expression vector) linked to a promoter that can be expressed in the cells of an animal into which the DNA is to be introduced. The recombinant gene construct of the present invention can be constructed by inserting the above-described DNA that suppresses gene expression into a vector that can be cloned using an appropriate host and a promoter upstream thereof and cloning. it can.
[0074] 本発明に利用することができるプロモーターとしては、動物細胞で発現可能なプロ モーターであれば特に制限されず、例えば、哺乳動物細胞由来のプロモーターや、 サイトメガロウィルス、レトロウイルス、ポリオ一マウィルス、アデノウイルス、シミアンウイ ルス 40 (SV40)等のウィルスプロモーターを挙げることができる。例えば、 SV40のプロ モーターを使用する場合は、 Mulliganらの方法（Nature (1990) 277， 108)に従って、 上記コンストラクトを作製することができる。  [0074] The promoter that can be used in the present invention is not particularly limited as long as it is a promoter that can be expressed in animal cells. For example, promoters derived from mammalian cells, cytomegalovirus, retrovirus, polio Examples include virus promoters such as mavirus, adenovirus, and simian virus 40 (SV40). For example, when the SV40 promoter is used, the above construct can be prepared according to the method of Mulligan et al. (Nature (1990) 277, 108).
[0075] また、本発明に使用可能なベクターとしては、導入遺伝子を動物の生体内で広範 囲に発現誘導でき得るものであればよぐ当業者において周知の発現ベクターを利 用すること力 Sできる。  [0075] In addition, as a vector that can be used in the present invention, any expression vector known to those skilled in the art may be used as long as it can induce expression of a transgene in a wide range in an animal body. it can.
[0076] 適当な制限酵素によって前記ベクターから切り出した組み換え遺伝子コンストラクト は、十分に精製されトランスジエニック動物の作製に用いられる。通常、トランスジェニ ック動物は、未受精卵、受精卵、精子およびその始原細胞を含む胚芽細胞などに、 前記コンストラクトを導入することによって作製される。コンストラクトを導入する細胞と しては、通常、非ヒト哺乳動物の発生における胚発生の段階、より具体的には単細胞 あるいは受精卵細胞の段階で、通常 8細胞期以前のものが利用される。上記コンスト ラタトの導入方法としては、例えば、リン酸カルシウム法、エレクト口ポレーシヨン法、リ ポフエクシヨン法、凝集法、マイクロインジェクション法、パーティクルガン法、 DEAE— デキストラン法等が公知である。さらに、こうして得られた形質転換細胞を上述の胚芽 細胞と融合させることによりトランスジヱニック動物を作製することも可能である。 [0076] The recombinant gene construct excised from the vector with an appropriate restriction enzyme is sufficiently purified and used to produce a transgenic animal. Usually, a transgenic animal is produced by introducing the construct into an unfertilized egg, a fertilized egg, a sperm and a germ cell containing a primordial cell thereof. The cells into which the construct is introduced are usually used at the stage of embryonic development in the development of non-human mammals, more specifically at the stage of single cells or fertilized egg cells, and usually those before the 8-cell stage. Const above As a method for introducing ratato, for example, the calcium phosphate method, the electopore position method, the repofusion method, the aggregation method, the microinjection method, the particle gun method, the DEAE-dextran method and the like are known. Furthermore, it is also possible to produce a transgenic animal by fusing the thus obtained transformed cell with the above-mentioned embryo cell.
[0077] マウスの場合、例えば、排卵誘発剤を投与したメスのマウスに正常なォスのマウスを 交配させることにより、コンストラクトの導入が可能な受精卵を回収することができる。 マウス受精卵では、一般に雄性前核へのマイクロインジェクションによりコンストラクト が導入される。コンストラクトを導入した細胞は、体外での培養の後、導入に成功した と思われる細胞が代理母の卵管に移植され、トランスジエニックキメラ動物が誕生する 。代理母には、通常、精管を切断したォスと交配させて偽妊娠状態としたメスが利用 される。 In the case of a mouse, for example, by mating a normal male mouse with a female mouse administered with an ovulation inducing agent, a fertilized egg capable of introducing a construct can be collected. In mouse fertilized eggs, the construct is generally introduced by microinjection into the male pronucleus. The cells into which the construct has been introduced are cultured outside the body, and then the cells that appear to have been successfully introduced are transplanted into the surrogate mother's fallopian tube, and a transgenic chimeric animal is born. A surrogate mother is usually a female that has been pseudopregnant by mating with males that have had their vagina cut.
[0078] 生まれたトランスジヱニックキメラ動物は、上記の遺伝子発現を抑制する DNAが組 み込まれていることを確認した上で、 F1動物の誕生のために正常な動物と交配させ る。一般にコンストラクトとして導入した外来 DNAは、ゲノムの同一の部分に複数コピ 一が直列に組み込まれる。通常はこの組み込みコピー数が多いほど、多量の遺伝子 発現につながり、より明瞭な表現型が期待できる。体細胞ゲノムにおいて、上記の遺 伝子発現を抑制する DNAが組み込まれていることは、コンストラクトに特異的なプライ マーを用いた PCRや特異的なプローブを用いたサザンブロット法によって確認するこ とがでさる。  [0078] Transborn chimeric animals are bred with normal animals for the birth of F1 animals after confirming that the DNAs that suppress the above gene expression have been incorporated. In general, foreign DNA introduced as a construct has multiple copies integrated in series in the same part of the genome. In general, the higher the number of integrated copies, the greater the expression of the gene, and a clearer phenotype can be expected. The incorporation of DNA that suppresses the above gene expression in the somatic cell genome can be confirmed by PCR using a primer specific for the construct or Southern blotting using a specific probe. It is out.
[0079] この交配の結果誕生する F1動物の中で、体細胞に外来遺伝子（上記の遺伝子発 現を抑制する DNA)を有するもの（ヘテロザィゴート）は、生殖細胞に外来遺伝子を伝 えることができるトランスジエニック動物である。 F1動物の中から体細胞に外来遺伝子 を保持するものを選び、これらを両親とすることによって、 F2動物であるホモザィゴー トを得ること力 Sできる。  [0079] Among F1 animals born as a result of this mating, those that have foreign genes in the somatic cells (DNA that suppresses the above gene expression) (heterozygotes) can transmit foreign genes to germ cells. Transgenic animal. The ability to obtain homozygotes that are F2 animals can be obtained by selecting F1 animals that have foreign genes in somatic cells and using them as parents.
[0080] また、本発明においては、トランスジヱニック技術を用いて、癌遺伝子をコードする D NAを所望の非ヒト動物に導入することで、癌遺伝子の機能が促進されている非ヒト動 物を作製することもできる。作製された非ヒト動物と GsdmA遺伝子の機能が抑制され ている非ヒト動物を交配することで、 GsdmA遺伝子の機能が抑制され、癌遺伝子の機 能が促進された非ヒト動物を作製することができる。 [0080] Furthermore, in the present invention, non-human kinetics in which the function of an oncogene is promoted by introducing DNA encoding the oncogene into a desired non-human animal using transgene technology. Things can also be made. By mating the prepared non-human animal with a non-human animal in which the function of the GsdmA gene is suppressed, the function of the GsdmA gene is suppressed and the Non-human animals with enhanced performance can be produced.
[0081] また、本発明においては、トランスジヱニック技術を用いて、癌遺伝子をコードする D NAを、 GsdmA遺伝子の機能が抑制されている非ヒト動物に導入することで、 GsdmA 遺伝子の機能が抑制され、癌遺伝子の機能が促進された非ヒト動物を作製することも できる。 [0081] Further, in the present invention, the function of the GsdmA gene is obtained by introducing DNA encoding an oncogene into a non-human animal in which the function of the GsdmA gene is suppressed, using transgene technology. It is also possible to produce non-human animals in which the function of oncogenes is promoted.
[0082] また、本発明は GsdmA遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進 または抑制されている動物細胞（GsdmA遺伝子の機能が抑制され、癌遺伝子の機能 が促進されている動物細胞、または GsdmA遺伝子の機能が抑制され、癌抑制遺伝 子の機能が抑制されている動物細胞）に関する。本発明の動物細胞が由来する生物 種は、特に制限はなぐヒトを含む種々の生物種由来の細胞である。また、本発明の 動物細胞としては、例えば、体細胞、受精卵、 ES細胞、本発明の非ヒト動物から樹立 された細胞が挙げられる力 これらに限定されるものではない。上記非ヒト動物由来 の細胞株を樹立する方法としては、公知の方法を利用することができる。例えば、げ つ歯類においては、胎仔細胞の初代培養の方法を用いることが可能である（新生化 学実験講座、 18卷、 125頁〜 129頁東京化学同人、およびマウス胚の操作マニュアル 、 262頁〜 264頁、近代出版)。  [0082] Further, the present invention relates to an animal cell in which the function of the GsdmA gene is suppressed and the function of the canceration-related gene is promoted or suppressed (an animal in which the function of the GsdmA gene is suppressed and the function of the oncogene is promoted). Cell or animal cell in which the function of the GsdmA gene is suppressed and the function of the tumor suppressor gene is suppressed. The biological species from which the animal cell of the present invention is derived is a cell derived from various biological species including humans without particular limitation. Further, the animal cell of the present invention is not limited to these, including, for example, a somatic cell, a fertilized egg, an ES cell, and a cell established from the non-human animal of the present invention. As a method for establishing the cell line derived from the non-human animal, a known method can be used. For example, in rodents, it is possible to use a method of primary culture of fetal cells (Neochemistry Laboratory, 18 pp. 125-129, Tokyo Chemistry Doujin, and Mouse Embryo Operation Manual, 262 Pp. 264, modern publication).
[0083] 本発明は、本発明の非ヒト動物の表現型を相補する化合物のスクリーニング方法を 提供する。  [0083] The present invention provides a screening method for a compound that complements the phenotype of the non-human animal of the present invention.
本発明の方法における「被験試料」としては、特に制限はなぐ例えば、天然化合 物、有機化合物、無機化合物、タンパク質、ペプチド、抗体等の単一化合物、並びに 、化合物ライブラリー、遺伝子ライブラリー、抗体ライブラリーの発現産物、細胞抽出 物、細胞培養上清、発酵微生物産生物、海洋生物抽出物、植物抽出物、原核細胞 抽出物、真核単細胞抽出物もしくは動物細胞抽出物等を挙げることができる。上記 被験試料は必要に応じて適宜標識して用いることができる。標識としては、例えば、 放射標識、蛍光標識等を挙げることができる。  The “test sample” in the method of the present invention is not particularly limited, for example, a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, a peptide, or an antibody, and a compound library, gene library, antibody Examples include library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts. . The test sample can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.
[0084] また、本発明におけるスクリーニング方法の第一の態様は、本発明の非ヒト動物の 表現型を相補する化合物のスクリーニングに関するものである。第一の態様としては 、まず、被検化合物を本発明の非ヒト動物に投与する。本発明の非ヒト動物への被検 化合物の投与は、例えば、経口的または非経口的に行うことができるが、それらに限 定されない。被検化合物がタンパク質である場合には、例えば、該タンパク質をコー ドする遺伝子を有するウィルスベクターを構築し、その感染力を利用して、本発明の 非ヒト動物に該遺伝子を導入することも可能である。第一の態様としては、次いで、被 検化合物が非ヒト動物の表現型を相補しているか否かを判定する。次いで、被検化 合物を投与してレ、なレ、場合と比較して、非ヒト動物の表現型を相補する化合物を選 択する。 [0084] The first aspect of the screening method of the present invention relates to the screening of compounds that complement the phenotype of the non-human animal of the present invention. In the first embodiment, first, a test compound is administered to the non-human animal of the present invention. Examination of non-human animals of the present invention Administration of the compound can be performed, for example, orally or parenterally, but is not limited thereto. When the test compound is a protein, for example, a viral vector having a gene encoding the protein can be constructed, and the gene can be introduced into the non-human animal of the present invention using its infectivity. Is possible. In the first embodiment, it is then determined whether or not the test compound complements the phenotype of the non-human animal. Then, a test compound is administered, and a compound that complements the phenotype of the non-human animal as compared with the case is selected.
[0085] 非ヒト動物の表現型としては、癌 (例えば、皮膚癌、喉頭癌、舌癌、食道癌、胃癌、 乳癌、大腸癌、卵巣癌、膀胱癌、子宮癌、膝臓癌、腎臓癌、肺癌、メラノーマ、白血 病等）の発症、進行、転移等、特に皮膚癌の発症、進行、転移等が例示できる。また 、非ヒト動物の表現型の相補としては、癌の発症、進行、転移等の抑制が挙げられる  [0085] Non-human animal phenotypes include cancer (eg, skin cancer, laryngeal cancer, tongue cancer, esophageal cancer, gastric cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, uterine cancer, knee cancer, kidney cancer. Onset, progression, metastasis, etc. of lung cancer, melanoma, leukemia etc., especially onset, progression, metastasis, etc. of skin cancer. In addition, examples of complementation of the phenotype of non-human animals include suppression of cancer onset, progression, metastasis and the like.
[0086] 本発明におけるスクリーニング方法の第二の態様は、本発明の動物細胞の表現型 を相補する化合物のスクリーニングに関する。第二の態様としては、まず、被検化合 物を本発明の動物細胞に接触させる。 [0086] The second embodiment of the screening method of the present invention relates to screening for compounds that complement the phenotype of animal cells of the present invention. In the second embodiment, first, the test compound is brought into contact with the animal cell of the present invention.
[0087] 本発明において「接触」は、例えば、細胞の培養液に被験試料を添加することによ り行うことができる。被験試料がタンパク質の場合には、例えば、該タンパク質をコー ドする DNAを含むベクターを、該細胞へ導入することも可能である。  In the present invention, “contact” can be performed, for example, by adding a test sample to a cell culture medium. When the test sample is a protein, for example, a vector containing DNA encoding the protein can be introduced into the cell.
[0088] 次レ、で、被検化合物が動物細胞の表現型を相補してレ、るか否かを判定する。次レ、 で、被検化合物を投与していない場合と比較して、動物細胞の表現型を相補する化 合物を選択する。  [0088] Next, whether or not the test compound complements the phenotype of the animal cell is determined. In the next step, a compound that complements the phenotype of the animal cell is selected as compared to the case where the test compound is not administered.
本発明の細胞の表現型としては、増殖異常、分化異常、接着異常、移動能の亢進 等が例示できる。また、細胞の表現型の相補としては、増殖異常、分化異常、接着異 常、移動能の亢進等の抑制が挙げられる。  Examples of the cell phenotype of the present invention include abnormal growth, abnormal differentiation, abnormal adhesion, and enhanced mobility. In addition, complementation of cell phenotype includes suppression of proliferation abnormality, differentiation abnormality, adhesion abnormality, enhancement of mobility and the like.
[0089] また、本発明は、 GsdmAl、 GsdmA2、 GsdmA3、 GSDMA、もしくは、これらタンパク質 の変異体、バリアント、ホモログ（以下 GsdmAl、 GsdmA2、 GsdmA3、または GSDMA等 と称す)、または、これらの断片に結合する抗体を提供する。本発明の抗体は、癌の 検査や、癌の研究等において、上記タンパク質の検出に利用することができる。該抗 体の種類には、特に制限はなレ、が、例えばモノクローナル抗体、またはポリクローナ ル抗体の両方を利用することができる。該抗体は、当業者に公知の方法により調製 することが可能である。ポリクローナル抗体であれば、例えば、次のようにして取得す ることができる。 GsdmAl、 GsdmA2、 GsdmA3または GSDMA等、これらと GSTとの融合 タンパク質として大腸菌等の微生物において発現させたリコンビナントタンパク質、ま たはその部分断片をゥサギ等の小動物に免疫し血清を得る。これを、例えば、硫安 沈殿、プロテイン A、プロテイン Gカラム、 DEAEイオン交換クロマトグラフィー、 GsdmAl 、 GsdmA2、 GsdmA3または GSDMA等や合成ペプチドをカップリングしたァフィ二ティ 一力ラム等により精製することにより調製する。また、モノクローナル抗体であれば、例 えば、 GsdmAl、 GsdmA2、 GsdmA3または GSDMA等またはその部分断片をマウス等 の小動物に免疫を行い、同マウスより脾臓を摘出し、これをすりつぶして細胞を分離 し、該細胞とマウスミエローマ細胞とをポリエチレングリコール等の試薬を用いて融合 させ、これによりできた融合細胞（ノヽイブリドーマ）の中から、 GsdmAl, GsdmA2、 Gsd mA3または GSDMA等に結合する抗体を産生するクローンを選択する。次いで、得ら れたハイブリドーマをマウス腹腔内に移植し、同マウスより腹水を回収し、得られたモ ノクローナル抗体を、例えば、硫安沈殿、プロテイン A、プロテイン Gカラム、 DEAEィォ ン交換クロマトグラフィー、 GsdmAl、 GsdmA2、 GsdmA3または GSDMA等や合成ぺプ チドをカップリングしたァフィ二ティーカラム等により精製することで、調製することが可 能である。 [0089] Further, the present invention binds to GsdmAl, GsdmA2, GsdmA3, GSDMA, or mutants, variants, homologs (hereinafter referred to as GsdmAl, GsdmA2, GsdmA3, or GSDMA) of these proteins, or fragments thereof. An antibody is provided. The antibody of the present invention can be used for detection of the protein in cancer screening, cancer research, and the like. The anti There are no particular restrictions on the type of body. For example, both monoclonal antibodies and polyclonal antibodies can be used. The antibody can be prepared by methods known to those skilled in the art. For example, a polyclonal antibody can be obtained as follows. GsdmAl, GsdmA2, GsdmA3, GSDMA, and the like, and recombinant proteins expressed in microorganisms such as Escherichia coli as a fusion protein of these with GST, or a partial fragment thereof, are immunized to small animals such as rabbits to obtain serum. This is prepared, for example, by purifying with ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, GsdmAl, GsdmA2, GsdmA3, GSDMA, etc., or a affinity ram with a synthetic peptide coupled. . In the case of a monoclonal antibody, for example, GsdmAl, GsdmA2, GsdmA3, or GSDMA or a partial fragment thereof is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to isolate cells. A clone that produces an antibody that binds to GsdmAl, GsdmA2, Gsd mA3, GSDMA, etc. from the fused cells (neubridoma) produced by fusing the cells with mouse myeloma cells using a reagent such as polyethylene glycol. Select. Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is purified by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography. GsdmAl, GsdmA2, GsdmA3, GSDMA or the like, or a purification column coupled with a synthetic peptide can be used for the preparation.
[0090] 上記タンパク質の断片は、実施例に記載の断片、また、例えば、 GsdmAl, GsdmA2 、 GsdmA3または GSDMA等の C末端領域であり、このような領域としては、 GsdmA3の アミノ酸第 343番目から第 451番目、 GsdmAlのアミノ酸第 334番目から第 442番目、 Gs dmA2のアミノ酸第 352番目から第 460番目、 GSDMAのアミノ酸第 333番目から第 441 番目が挙げられる力 これらに限定されるものではない。  [0090] The protein fragment is a fragment described in the examples, or a C-terminal region such as GsdmAl, GsdmA2, GsdmA3, or GSDMA. Examples of such a region include the 343rd to 343th amino acids of GsdmA3. 451st, amino acids 334 to 442 of GsdmAl, amino acids 352 to 460 of GsdmA2, amino acids 333 to 441 of GSDMA, and the like, but not limited thereto.
なお、本明細書において引用された全ての先行技術文献は、参照として本明細書 に組み入れられる。  It should be noted that all prior art documents cited in this specification are incorporated herein by reference.
実施例  Example
[0091] 以下、本発明を実施例により、さらに具体的に説明するが本発明はこれら実施例に 制限されるものではない。 [0091] The present invention will be described more specifically with reference to the following examples. It is not limited.
材料と方法  Materials and methods
1.実験動物（マウス）  1. Experimental animal (mouse)
C57BL/6J, C57BL/10Jは、 Jackson Laboratoryより購入した。突然変異マウス ' Rim 3は、国立遺伝学研究所において独自に系統化され、 p53 (Trp53)ノックアウトマウス は、相沢慎一博士 (現 ·理化学研究所 ·発生再生科学総合研究センター)より提供さ れた。これらのマウス系統は、国立遺伝学研究所'系統生物研究センターにおいて 飼育管理され、実験には系統生物研究センターにおいて繁殖させた動物を用いた。 なお動物実験は、国立遺伝学研究所'動物実験委員会の承認を得ると共にそのガイ ドラインに則って行った。  C57BL / 6J and C57BL / 10J were purchased from Jackson Laboratory. Mutant mice '' Rim 3 was independently systematized at the National Institute of Genetics, and p53 (Trp53) knockout mice were provided by Dr. Shinichi Aizawa (currently RIKEN Research Center for Developmental Sciences) . These mouse strains were bred and managed at the National Institute of Genetics' Center for Biological Biology, and animals bred at the Center for Biological Biology were used for experiments. Animal experiments were approved in accordance with the guidelines of the National Institute of Genetics' Animal Experimentation Committee.
[0092] 2. Rim3ヘテロ ·ρ53ノックアウトヘテロマウス  [0092] 2. Rim3 heterozygous ρ53 knockout heterozygous mouse
Rim3ヘテロ · ρ53ノックアウトヘテロ個体 (GsdmA3Rim3/+;TYp53—/+)の作出には、 Rim3 ヘテロ個体 (GsdmA3Rim3/+)と p53ノックアウトヘテロ個体 (TYp53—/+)を自然交配すること により得た。この親系統の組み合わせにより得られる子供の遺伝型は、 Rim3ヘテロ · ρ 53ノックアウトヘテロ個体以外に Rim3ヘテロ個体（GsdmA3Rim3/+;TYp53+/+)、 p53ノッ クアウトへテロ個体（GsdmA3+/+;TYp53—/+)、正常個体（GsdmA3+/+;TYp53+/+)であ る。これら全ての産子は、同一条件下（明暗条件、温度、湿度、飼料、飲料水、床敷 き等）で飼育し、皮膚癌発症の割合を経時的に観察した。 Rim3 heterozygous · ρ53 knockout heterozygous individuals (GsdmA3 Rim3 / +; TYp53 — / +) were created by natural mating of Rim3 heterozygous individuals (GsdmA3 Rim3 / +) and p53 knockout heterozygous individuals (TYp53 — / +). It was. In addition to Rim3 hetero · ρ53 knockout heterozygous individuals, the genotypes of children obtained from this parental lineage combination include Rim3 heterozygous individuals (GsdmA3 Rim3 / +; TYp53 + / +), p53 knockout heterozygous individuals (GsdmA3 + / +; TYp53 — / +), Normal individuals (GsdmA3 + / +; TYp53 + / +). All these offspring were raised under the same conditions (light / dark conditions, temperature, humidity, feed, drinking water, flooring, etc.), and the rate of skin cancer was observed over time.
[0093] 3.細胞系及び細胞培養  [0093] 3. Cell lines and cell cultures
4種の皮膚癌細胞株： HSC1 (皮膚扁平上皮癌）、 HSC4 (舌扁平上皮癌）、 HSC5 (皮 膚扁平上皮癌）、及び A431 (外陰部類上皮癌）はヒューマンサイエンス振興財団'ヒュ 一マンサイエンス研究資源バンク力 購入した。 HSC1は、 20%胎仔ゥシ血清、及び ペニシリン一ストレプトマイシンが補足された DMEM、 HSC4は、 10%胎仔ゥシ血清、 及びペニシリン—ストレプトマイシンが補足された MEM、 HSC5は、 10%胎仔ゥシ血清 、及びペニシリン—ストレプトマイシンが補足された ISC0ve' s改変 DMEM、 A431は 10 %胎仔ゥシ血清、及びペニシリン—ストレプトマイシンが補足された DMEMの中で培 養を行った。 Four skin cancer cell lines: HSC1 (squamous cell carcinoma of the skin), HSC4 (squamous cell carcinoma of the tongue), HSC5 (squamous cell carcinoma of the skin), and A431 (epithelioid carcinoma of the vulva) are human science promotion foundation Science Research Resource Bank Power purchased. HSC1 is 20% fetal urine serum and DMEM supplemented with penicillin-streptomycin, HSC4 is 10% fetal urine serum, and MEM supplemented with penicillin-streptomycin, HSC5 is 10% fetal urine serum, and penicillin - streptomycin I SC0ve 's modified DMEM supplemented, A431 10% fetal © shea serum and penicillin - streptomycin was cultured in DMEM supplemented.
[0094] 4. RT-PCR解析（RT-PCR) 皮膚癌培養細胞からの全 RNAの調製は、 ISOGEN (日本ジーン社)を使用し、供給 元の推奨する手順に従って行った。ヒト正常皮膚全 RNAは、 Invitrogen社より購入した 。各皮膚癌培養細胞から得られた全 RNAは、 37°Cで 30分間、 DNA分解酵素で処理し 、サンプル中のゲノム DNAを除去した。全 RNAから cDNAの合成の手順は、以下の通 りである。各皮膚癌培養細胞全 RNA、並びにヒト正常皮膚全 RNA、 7.5 x gに 10mM d NTP (lOmM dATP, lOmM dCTP, lOmM dGTP, lOmM dTTP)を 3 μ 1、 Oligo(dT) プ [0094] 4. RT-PCR analysis (RT-PCR) Preparation of total RNA from cultured skin cancer cells was performed using ISOGEN (Nippon Gene) according to the procedure recommended by the supplier. Human normal skin total RNA was purchased from Invitrogen. Total RNA obtained from each cultured skin cancer cell was treated with a DNA-degrading enzyme at 37 ° C. for 30 minutes to remove genomic DNA in the sample. The procedure for synthesizing cDNA from total RNA is as follows. Total RNA of cultured skin cancer cells and human normal skin total RNA, 7.5 μg, 10 μm dNTP (lOmM dATP, lOmM dCTP, lOmM dGTP, lOmM dTTP) 3 μ1, Oligo (dT)
12-18 ライマーを 1500ngカ卩え、 RNA分解酵素除去処理を行った蒸留水で、最終量 39 μ ΐに 調製した。この溶液を 65°Cで 5分間処理し、その後氷中にて急冷した。十分に氷温ま で下がった溶液に 5x First strand buffer (250mM Tris- HC1 pH 8.3, 375mM KC1, 15 mM MgCl )を 12 μ 1、 lOOmM DTTを 3 μ 1、 RNA Inhibitorを 3 μ 1、逆転写酵素 (SuperSc  12-18 Limer was added in an amount of 1500 ng, and the final volume was adjusted to 39 μΐ with distilled water from which RNAse was removed. This solution was treated at 65 ° C for 5 minutes and then rapidly cooled in ice. 5x First strand buffer (250 mM Tris-HC1 pH 8.3, 375 mM KC1, 15 mM MgCl) 12 μ1, lOOmM DTT 3 μ1, RNA Inhibitor 3 μ1, reverse transcriptase (SuperSc
2  2
ript III Reverse Transcriptase: Invitrogen社)を 3 μ 1カロえ、 55°Cで 50分間、反応させた 。反応終了後、更に 70°Cで 15分間保温し、逆転写酵素の酵素活性除去を行って、こ の最終反応物を PCRに用いる各皮膚癌培養細胞、並びにヒト正常皮膚全 RNAの cDN Aサンプルとした。  ript III Reverse Transcriptase: Invitrogen) was reacted with 3 μl and reacted at 55 ° C. for 50 minutes. After completion of the reaction, incubate at 70 ° C for 15 minutes to remove the enzyme activity of reverse transcriptase, and use this final reaction product for each cultured skin cancer cell and PCR sample of human normal skin total RNA. It was.
[0095] 各皮膚癌培養細胞、及びヒト正常皮膚サンプルにおける GSDMA発現の確認には、 PCR法により行った。使用したプライマーは、（GSDMAFATGACCATGTTTGAAAAT GTC (配列番号： 13) )と (GSDMAR:TTAGGAGGCCTTGGTAAGCTG (配列番号 14) )のセットである。反応条件は、 DNA変性反応が 94°Cで 30秒間、アニーリング反応が 5 7°Cで 30秒間、伸長反応が 72°Cで 1分 30秒間、反応サイクルは 35サイクル、使用した 機械は、バイオメトラ社の DNA増幅装置である。反応終了後の PCRサンプルは、 1% ァガロースゲルにより電気泳動し、各サンプルにおける GSDMA発現の評価を行った  [0095] The GSDMA expression in each cultured skin cancer cell and human normal skin sample was confirmed by PCR. The primer used is a set of (GSDMAFATGACCATGTTTGAAAAT GTC (SEQ ID NO: 13)) and (GSDMAR: TTAGGAGGCCTTGGTAAGCTG (SEQ ID NO: 14)). The reaction conditions were DNA denaturation reaction at 94 ° C for 30 seconds, annealing reaction at 57 ° C for 30 seconds, extension reaction at 72 ° C for 1 minute 30 seconds, reaction cycle of 35 cycles, and the machine used was The company's DNA amplification device. After completion of the reaction, PCR samples were electrophoresed on a 1% agarose gel to evaluate GSDMA expression in each sample.
[0096] 5.抗 GsdmA/GSDMA抗体の作製（抗体作製） [0096] 5. Production of anti-GsdmA / GSDMA antibody (antibody production)
抗 GsdmA/GSDMA抗体は、 GsdmAlタンパク質の 37番目から 12アミノ酸（CLVLRKR KSTLF (配列番号： 15) )、並びに GsdmA3タンパク質の 248番目から 14アミノ酸（QMIS EEPEEEKLIG (配列番号： 16) )に相当する領域のペプチドを合成し、ニヮトリに免疫 することにより作製した。抗体の力価測定は、 ELISA法により行った。得られた抗体を Western blot法によりその抗原特異性について検証したところ、これらの抗体はマウ ス GsdmAl、 GsdmA2、 GsdmA3及びヒト GSDMAを全て特異的に認識する事が確認さ れた。 The anti-GsdmA / GSDMA antibody has a region corresponding to the 37th to 12th amino acids (CLVLRKR KSTLF (SEQ ID NO: 15)) of the GsdmAl protein and the 248th to 14th amino acids (QMIS EEPEEEKLIG (SEQ ID NO: 16)) of the GsdmA3 protein. Peptides were synthesized and prepared by immunizing chickens. Antibody titer was measured by ELISA. When the obtained antibodies were verified for their antigen specificity by Western blotting, these antibodies It was confirmed that GsdmAl, GsdmA2, GsdmA3 and human GSDMA were all specifically recognized.
[0097] 6.免疫組織化学 (免疫染色）  [0097] 6. Immunohistochemistry (immunostaining)
皮膚癌を自然発症した Rim3ヘテロ · ρ53ノックアウトヘテロマウス個体の皮膚サンプ ルは、採取後 4 %ホルムアルデヒド溶液で 12時間から 16時間、固定を行った。皮膚組 織はパラフィンで包埋し、厚さ 5 z mに薄切した。ヒト正常皮膚、並びに皮膚扁平上皮 癌パラフィン切片は、 BIOCHAIN社の製品を購入して実験に使用した。脱パラフィン 化したサンプルは、抗原の賦活処理として、 10 mMのクェン酸バッファ一中で 5分間 のマイクロウェーブ処理、もしくは 37°Cで 5分間、 0.05 % Trypsin処理を行った。また、 内在性ペルォキシターゼ活性を抑えるために 0.3 % H 0 /MtOHで室温 30分間、処 理を行った。一次抗体は、抗 GsdmA/GSDMA (50倍希釈)を、二次抗体にはペルォキ シダーゼ標識ロバ抗ニヮトリ IgYポリクローナル抗体（500倍希釈， Jacson ImmunoRese ach)を用いた。可視化には 3' 3-ジァミノべンシジン四塩酸塩（和光純薬）を用いた。 抗体染色後の標本は、ガバーガラスをかけて正立型光学顕微鏡 (OLYMPUS BX51) を用いて観察を行った。  Skin samples of Rim3 heterozygous ρ53 knockout heterozygous mice that spontaneously developed skin cancer were fixed with 4% formaldehyde solution for 12 to 16 hours after collection. The skin tissue was embedded in paraffin and sliced to a thickness of 5 zm. Normal human skin as well as cutaneous squamous cell carcinoma paraffin sections were purchased from BIOCHAIN and used for experiments. The deparaffinized samples were subjected to microwave treatment for 5 minutes in 10 mM citrate buffer or 0.05% Trypsin treatment at 37 ° C for 5 minutes as an antigen activation treatment. In order to suppress the endogenous peroxidase activity, treatment was performed with 0.3% H 0 / MtOH for 30 minutes at room temperature. Anti-GsdmA / GSDMA (diluted by 50 times) was used as the primary antibody, and peroxidase-labeled donkey anti-nitrate IgY polyclonal antibody (diluted by 500 times, Jacson ImmunoResearch) was used as the secondary antibody. For visualization, 3 '3-daminobencidine tetrahydrochloride (Wako Pure Chemical Industries) was used. The specimen after antibody staining was observed with an upright light microscope (OLYMPUS BX51) covered with a rubber glass.
[0098] 〔実施例 1〕皮膚癌における GSDMAの消失  [0098] [Example 1] Loss of GSDMA in skin cancer
マウス GsdmAクラスターのヒト相同遺伝子： GSDMAは、胃癌、食道癌においてその 発現が消失し、また癌抑制能を持つことが明らかにされていた。し力 ながら皮膚に ぉレ、て、同様な発現様式並びに機能を持つかにつレ、ては明らかにされてレ、なレ、。  Human homologous gene of mouse GsdmA cluster: GSDMA has been shown to have its gene expression suppressed in gastric cancer and esophageal cancer, and to have tumor suppressive ability. However, it has been clarified in the skin, and it has been revealed whether it has the same expression pattern and function.
[0099] そこで、ヒト GSDMA遺伝子の皮膚での機能を明らかにする第一段階として、正常皮 膚、皮膚癌における GSDMA遺伝子発現変化の解析を行った。正常皮膚と 4種類の 皮膚癌細胞株： HSC1 (皮膚扁平上皮癌）、 HSC4 (舌扁平上皮癌）、 HSC5 (皮膚扁平 上皮癌）、及び A431 (外陰部類上皮癌）を用いて GSDMA遺伝子の発現を RT-PCR法 により調べた結果、ヒト正常皮膚には GSDMA遺伝子の発現が観察されるにもかかわ らず、全ての癌細胞株においては GSDMA遺伝子の発現が消失している事が明らか となった（図 1)。このことは、胃並びに胃癌における GSDMA遺伝子の発現様式と同 様であり、皮膚においても GSDMA遺伝子が癌抑制機能を持つ可能性があることを示 唆する。 [0100] 〔実施例 2〕 Rim3ヘテロ · ρ53ノックアウトヘテロ個体は皮膚癌を自然発症する マウスにおいて GsdmAクラスター遺伝子（マウス 11番染色体に存在）の内、 GsdmAl と GsdmA3が皮膚において発現している。上皮形態形成異常突然変異マウス Rim3の 原因遺伝子は GsdmA3であり、 Rim3の表現型から考えて、 GsdmA3遺伝子の機能は、 細胞増殖 ·分化に深く関わっていることが予想される。これらの事とヒト皮膚癌におい て GSDMA遺伝子発現が消失することなどから考えて、本発明者らはマウス皮膚にお いて GsdmA3は癌抑制能を持つのではないかと仮説を立てた。この仮説を確かめる ために、本発明者らは GsdmA3が原因遺伝子である上皮形態異常突然変異マウス · R im3に、癌抑制遺伝子として知られている p53 (T卬 53)のノックアウトァリルを導入した R im3ヘテロ · ρ53ノックアウトヘテロ個体（GsdmA3Rim3/+;TYp53—/+)を作製し、その動物 個体における皮膚癌発症率の検証を行った。実験に用いた掛合わせの模式図を図 2に示す。 Rim3ヘテロ個体（GsdmA3Rim3/+)と p53ノックアウトヘテロ個体（TYp53—/+)を 交配することにより Rim3ヘテロ · ρ53ノックアウトヘテロ個体、 Rim3ヘテロ個体、 p53ノッ クアウトへテロ個体、正常個体が、理論値通りの割合で得られた。これら全ての遺伝 型個体を同一条件下かつ発ガン性物質の接触なしで飼育し、経時観察を行った。こ れらの動物において皮膚癌が発症したならば、その原因は遺伝型のみによる自然発 症であると言える。経時観察の結果、全ての遺伝型において、生後 6ヶ月までは皮膚 癌の発症は観察されな力つた。生後 7ヶ月になると初めて Rim3ヘテロ ·ρ53ノックアウト ヘテロ個体においてのみ、皮膚癌の発症が観察された。ダブルへテロ個体では、カロ 齢と共にその発症率が上昇し、 8ヶ月齢では 38% (8/21)、 10ヶ月齢では 62% (13/21) 、 12ヶ月齢では 81% (17/21)の個体で皮膚癌が発症した（図 3)。興味深いことに、そ の皮膚癌は一個体につき複数個発症し、発症部位はヒトの皮膚癌と同様に頭部と頸 部に集中していた（表 1)。 Rim3ヘテロ ·ρ53ノックアウトヘテロ個体以外の遺伝型個体 ： Rim3ヘテロ個体、 p53ノックアウトヘテロ個体、正常個体においては、 Rim3ヘテロ個 体のみに、生後 11ヶ月齢で皮膚癌の発症がみられたが、その発症率は生後 12ヶ月 齢となつても約 10%と非常に低レ、値であった。 [0099] Therefore, as a first step to clarify the function of the human GSDMA gene in the skin, analysis of changes in GSDMA gene expression in normal skin and skin cancer was performed. GSDMA gene expression using normal skin and four skin cancer cell lines: HSC1 (squamous squamous cell carcinoma), HSC4 (squamous carcinoma of the tongue), HSC5 (squamous cell carcinoma of the skin), and A431 (epidermoid carcinoma) As a result of examining the RT-PCR method, it was revealed that the expression of GSDMA gene was lost in all cancer cell lines even though GSDMA gene expression was observed in normal human skin. (Fig. 1). This is similar to the expression pattern of the GSDMA gene in the stomach and stomach cancer, and suggests that the GSDMA gene may also have a tumor suppressor function in the skin. [Example 2] Rim3 heterozygote · ρ53 knockout heterozygote spontaneously develops skin cancer In mice, GsdmAl and GsdmA3 are expressed in the skin among the GsdmA cluster genes (present on mouse chromosome 11). The causative gene of epithelial morphogenesis mutant mouse Rim3 is GsdmA3. Considering the phenotype of Rim3, the function of GsdmA3 gene is expected to be deeply involved in cell growth and differentiation. Considering these things and the disappearance of GSDMA gene expression in human skin cancer, the present inventors hypothesized that GsdmA3 may have cancer suppressive ability in mouse skin. In order to confirm this hypothesis, the present inventors introduced a knockout allele of p53 (T) 53), which is known as a tumor suppressor gene, to the epithelial morphogenic mutant mouse Rim3, which is a causative gene of GsdmA3. Rim3 heterozygous ρ53 knockout heterozygous individuals (GsdmA3 Rim3 / +; TYp53 — / +) were prepared, and skin cancer incidence in the animal individuals was verified. Figure 2 shows a schematic diagram of the crossing used in the experiment. Rim3 heterozygous individuals (GsdmA3 Rim3 / +) and p53 knockout heterozygous individuals (TYp53 — / +) can be crossed to obtain Rim3 heterozygous, ρ53 knockout heterozygous individuals, Rim3 heterozygous individuals, p53 knockout heterozygous individuals, and normal individuals. Obtained in street proportions. All these genotype individuals were bred under the same conditions and without contact with carcinogens, and observed over time. If skin cancer develops in these animals, it can be said that the cause is a spontaneous disease caused only by the genotype. As a result of time-lapse observation, the development of skin cancer was not observed until 6 months after birth in all genotypes. Skin cancer was observed only in Rim3 heterozygous ρ53 knockout heterozygous animals for the first time at 7 months of age. In double heterozygous individuals, the incidence increases with age of caro, with 38% (8/21) at 8 months, 62% (13/21) at 10 months, and 81% (17/21) at 12 months. ) Individuals developed skin cancer (Fig. 3). Interestingly, multiple skin cancers occurred in each individual, and the affected sites were concentrated in the head and neck as in human skin cancer (Table 1). Rim3 heterozygous individuals other than ρ53 knockout heterozygous individuals: In Rim3 heterozygous individuals, p53 knockout heterozygous individuals, and normal individuals, only Rim3 heterozygous individuals developed skin cancer at 11 months of age. The incidence was very low, about 10% even at 12 months of age.
[0101] [表 1]
Figure imgf000032_0001
[0101] [Table 1]
Figure imgf000032_0001
表 1は Rim3/+マウスと TYp53—/+マウスの交酉己から得られた交雑マウスにおける自然 発生皮膚ガンの発症率を示す。  Table 1 shows the incidence of spontaneous skin cancer in cross-breed mice obtained from crosses of Rim3 / + and TYp53-/ + mice.
〔実施例 3〕 Rim3ヘテロ · ρ53ノックアウトヘテロマウス発症皮膚癌の組織化学的観察 より詳しく Rim3ヘテロ ·ρ53ノックアウトヘテロ個体において発症した皮膚癌を調べる ために、組織切片を作製した。図 4の Αは、 Rim3ヘテロ ·ρ53ノックアウトヘテロ個体に ぉレ、て発症した皮膚癌の全体像を示し、図 4の Βはその個体の癌の切片像である。 図 4Α中の白線は、図 4Βの切断面と一致する。 Rim3ヘテロ ·ρ53ノックアウトヘテロ個 体における皮膚癌は、非常にケラチン化した領域が多ぐケラトアカントーマ様であつ た（図 4Β)。また癌組織領域を病理学的に調べると、この皮膚癌は典型的な扁平上 皮癌であることが明らかとなった（図 4D)。同時に、癌に隣接した非癌部領域では、 細胞の過増殖が観察された（図 4C)。組織化学的観察において、最も興味深いこと は、 Rim3ヘテロ ·ρ53ノックアウトヘテロ個体所属リンパ節における知見である。癌発症 Rim3ヘテロ ·ρ53ノックアウトヘテロ個体の所属リンパ節を調べたところ、そのリンパ節 内には上皮構造が形成されており、また過度な角化 (ケラチン化)像が観察された（ 図 4Ε)。このことは、 Rim3ヘテロ ·ρ53ノックアウトヘテロ個体において発生した皮膚癌 細胞が所属リンパ節へ転移し、そのリンパ節内に皮膚様構造を形成した事を意味す る。 [Example 3] Histochemical observation of Rim3 hetero-ρ53 knockout heteromouse skin cancer To examine skin cancer that developed in Rim3 heterozygous ρ53 knockout heterozygotes in detail, tissue sections were prepared. The Α in Fig. 4 shows an overall image of skin cancer that developed in a Rim3 heterozygous ρ53 knockout heterozygous individual, and the Α in Fig. 4 is a slice image of the individual's cancer. The white line in Fig. 4Α coincides with the cut surface in Fig. 4Β. Skin cancer in the Rim3 heterozygous ρ53 knockout heterozygote was keratoacantoma-like, with many highly keratinized regions (Fig. 4Β). Pathological examination of the cancer tissue area revealed that this skin cancer was a typical squamous cell carcinoma (Fig. 4D). At the same time, cell overgrowth was observed in the non-cancerous region adjacent to the cancer (Fig. 4C). What is most interesting in histochemical observations is the findings in the lymph nodes of Rim3 heterozygous / ρ53 knockout heterozygous individuals. Cancer onset Rim3 heterozygous ・ When investigating the regional lymph nodes of ρ53 knockout heterozygous individuals, an epithelial structure was formed in the lymph nodes, and excessive keratinization was observed (Fig. 4Ε). . This means that skin cancer cells that developed in Rim3 heterozygous and ρ53 knockout heterozygous metastasized to regional lymph nodes and formed skin-like structures in the lymph nodes.
[0103] 〔実施例 4〕皮膚癌における GsdmA/GSDMAタンパク質の消失  [Example 4] Disappearance of GsdmA / GSDMA protein in skin cancer
ヒト皮膚癌培養細胞では、 GSDMA遺伝子の発現は消失していた。し力 ながらこの 結果は、あくまで培養細胞系 (in vitro)の実験結果である。そこでヒト並びに動物個体 ( in vivo)での癌部における GSDMAの発現が消失しているか否かについて調べる手始 めとして、本発明者らは GsdmA/GSDMA抗体を作製した。 GsdmA/GSDMAに対する 合成ペプチドを作製し、ニヮトリに免疫することにより抗 GsdmA/GSDMAポリクローナ ル抗体の作出に本発明者らは成功した。ウェスタンブロットによる抗 GsdmA/GSDMA ポリクローナル抗体の特異性を図 5に示す。今回作製した GsdmA/GSDMAポリクロー ナル抗体は、確実に GsdmA/GSDMA (マウス GsdmAl〜A3、並びにヒト GSDMAタンパ ク質)を認識する。  In cultured human skin cancer cells, GSDMA gene expression was lost. However, these results are only experimental results of cultured cell systems (in vitro). Therefore, the present inventors prepared a GsdmA / GSDMA antibody as a starting point for examining whether or not the expression of GSDMA in the cancer site in humans and animal individuals (in vivo) has disappeared. The present inventors succeeded in producing an anti-GsdmA / GSDMA polyclonal antibody by producing a synthetic peptide against GsdmA / GSDMA and immunizing a chicken. The specificity of the anti-GsdmA / GSDMA polyclonal antibody by Western blot is shown in FIG. The GsdmA / GSDMA polyclonal antibody prepared this time surely recognizes GsdmA / GSDMA (mouse GsdmAl to A3 and human GSDMA protein).
[0104] この抗体を用いて Rim3ヘテロ ·ρ53ノックアウトヘテロ個体に発症した皮膚癌を調べ たところ、非癌部上皮組織にぉレ、ては GsdmAタンパク質の存在が有棘細胞層から顆 粒層、透明層にかけて確認されたが（図 6A、 6B)、典型的な扁平上皮癌細胞領域に ぉレ、て GsdmAタンパク質は消失してレ、ることが明らかとなった（図 6C、 6D中 *印）。 同様に、ヒト正常皮膚、扁平上皮癌において GSDMAタンパク質の検出を行った。ヒト 正常皮膚において GSDMAタンパク質は、マウス正常皮膚と同様に基底膜細胞層に は発現がなぐ有棘細胞層から顆粒層、透明層にかけて発現していた（図 7A)。また 、ヒトにおける GSDMAタンパク質の毛胞での発現もマウスと同様であった（図 7B)。ヒ ト皮膚癌においては、扁平上皮癌、基底細胞癌共に GSDMAタンパク質の発現が消 失していることが見出された（図 7C、 7D) 0以上の結果から、培養癌細胞株のみなら ずヒト、マウス個体においても GsdmA/GSDMAタンパク質は、癌化に伴い消失するこ とが明らかとなった。 [0104] Using this antibody, we examined skin cancer that developed in Rim3 heterozygous ρ53 knockout heterozygous individuals, and found that the presence of GsdmA protein in the non-cancerous epithelial tissue, from the spinous cell layer to the condylar layer, Although it was confirmed over the transparent layer (Figs. 6A and 6B), it was found that the GsdmA protein disappeared in the typical squamous cell carcinoma cell region (Figs. 6C and 6D). ). Similarly, GSDMA protein was detected in human normal skin and squamous cell carcinoma. In normal human skin, GSDMA protein was expressed from the spinous cell layer, which is not expressed in the basement membrane cell layer, to the granular layer and the transparent layer, as in normal mouse skin (FIG. 7A). In addition, the expression of GSDMA protein in human hair follicles was similar to that in mice (FIG. 7B). In human skin cancer, squamous cell carcinoma, expression of GSDMA protein basal cell carcinoma both were found to be deleted extinction (Fig. 7C, 7D) of zero or more results, not only cultured cancer cell lines It became clear that GsdmA / GSDMA protein disappears with canceration in human and mouse individuals.
[0105] 本発明者らは、 GsdmA3に変異が存在する上皮形態形成異常突然変異マウス Rim3 に p53 (TYp53)ノックアウトァリルを導入することにより、 自然発症で皮膚癌を誘発する 新たな皮膚癌モデル動物を開発した。 Rim3ヘテロ ·ρ53ノックアウトヘテロ個体は、生 後約 7ヶ月齢前後で扁平上皮癌を発症し、生後 12ヶ月齢になるとその割合は、 80% を超えるものである。その皮膚癌発症部位は、ヒト皮膚癌において発症が多い頭部と 頸部に集中し、また 1個体につき複数の癌の発生が観察される。更にその癌細胞は、 驚くべき事に所属リンパ節へ転移することが確かめられた。  [0105] The present inventors have introduced a new skin cancer model that induces spontaneous skin cancer by introducing p53 (TYp53) knockout aryl into Rim3, an epithelial dysplasia mutant mouse having a mutation in GsdmA3. An animal was developed. Rim3 heterozygous ρ53 knockout heterozygous individuals develop squamous cell carcinoma around 7 months of age, and the proportion exceeds 80% at 12 months of age. The site of skin cancer is concentrated in the head and neck where human skin cancer is common, and multiple cancers are observed per individual. Surprisingly, the cancer cells were confirmed to metastasize to the regional lymph nodes.
[0106] 皮膚癌の発症率が高ぐ 1個体につき複数の癌が発症することは、皮膚癌の発症メ 力二ズムを解析する上で、非常に有用である。また、化学誘導なしに自然発症で皮 膚癌を誘発することも特筆すべき事である。 Rim3ヘテロ ·ρ53ノックアウトヘテロ個体は 、その遺伝型のみの影響により加齢と共に皮膚癌を発症する。  [0106] Incidence of skin cancer is high The occurrence of multiple cancers per individual is very useful in analyzing the onset mechanism of skin cancer. It is also noteworthy that it induces skin cancer spontaneously without chemical induction. Rim3 heterozygous ρ53 knockout heterozygous individuals develop skin cancer with aging due to the effect of their genotype alone.
[0107] 〔実施例 5〕  [Example 5]
Rim3単独での皮膚癌発症は、非常に低ぐ今回の実験では約 10%であった。また 、その癌も単独で発症した。データには示さないが、 Rim3ホモ個体においても今回実 験を行った Rim3ヘテロ個体とほぼ同様に癌の発生率は非常に低い。 p53 (Trp53)ノッ クアウトマウスは、これまでにヘテロ、ホモ個体共に皮膚癌の自然発症報告はない。 今回行った実験において p53ノックアウトヘテロマウスで皮膚癌が発症しなかったのは 、これまでの報告 (皮膚癌は自然発症しない）と一致する。この 2つの遺伝子に同時 に変異が存在する時にのみ皮膚癌が発症する理由を、以下考察する。  The incidence of skin cancer with Rim3 alone was about 10% in this experiment, which is very low. The cancer also developed alone. Although not shown in the data, the incidence of cancer in the Rim3 homozygous individuals is very low, as is the case with the Rim3 heterozygous individuals tested in this study. No p53 (Trp53) knockout mice have ever been reported to spontaneously develop skin cancer in both hetero and homozygous mice. The fact that skin cancer did not develop in p53 knockout hetero mice in this experiment is consistent with previous reports (skin cancer does not spontaneously develop). The reason why skin cancer develops only when there are mutations in these two genes at the same time is discussed below.
[0108] 癌化関連遺伝子である癌遺伝子（oncogene)や癌抑制遺伝子（tumor suppressor g ene)は、本来正常遺伝子に由来し、細胞の増殖 ·分化調節など極めて重要な役割を 担うものが多い。何らかの原因でこの癌化関連遺伝子に変異が生じると、遺伝子の 異常な機能亢進 (癌遺伝子)や機能低下 (癌抑制遺伝子）が生じ、細胞増殖シグナ ルの異常亢進や細胞周期異常を引き起こす。その結果、遺伝子修復などが不能とな り、更に複数の遺伝子に変異が蓄積して、最終的に細胞が癌化すると考えられる。事 実多くの場合、癌においては、複数の癌関連遺伝子に変異が蓄積している。 [0108] Oncogene, which is a canceration-related gene, and tumor suppressor g ene) is originally derived from normal genes, and many of them play extremely important roles such as cell growth / differentiation regulation. If the canceration-related gene is mutated for any reason, abnormal gene hyperfunction (oncogene) or functional decline (tumor suppressor gene) occurs, leading to abnormal cell proliferation signal abnormalities or cell cycle abnormalities. As a result, gene repair or the like becomes impossible, and mutations accumulate in more than one gene, leading to cancerous cells. In fact, in many cases, mutations accumulate in multiple cancer-related genes in cancer.
[0109] まさに Rim3ヘテロ ·ρ53ノックアウトヘテロ個体は、この癌発生機序を反映するもので あると考えられる。 GsdmA3、もしくは p53遺伝子単独の変異では皮膚癌を発症しない 。このことは、 GsdmA3遺伝子に変異が存在する場合（Rim3ホモ/ヘテロ個体）にお いては、 p53遺伝子により癌発生が押さえられ、逆に p53遺伝子に変異が存在する場 合 (p53ノックアウトホモ/ヘテロ個体）には、 GsdmA3遺伝子が癌発生を押さえている と推測される。両遺伝子に変異が存在し、かつ加齢を伴うことで初めて皮膚癌が発症 する。そもそも Rim3ヘテロ ·ρ53ノックアウトヘテロ個体では、 GsdmA3遺伝子、 p53遺伝 子共に 1ァリルは正常タイプである。即ち、野生型の半分ではあるが正常な機能を持 つ。おそらく皮膚癌が発症するまでの 6ヶ月の間に半分量の正常な機能では遺伝子 修復などが正常に行えなくなり、多くの癌化関連遺伝子に変異が蓄積して皮膚癌発 症へと繋がるものと考えられる。  [0109] The Rim3 heterozygote · ρ53 knockout heterozygote is considered to reflect this mechanism of cancer development. Mutation of GsdmA3 or p53 gene alone does not cause skin cancer. This means that in the presence of mutations in the GsdmA3 gene (Rim3 homo / hetero individuals), the occurrence of cancer is suppressed by the p53 gene, and conversely, if there is a mutation in the p53 gene (p53 knockout homo / hetero). It is speculated that the GsdmA3 gene suppresses cancer development in individuals. Skin cancer develops only when mutations exist in both genes and age increases. In the first place, in Rim3 heterozygous ρ53 knockout heterozygous individuals, both GsdmA3 gene and p53 gene are normal types. That is, it is half the wild type but has a normal function. Perhaps half of the normal function during the six months until skin cancer develops, gene repair, etc. cannot be performed normally, and mutations accumulate in many canceration-related genes, leading to skin cancer. Conceivable.
[0110] また、 Rim3ヘテロ ·ρ53ノックアウトヘテロ個体では、 GsdmA3遺伝子、 T卬 53遺伝子 共に 1ァリルは正常タイプであるので、その表現型は緩やかなものであると考えられる 。よって、 Rim3ホモ ·ρ53ノックアウトヘテロ個体 (GsdmA3Rim3/Rim3;T卬 537+)、 Rim3へテ 口 ·ρ53ノックアウトホモ個体 (GsdmA3Rim3/+;Trp53—ん)は、 Rim3ヘテロ ·ρ53ノックアウト ヘテロ個体よりも早く皮膚癌が発症し、 Rim3ホモ ·ρ53ノックアウトホモ個体 (GsdmA3Rim 3/Rim3;Trp537— )は、更に早く皮膚癌が発症するものと考えられる。 [0110] Moreover, in the Rim3 heterozygous ρ53 knockout heterozygous individual, both GsdmA3 gene and T 卬 53 gene are normal types, so the phenotype is considered to be moderate. Therefore, Rim3 homo · ρ53 knockout heterozygous individuals (GsdmA3 Rim3 / Rim3 ; T 卬 537+), Rim3 head · ρ53 knockout homozygous individuals (GsdmA3 Rim3 / +; Trp53—n) Skin cancer develops earlier, and Rim3 homo · ρ53 knockout homozygous individuals (GsdmA3 Rim 3 / Rim3 ; Trp537—) are considered to develop skin cancer earlier.
[0111] Rim3ヘテロ ·ρ53ノックアウトヘテロ個体の皮膚癌における変異蓄積癌化関連遺伝 子の候補の 1つは、 GsdmAlである。 Rim3ヘテロ ·ρ53ノックアウトヘテロ個体において GsdmAl遺伝子に変異は存在していなレ、。しかしながら、抗 GsdmA/GSDMA抗体を 用いた免疫染色の結果は、 Rim3ヘテロ ·ρ53ノックアウトヘテロ個体の皮膚癌における 、本来皮膚で発現している GsdmAl、 GsdmA3両タンパク質が消失している事を明ら 力とした。 GsdmA3のみならず、変異の存在しない GsdmAlの消失は非常に興味深い 。 GsdmAlと GsdmA3は、非常に相同性が高ぐ機能的な重複性が考えられる。 Gsdm A3のみに変異が存在する段階において（Rim3ホモもしくはヘテロ個体）は GsdmAlが その機能を補って癌化を抑えていると思われる。癌化には両遺伝子 GsdmAl、 Gsdm A3の消失が必要なのであろう。また、 GsdmAl、 GsdmA3共に発現が抑制されるの力 についても興味が持たれ、両遺伝子の転写調節に関わる因子も皮膚癌における変 異蓄積癌化関連遺伝子の候補と考えられる。 [0111] One candidate for a mutation-accumulating canceration-related gene in skin cancer of Rim3 heterozygous ρ53 knockout heterozygous is GsdmAl. Rim3 heterozygous ・ No mutation exists in the GsdmAl gene in ρ53 knockout heterozygous individuals. However, the results of immunostaining with anti-GsdmA / GSDMA antibody revealed that both GsdmAl and GsdmA3 proteins originally expressed in the skin disappeared in skin cancer of Rim3 hetero-ρ53 knockout hetero individuals. Power. Not only GsdmA3 but also the disappearance of GsdmAl without mutation is very interesting. GsdmAl and GsdmA3 are considered to be functionally redundant with very high homology. At the stage where mutations exist only in Gsdm A3 (Rim3 homozygous or heterozygous individuals), it seems that GsdmAl supplements its function and suppresses canceration. For canceration, the disappearance of both genes GsdmAl and Gsdm A3 may be necessary. In addition, we are interested in the ability to suppress the expression of both GsdmAl and GsdmA3, and the factors involved in the transcriptional regulation of both genes are considered to be candidates for mutation-accumulating canceration-related genes in skin cancer.
[0112] 今回の実験において本発明者らは、皮膚特異的に発現する GsdmA3に変異の存在 する Rim3に p53ノックアウトァリルを導入することにより、皮膚癌自然発症モデル動物 を作製した。 GsdmA3はパラログとして GsdmAl、 GsdmA2を持つ。 GsdmAlは皮膚、食 道、前胃領域の扁平上皮に、 GsdmA2は胃腺上皮に、それぞれ特異的に発現してい る。 GsdmAl,もしくは GsdmA2に変異を導入したトランスジエニック、又はノックインマ ウスを作製し、さらに p53ノックアウトァリルを導入することにより皮膚癌、食道癌などを 発症する扁平上皮癌モデルマウス（GsdmAl変異/ p53変異）や胃腺上皮に癌を生じ る胃癌モデルマウス（GsdmA2変異/ p53変異）を作製可能である。 [0112] In this experiment, the present inventors produced a skin cancer spontaneous model animal by introducing a p53 knockout aryl into Rim3 having a mutation in GsdmA3 expressed specifically in the skin. GsdmA3 has GsdmAl and GsdmA2 as paralogs. GsdmAl is specifically expressed in the squamous epithelium in the skin, esophagus and foregut region, and GsdmA2 is expressed specifically in the gastric gland epithelium. Squamous cell carcinoma model mice that develop skin cancer, esophageal cancer, etc. by creating a transgenic or knock-in mouse with mutations introduced into GsdmAl or GsdmA2 and then introducing p53 knockout aryl (GsdmAl mutation / p53 mutation) ) And gastric cancer model mice (GsdmA2 mutation / p53 mutation) that produce cancer in the gastric gland epithelium.
産業上の利用可能性  Industrial applicability
[0113] これまで、皮膚癌発症モデル動物は、遺伝子欠損（ノックアウト）、遺伝子導入（トラ ンスジエニック）技術を用いて数種類作製されているが、その多くは、皮膚癌の発症 に化学誘導等が必要であり、リンパ節への転移は報告されていない（Capozza et al , Am J Pathol. 162(6). 2029-2039. 2003、 Ishikawa et al , Mutat Res. 477(1-2). 41-49. 2001、 Nicolas et al., Nat Genet. 33(3). 416-421. 2003)。  [0113] To date, several types of animal models of skin cancer have been created using gene deficiency (knockout) and gene transfer (transgeneic) technologies, but many of them require chemical induction to develop skin cancer. No metastasis to lymph nodes has been reported (Capozza et al, Am J Pathol. 162 (6). 2029-2039. 2003, Ishikawa et al, Mutat Res. 477 (1-2). 41-49 2001, Nicolas et al., Nat Genet. 33 (3). 416-421. 2003).
[0114] p53のノックアウトホモマウスは生後 3ヶ月力ら 6ヶ月、 p53のノックアウトヘテロマウスは 生後 10ヶ月でリンパ癌を自然発症するが、皮膚癌は発症しない（Donehower et al., N ature. 356， 215-221· 1992·)。また、本発明者らは、 Rim3 (GsdmA3Rim3/+並びに Gsdm A3Rim3/Rim3)における皮膚癌の自然発症率は極めて低いことを見出している。 [0114] p53 knockout homozygous mice are 3 months old and 6 months old, and p53 knockout heterozygous mice spontaneously develop lymphoma at 10 months of age, but do not develop skin cancer (Donehower et al., Nature. 356). , 215-221 · 1992 ·). The present inventors have also found that the spontaneous incidence of skin cancer in Rim3 (GsdmA3 Rim3 / + and Gsdm A3 Rim3 / Rim3 ) is extremely low.
[0115] これに対し、本発明者らによって提供された Rim3ヘテロ ·ρ53ノックアウトヘテロマウ ス（GsdmA3Rim3/+;TYp53—/+)では、生後 7ヶ月より自然発症で多発的に皮膚扁平上皮 癌を引き起こし、生後 12ヶ月では 8割以上の個体において同様な扁平上皮癌の発症 が観察された。更に驚くべき事に、扁平上皮癌が所属リンパ節へ転移することが確か められた。 [0115] In contrast, the Rim3 heterozygous ρ53 knockout heteromaus (GsdmA3 Rim3 / +; TYp53 — / +) provided by the inventors of the present invention is a spontaneous onset of squamous cell carcinoma of the skin more frequently than 7 months after birth. The development of similar squamous cell carcinoma in more than 80% of individuals at 12 months of age Was observed. More surprisingly, it was confirmed that squamous cell carcinoma metastasized to regional lymph nodes.
〃高頻度〃で〃多発的〃に扁平上皮癌を"自然発症〃し、かつリンパ節への転移を起こ すことは、本発明の Rim3ヘテロ · ρ53ノックアウトヘテロマウス力 扁平上皮癌モデル 動物として有用であり、また、扁平上皮癌発生'メカニズムの解明、並びに創薬、診断 薬の開発に有用であることを示している。  It is useful for Rim3 heterozygous ρ53 knockout heterozygous mouse model of squamous cell carcinoma of the present invention to “spontaneously develop squamous cell carcinoma in a high frequency and frequently occurring” and to metastasize to lymph nodes. It is also useful for elucidating the mechanism of 'squamous cell carcinoma development' and for developing drug discovery and diagnostics.

Claims

請求の範囲 The scope of the claims
[I] GsdmA遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進または抑制された 非ヒト動物。  [I] A non-human animal in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene is promoted or suppressed.
[2] 癌化関連遺伝子が、 Trp53遺伝子、 Rb遺伝子、 Ras遺伝子、 Cyclin-D遺伝子、 Cdk遺 伝子、 TNF- a遺伝子、および TGF- β遺伝子からなる群より選択される少なくとも 1つ の遺伝子である、請求項 1に記載の非ヒト動物。  [2] The canceration-related gene is at least one gene selected from the group consisting of Trp53 gene, Rb gene, Ras gene, Cyclin-D gene, Cdk gene, TNF-a gene, and TGF-β gene The non-human animal according to claim 1, wherein
[3] 癌のモデル動物である、請求項 1または 2に記載の非ヒト動物。 [3] The non-human animal according to claim 1 or 2, which is a model animal for cancer.
[4] GsdmAl遺伝子、 GsdmA2遺伝子および GsdmA3遺伝子からなる群より選択される少 なくとも 1つの遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進または抑制 されたマウス。 [4] A mouse in which the function of at least one gene selected from the group consisting of the GsdmAl gene, GsdmA2 gene, and GsdmA3 gene is suppressed, and the function of a canceration-related gene is promoted or suppressed.
[5] 癌化関連遺伝子が、 Trp53遺伝子、 Rb遺伝子、 Ras遺伝子、 Cyclin-D遺伝子、 Cdk遺 伝子、 TNF- a遺伝子、および TGF- β遺伝子力 なる群より選択される少なくとも 1つ の遺伝子である、請求項 4に記載のマウス。  [5] At least one gene selected from the group consisting of Trp53 gene, Rb gene, Ras gene, Cyclin-D gene, Cdk gene, TNF-a gene, and TGF-β gene force. The mouse according to claim 4, wherein
[6] GsdmA3遺伝子および Trp53遺伝子の機能が抑制されたマウス。  [6] A mouse in which the functions of the GsdmA3 gene and the Trp53 gene are suppressed.
[7] 癌のモデル動物である、請求項 4〜6のいずれかに記載のマウス。  7. The mouse according to any one of claims 4 to 6, which is a cancer model animal.
[8] 癌が皮膚癌である、請求項 7に記載のマウス。  8. The mouse according to claim 7, wherein the cancer is skin cancer.
[9] GsdmA遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進または抑制された 動物細胞。  [9] An animal cell in which the function of a GsdmA gene is suppressed and the function of a canceration-related gene is promoted or suppressed.
[10] GsdmAl遺伝子、 GsdmA2遺伝子および GsdmA3遺伝子からなる群より選択される少 なくとも 1つの遺伝子の機能が抑制され、癌化関連遺伝子の機能が促進または抑制 されたマウス細胞。  [10] A mouse cell in which the function of at least one gene selected from the group consisting of the GsdmAl gene, the GsdmA2 gene, and the GsdmA3 gene is suppressed, and the function of a canceration-related gene is promoted or suppressed.
[I I] 癌化関連遺伝子が、 Trp53遺伝子、 Rb遺伝子、 Ras遺伝子、 Cyclin-D遺伝子、 Cdk遺 伝子、 TNF- a遺伝子、および TGF- β遺伝子からなる群より選択される少なくとも 1つ の遺伝子である、請求項 9または 10に記載の細胞。  [II] The canceration-related gene is at least one gene selected from the group consisting of Trp53 gene, Rb gene, Ras gene, Cyclin-D gene, Cdk gene, TNF-a gene, and TGF-β gene The cell according to claim 9 or 10, which is
[12] GsdmA3遺伝子および ΤΥρ53遺伝子の機能が抑制されたマウス細胞。  [12] A mouse cell in which the functions of the GsdmA3 gene and the ΤΥρ53 gene are suppressed.
[13] 請求項 1もしくは 2に記載の非ヒト動物、または 4〜6のいずれかに記載のマウスから 樹立された細胞。 [13] A cell established from the non-human animal according to claim 1 or 2, or the mouse according to any one of 4 to 6.
[14] 以下の（a)〜（c)の工程を含む、請求項 1もしくは 2に記載の非ヒト動物、または 4〜6 のいずれかに記載のマウスの表現型を相補する化合物のスクリーニング方法。[14] The non-human animal according to claim 1 or 2, or 4 to 6, comprising the following steps (a) to (c): A screening method for a compound that complements the mouse phenotype according to any one of the above.
(a)請求項 1もしくは 2に記載の非ヒト動物、または 4〜6のいずれかに記載のマウスに 投与する工程 (a) a step of administering to the non-human animal according to claim 1 or 2 or the mouse according to any one of 4 to 6;
(b)該非ヒト動物の表現型を測定する工程  (b) measuring the phenotype of the non-human animal
(c)被検化合物を投与してレ、なレ、場合と比較して、表現型を相補する化合物を選択 する工程  (c) A step of administering a test compound and selecting a compound that complements the phenotype compared to the case.
以下の（a)〜（c)の工程を含む、請求項 9〜： 13のいずれかに記載の動物細胞の表 現型を相補する化合物のスクリーニング方法。 14. The screening method for a compound that complements the expression type of an animal cell according to any one of claims 9 to 13, comprising the following steps (a) to (c).
(a)請求項 9〜： 13のいずれかに記載の動物細胞に被検化合物を接触させる工程 (a) A step of bringing the test compound into contact with the animal cell according to any one of claims 9 to 13
(b)該細胞の表現型を測定する工程 (b) measuring the phenotype of the cell
(c)被験試料を接触させないときに比較して、表現型を相補する化合物を選択する 工程  (c) selecting a compound that complements the phenotype compared to when the test sample is not contacted
以下の（a)〜（e)のレ、ずれかに記載のタンパク質に結合する抗体。 An antibody that binds to the protein according to any one of the following (a) to (e):
(a)配列番号： 1、 3、 5、 7のいずれかに記載の塩基配列からなる DNAよりコードされ るタンパク質  (a) a protein encoded by DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, and 7
(b)配列番号： 2、 4、 6、 8のいずれかに記載のアミノ酸配列からなるタンパク質 (b) a protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, and 8
(c)配列番号： 2、 4、 6、 8のいずれかに記載のアミノ酸配列において 1若しくは複数 のアミノ酸が置換、欠失、揷入、および/または付加したアミノ酸配列からなる天然由 来のタンパク質であって、配列番号： 2、 4、 6、 8のいずれかに記載のアミノ酸配列か らなるタンパク質と機能的に同等なタンパク質 (c) a naturally occurring protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8 A protein functionally equivalent to a protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, and 8.
(d)配列番号： 1、 3、 5、 7のいずれかに記載の塩基配列からなる DNAとストリンジヱン トな条件下でハイブリダィズする天然由来の DNAよりコードされるタンパク質であって (d) a protein encoded by a naturally occurring DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 7;
、配列番号： 2、 4、 6、 8のいずれかに記載のアミノ酸配列からなるタンパク質と機能 的に同等なタンパク質 A protein functionally equivalent to the protein consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, and 8.
(e) (a)〜（d)のレ、ずれかに記載のタンパク質の部分タンパク質  (e) A partial protein of the protein according to (a) to (d)
PCT/JP2005/015126 2004-08-20 2005-08-19 NONHUMAN ANIMAL WITH INHIBITED GdsmA GENE FUNCTION AND PROMOTED OR INHIBITIED CANCERATION-ASSOCIATED GENE FUNCTION WO2006019153A1 (en)

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