WO2002011530A9 - Transgenic animal - Google Patents

Transgenic animal

Info

Publication number
WO2002011530A9
WO2002011530A9 PCT/JP2001/006826 JP0106826W WO0211530A9 WO 2002011530 A9 WO2002011530 A9 WO 2002011530A9 JP 0106826 W JP0106826 W JP 0106826W WO 0211530 A9 WO0211530 A9 WO 0211530A9
Authority
WO
WIPO (PCT)
Prior art keywords
gene
mmp
rat
extracellular matrix
animal
Prior art date
Application number
PCT/JP2001/006826
Other languages
French (fr)
Japanese (ja)
Other versions
WO2002011530A1 (en
Inventor
Koji Yoshimura
Atsushi Nishimura
Mayumi Nishida
Kazuhiro Hosono
Original Assignee
Takeda Chemical Industries Ltd
Koji Yoshimura
Atsushi Nishimura
Mayumi Nishida
Kazuhiro Hosono
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Chemical Industries Ltd, Koji Yoshimura, Atsushi Nishimura, Mayumi Nishida, Kazuhiro Hosono filed Critical Takeda Chemical Industries Ltd
Priority to AU2001277716A priority Critical patent/AU2001277716A1/en
Publication of WO2002011530A1 publication Critical patent/WO2002011530A1/en
Publication of WO2002011530A9 publication Critical patent/WO2002011530A9/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • 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
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • 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

Definitions

  • the present invention relates to an MMP-19 transgenic non-human mammal.
  • the extracellular matrix is a cell-supporting tissue surrounding cells of the tissue, and is composed of fibrous proteins such as collagen-elastin, glycoproteins such as proteodarican, fibronectin, laminin, and carbohydrates such as hyaluronic acid.
  • Extracellular matrices have a significant effect on cell activities such as cell morphology, metabolism, migration, proliferation, and differentiation, and are associated with many biological phenomena such as development, aging, inflammation, wound healing, immunity, and tumors. It is known. It is also known that abnormal degradation of extracellular matrix occurs in various diseases such as rheumatoid arthritis, osteoarthritis, osteoporosis, cancer metastasis, invasion, arteriosclerosis, and corneal ulcer.
  • MMP matrix metalloproteinase
  • Human MMP-19 (also called MMP-18 or rasi-1 but the same protein) is a 508 amino acid protein that forms a new subfamily that does not belong to the above subfamily because of its structure. Attention has been drawn (J. Cossins et al., Biochem. Biophys. Res. Co. thigh un. 228: 494-498, 1996, (denoted as MMP-18), BA. M. Pendas et al. , J. Biol. Cheni. 272: 4281-4286, 1997, I. Massova et al., FASEB J. 12: 1075-1095, 1998). However, only the relationship between ovulation and the physiological function of MMP-19 has been pointed out (A.
  • New transgenic animals have been thought to enable the development of new medicines that can help prevent and treat joint diseases such as rheumatoid arthritis and osteoarthritis.
  • transgenic non-human animals in which degradation of cartilage matrix observed in joint diseases such as rheumatoid arthritis and osteoarthritis are observed (hereinafter sometimes referred to as transgenic animals) ), And the development of a method for mass-producing animal models of joint disease was desired. Disclosure of the invention
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, have produced a novel transgenic rat expressing MMP-19 under the control of type II collagen promoter overnight. As a result, they found that the extracellular matrix of cartilage was degraded and exhibited a phenotype such as shortening of limbs.
  • the exogenous MMP-19 gene has the nucleotide sequence represented by SEQ ID NO: 12.
  • test substance is applied to the animal or a part of the organism described in any of (1) to (6) above, and the effect of ameliorating a disease caused by abnormal degradation of extracellular matrix is assayed. Screening methods for substances used for the prevention and treatment of diseases caused by abnormal degradation of extracellular matrix,
  • Diseases caused by abnormal degradation of extracellular matrix include chondrodysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis, eye disease and The screening method according to the above (13), which is a malignant tumor, (16) A disease caused by abnormal degradation of extracellular matrix containing a substance determined to have an improving effect on a disease caused by abnormal degradation of extracellular matrix by the method described in (13) above Prevention and treatment of medicines,
  • Diseases resulting from abnormal degradation of extracellular matrix include chondrodysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, eye disease and The method for preventing or treating malignant tumor according to the above (18),
  • DNA containing the exogenous MMP-19 gene or its mutant gene was introduced into female pseudopregnant rats mated with male rats after administration of luteinizing hormone-releasing hormone or its analogs.
  • FIG. 1 shows a schematic diagram of the plasmid pKS-MMPBcon_19 constructed in Example 2.
  • FIG. 2 shows the results of mRNA detection performed in Example 6.
  • Lanes 1 to 6 show wild-type rats used as controls, and lanes 7 to 12 show organs of transgenic rats.
  • Lanes 1 and 7 are the heart
  • lanes 2 and 8 are the lungs
  • lanes 3 and 9 are the liver
  • lanes 4 and 10 are the spleen
  • lanes 5 and 11 are the kidneys
  • lanes 6 and 12 are the Each shows cartilage.
  • FIG. 3 shows the results of analysis by the Southern hybridization method performed in Example 7. Lanes 1, 3 to 6, 8 to 13 indicate heterozygous, and lanes 2 and 7 indicate homozygous. BEST MODE FOR CARRYING OUT THE INVENTION
  • the transgenic non-human animal of the present invention is preferably used for embryo development during non-human mammal development (more preferably, for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their primordial cells).
  • gene transfer methods such as the calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc. It is created by introducing the desired exogenous MMP-19 gene or its mutant gene into the target cell.
  • the target gene can be introduced into somatic cells, organs of living organisms, tissue cells, and the like by the gene transfer method, and can be used for cell culture, tissue culture, and the like.
  • a transgenic animal can also be produced by fusing the germ cell with a known cell fusion method.
  • a part of the living body of the transgenic animal produced in this manner (for example, 1) cells, tissues, organs, etc. having DNA incorporating the exogenous MMP-19 gene or its mutant gene; The derived cells or tissues are cultured and passaged as necessary. (3) Various proteins or DNA that can be isolated from the transgenic animal) can also be used as the "exogenous MMP-19 gene or DNA" of the present invention.
  • a part of the living body of a non-human mammal having a DNA incorporating the mutant gene The non-human mammal having DNA incorporating the sex MMP-19 gene or its mutant gene can be used for the same purpose.
  • the tissue that is a part of the living body of the transgenic animal is preferably a joint tissue or the like.
  • Cells that are part of the living body of the transgenic animal are preferably cells that constitute joint tissues.
  • Non-human mammals that can be targeted in the present invention include porcupines, pigs, sheep, sheep, goats, puppies, dogs, cats, guinea pigs, hampus, rats, mice, and the like. Preferred are egrets, dogs, cats, cats, guinea pigs, hamsters, mice or rats, among which rodents (Rodentia) are preferred, especially rats (Wistar, SD, etc.), especially those of the Wistar strain. Rats are the most preferred target animals for disease model animals. In addition, birds and the like as bird animals can be used for the same purpose as the “non-human mammal” targeted in the present invention.
  • exogenous MMP-19 gene to be introduced into the target non-human mammal examples include, for example, human, bush, higgin, goat, rabbit, dog, cat, guinea pig, hamus, rat, mouse
  • a mammalian MMP-19 gene can be used.
  • the exogenous MMP-19 gene is a gene different from the endogenous gene of the animal into which the gene is to be introduced, and specifically, the MMP-19 gene isolated or purified from the mammal or a synthetic MMP-19 gene.
  • the MM P-19 gene is used.
  • the exogenous MMP-19 gene is preferably an MMP-19 gene that does not have an intron.
  • mutant gene of the exogenous MMP-19 gene of the present invention a mutation (for example, mutation, site-specific mutation, etc.) in the DNA of the present invention, specifically, the addition of a base Or a gene in which a deletion, substitution with another base, or the like has occurred. More specifically, as a result of the addition, deletion, or substitution of another base, 1 to 5 (preferably 1 or 2) amino acids are contained in the amino acid sequence constituting MMP-19. It is preferable to mutate so that substitution, addition or deletion occurs in the acid, and any mutation may be used as long as it does not lose the function of MMP-19.
  • the exogenous MMP-19 gene includes, for example, the amino acid sequence represented by SEQ ID NO: 13.
  • a gene encoding the human MMP-19 having the amino acid sequence and having the base sequence represented by SEQ ID NO: 12 is used.
  • the exogenous MMP-19 gene or a mutant gene thereof in the present invention may be of the same or different species as the non-human mammal to be introduced or expressed. It may be derived from any mammal.
  • the gene When introducing the gene into a target animal, it is generally advantageous to use the gene as a gene construct (eg, a vector, etc.) linked downstream of a promoter that can be expressed in the cells of the target animal.
  • a gene construct eg, a vector, etc.
  • various mammals having the MMP-l 19 gene having high homology to the human MMP-l9 gene Hamsters, rats, mice, etc.
  • a vector obtained by ligating the gene downstream of various promoters capable of expressing the human MMP119 gene preferably rats, etc.
  • Microinjection into a fertilized egg of a non-human mammal can produce a transgenic non-human mammal that highly expresses the desired human MMP-19 gene.
  • Examples of the expression vector for the MMP-19 gene include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, pateriophage such as ⁇ phage, retroviruses such as Moroni leukemia virus, vaccinia virus or Animal viruses such as baculovirus are used.
  • a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used, and a plasmid derived from Escherichia coli is particularly preferred.
  • Examples of the promoter that regulates the gene expression of the MMP-19 gene include, for example, promoters of genes derived from viruses (cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, etc.), various mammals (human, Genes from egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.
  • viruses cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, etc.
  • various mammals human, Genes from egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.
  • AMP-dependent protein kinase 3 I subunit, atrial natriuretic factor, dopamine
  • a Kuching promoter or the like can be used.
  • the promoter region of the type II collagen gene which is known to be expressed in cartilage, is effective.
  • the transgenic mammal has a sequence that terminates the transcription of the target messenger RNA (poly A, generally referred to as “Yuichi Mineta”).
  • poly A generally referred to as “Yuichi Mineta”.
  • it is derived from viruses, various mammals and birds Gene expression can be manipulated using the sequence of each gene.
  • the simian virus SV40 and the like are used.
  • the splicing signal of each gene, the enhancer region, and a part of the intron of the eukaryotic gene are transferred 5 'upstream of the promoter region, between the promoter region and the translation region. Alternatively, it may be linked to the 3 'downstream of the translation region depending on the purpose.
  • the translation region of MMP-19 is derived from DNA derived from the liver, kidney, fibroblasts, etc. of humans and various non-human mammals (eg, herons, dogs, cats, guinea pigs, hamsters, rats, mice, etc.).
  • a known method using all or part of the genomic DNA derived from various commercially available genomic DNA libraries as a raw material, or from RNA derived from liver, kidney, and fibroblasts of human and various non-human mammals Can be obtained using the complementary DNA prepared by the above as a raw material.
  • a translation region mutated by a point mutagenesis method or the like can also be prepared using the translation region of MMP-19 obtained from the above cells or tissues. These are all materials that can be used for transgenic animals.
  • the above-mentioned translation region is located downstream of the above promoter (preferably at the transcription termination site) as a gene construct (eg, a vector, etc.) that can be expressed in an introduced animal.
  • DNA incorporating the MMP-19 gene can be produced by the usual genetic engineering technique of ligation to the upstream.
  • the presence of the MMP-19 gene in the germ cells of the transgenic animal after gene transfer indicates that the progeny of the transgenic animal has the MMP-19 gene in all of its germ cells and somatic cells Means to do.
  • the offspring of this type of animal that has inherited the gene carry the MMP-19 gene in all of its germinal and somatic cells.
  • the fertilized egg used to introduce the exogenous MMP-19 gene into a target non-human mammal preferably a rat or the like, particularly preferably a Wistar strain rat or the like
  • a fertilized egg of its ancestor is Male non-human mammals of the same species (preferably male rats, etc., particularly preferably male rats of the Wistar strain) and female non-human mammals (preferably female rats, particularly preferably female rats of the Wistar strain) Are obtained by crossing.
  • Fertilized eggs can also be obtained by natural mating, but after artificially regulating the estrous cycle of female non-human mammals (preferably female rats, particularly preferably female rats of the Wistar strain), A method of crossing with mammals (preferably female rats, particularly preferably female rats of the Wistar strain) is preferred.
  • Methods for artificially regulating the estrous cycle of female non-human mammals include, for example, follicle-stimulating hormone (pregnant horse serum gonadotropin, generally abbreviated as PMSG) first, and then luteinizing hormone (human chorionic).
  • Gonadotropin generally abbreviated as hCG
  • hCG is preferably administered by, for example, intraperitoneal injection, but the preferred hormone dose and administration interval vary depending on the type of non-human mammal.
  • Non-human mammals are female rats (preferred In the case of female rats of the Wistar strain, it is usually preferable to administer luteinizing hormone about 48 hours after administration of follicle-stimulating hormone and obtain fertilized eggs by mating with male rats.
  • the dose of the hormone is about 20 to about 50 IU / person, preferably about 30 IUZ individuals, and the dose of luteinizing hormone is about 0 to about 10 IUZ individuals, preferably about 5 IUZ individuals. It is.
  • DNA is implanted and implanted artificially into a female non-human mammal, and DNA incorporating the exogenous gene is obtained.
  • a non-human mammal having the above is obtained.
  • L HRH luteinizing hormone-releasing hormone
  • the dose of LHRH or an analog thereof and the timing of mating with a male non-human mammal after its administration differ depending on the type of non-human mammal.
  • the non-human mammal is a female rat (preferably, a female rat of the Wistar strain)
  • 111111 is an analog thereof (eg, [3, 5-011]-11-1 ⁇ ).
  • the dose of LHRH or an analog thereof is usually about 10 to 60 individuals, preferably about 40 gZ. It is an individual.
  • a method for artificially regulating the estrous cycle of a female non-human mammal to obtain a fertilized egg and a method for artificially regulating the sexual cycle of the female non-human mammal It is preferable to use the method in combination with the method of transplantation and implantation.
  • the non-human mammal into which the MMP-19 gene of the present invention has been introduced (hereinafter sometimes abbreviated as the non-human mammal of the present invention) has cartilage destruction and has developed joint disease.
  • the non-human mammal of the present invention causes abnormal degradation of the extracellular matrix, and a disease caused by abnormal degradation of the extracellular matrix (eg, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis) , Rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye diseases, malignant tumors, etc.).
  • non-human mammals of the present invention include: 1) shortening and deformation of the limbs, 2) deformation of the skull, 3) malocclusion, 4) excessive maxillary and mandibular incisors, 4) insufficient calcification of the lumbar spine, 4) deformation of the tail vertebra, and And symptoms selected from caudal intervertebral disc defects.
  • non-human mammal of the present invention has the above-mentioned very unique features, it has the following useful uses.
  • the exogenous MMP-19 gene is highly expressed, and cartilage destruction is caused by the enzymatic activity of this MMP-19 gene.
  • Characteristic various diseases for example, extracellular matrices such as cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye diseases, malignant tumors It can be used to evaluate prophylactic or therapeutic agents for diseases caused by abnormal degradation of Rix.
  • the present invention is characterized in that a test substance is applied to the non-human mammal of the present invention or a part of the living body thereof, and the effect of ameliorating a disease caused by abnormal degradation of the extracellular matrix is assayed.
  • Prevention of diseases caused by abnormal degradation of extracellular matrix • Provide a method for screening substances used for treatment.
  • Abnormal degradation of the extracellular matrix includes that caused by an abnormal function of MMP-19.
  • test substance is administered to the non-human mammal of the present invention.
  • the test substance may be a known synthetic compound, peptide, protein, DNA library, or the like, or, for example, a warm-blooded mammal (eg, mouse, rat, bush, horsetail, hidge, monkey, human, etc.) Tissue extract, cell culture supernatant, etc. are used.
  • the cartilage degradation inhibitor can be evaluated by examining the drug evaluation or therapeutic effect using urinary and blood pyridinoline levels as indicators.
  • MMP tissue Inhibitor of Met alloproteinase
  • proteodalican collagen
  • keratan Sulfuric acid hyaluronic acid
  • osteocalcin calcium
  • phosphorus cytodynamics
  • blood growth factors etc.
  • drug evaluation or therapeutic effect can be examined using urinary cartilage and bone metabolism biochemical markers as indices. It is also useful to examine the degree of cartilage destruction by methods such as preparation of pathological specimens and staining with safranin O if necessary for experiments.
  • Various antigens eg, substrates such as type II collagen and aggrecan
  • bacteria eg, killed Mycobacterium tuberculosis, etc.
  • the virus eg, Epstein-Barr virus, etc.
  • Epstein-Barr virus is promoted by local, single, multiple or continuous administration of the virus systemically or jointly.
  • MMPs serine proteases such as trypsin, daltathione, active oxygen, nitrogen oxides such as N ⁇ , mercury compounds (eg, 4-aminophenylmercuric acetate, etc.), inflammation
  • the onset can be promoted by administering to the joints cytokines such as IL-1 and TN Fa that induce protease and express protease activity.
  • Non-human mammals in which the onset is promoted are not only non-human mammals in which the gene has not been introduced, but also have a greater absolute value of the above index than non-human mammals in which the onset has not been promoted. By performing the evaluation described in (1) using human animals, the evaluation can be performed with higher accuracy.
  • Cartilage fragments or chondrocytes collected from the non-human mammal of the present invention can be cultured and used for evaluation of MMP-19 inhibitors. Specifically, inhibitors can be evaluated by using these cartilage fragments or chondrocytes as a material for the MMP-19 enzyme and adding a synthetic peptide as a substrate. If necessary, it may be activated with a serine protease such as a mercury compound or trypsin.
  • a serine protease such as a mercury compound or trypsin.
  • the non-human mammal of the present invention is pregnant, and the pregnant animal is treated with cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic It can be used to evaluate prophylactic or therapeutic agents for arthropathy, eye diseases and malignancies, and their complications.
  • a test substance is administered to a pregnant non-human mammal of the present invention.
  • the non-human mammal of the present invention can be transformed with genes such as MMPs (eg, MMP-1, 8, 13 etc.), which are known to degrade cartilage collagen, and aggrecanase, which degrades aggrecan. Introduced non-human mammals are mated and the offspring obtained are diseases caused by the products (proteins) of these genes as well as MMP-19 (eg, cartilage dysplasia, bone dysplasia, osteoporosis, It is expected to develop osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye diseases and malignant tumors. Therefore, the offspring can be used for the evaluation of a prophylactic / therapeutic agent for such a disease by a method similar to (1) to (4) or a method analogous thereto.
  • MMPs eg, MMP-1, 8, 13 etc.
  • the test substance when it is determined that administration of a test substance has an effect of ameliorating a disease caused by abnormal degradation of the extracellular matrix, the test substance is extracellular as described above. It can be selected as a drug for prevention and treatment of diseases caused by abnormal degradation of matrix.
  • Substances selected as the above preventive and therapeutic drugs include, for example, sugar-coated tablets, forcepsels, elixirs, microforces, etc., orally, or water or other drugs. It can be used parenterally in the form of an injectable preparation such as a sterile solution with a liquid that is acceptable or a suspension.
  • an injectable preparation such as a sterile solution with a liquid that is acceptable or a suspension.
  • a substance selected as a prophylactic / therapeutic drug may be required to implement a generally accepted formulation with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. Can be prepared by mixing them in unit dosage form. The amount of the active ingredient in such preparations is to be adjusted so that an appropriate dose in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • Solubilizers for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 80 TM, HC 0-50, etc.) ) May be used together.
  • the oily liquid include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • buffers eg, phosphate buffer, sodium acetate buffer, etc.
  • soothing agents eg, benzalkonium chloride, proforce hydrochloride, etc.
  • stabilizers eg, human serum albumin, polyethylene glycol, etc.
  • a preservative eg, benzyl alcohol, phenol, etc.
  • the substance selected as a drug for prophylaxis or treatment is DNA
  • insert the DNA alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. After that, it can be administered to humans or warm-blooded animals according to conventional means.
  • the DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and can be administered using a gene gun or a catheter such as a hydrogel catheter.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, birds, higgies, bushes, dogs, dogs, Cats, dogs, monkeys, etc.).
  • the dose of a substance selected as a prophylactic or therapeutic drug may vary depending on the target disease, the subject of administration, the route of administration, and the like. ), the substance is administered from about 0.1 mg to about 0.1 mg per day, preferably from about 1.0 to 5 mg, more preferably from about 1.0 to 20 mg.
  • the single dose of the substance may vary depending on the target of administration, target disease, etc., for example, administration to adults (with a body weight of 6 O kg) in the form of injections for the treatment of arthritis
  • it is convenient to administer the substance by injecting about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day into the affected area. It is.
  • the dose can be administered in terms of 60 kg.
  • the non-human mammal of the present invention can be used for an experiment for gene therapy of a patient with an MMP-19 gene abnormality.
  • the above-described transgenic mammal of the present invention can also be used as a cell source for tissue culture. Further, for example, by directly analyzing DNA or RNA in the tissue of the transgenic rat of the present invention, or by analyzing the protein expressed by the gene, a transcription factor having a complex action of a nuclear receptor can be obtained. It is also possible to analyze the relationship between the two. Alternatively, culturing cells of a gene-bearing tissue by standard tissue culture techniques and using them to study the function of cells derived from tissues that are generally difficult to culture, such as cells forming cartilage tissue. Can also. Further, by using the cells, for example, it is possible to select a drug that enhances the function of the cells. In addition, if there is a high-expressing cell line, it is possible to isolate and purify MMP-19 in a large amount and to produce an antibody thereof.
  • the sequence numbers in the sequence listing of the present invention indicate the following sequences.
  • [SEQ ID NO: 1] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 1 described later.
  • [SEQ ID NO: 2] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 1 described later.
  • SEQ ID NO: 3 This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 1 described later.
  • SEQ ID NO: 4 This shows the base sequence of the primer used in PCR (polymerase chain reaction) method performed in Example 1 described later.
  • SEQ ID NO: 5 This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 4 described later.
  • SEQ ID NO: 6 This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 4 described later.
  • SEQ ID NO: 7 This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 6 described later.
  • SEQ ID NO: 8 This shows the base sequence of the primer used in the PCR (polymerase zetiein reaction) method performed in Example 6 described later.
  • SEQ ID NO: 10 This shows the base sequence of the enhancer region of rat type I collagen gene cloned in Example 1 described later.
  • SEQ ID NO: 11 This shows the base sequence of the DNA fragment containing the splicing site derived from pTB399 used for the construction of pKS-MMPB con-19 in Example 2 described later.
  • SEQ ID NO: 13 This shows the amino acid sequence of human MMP-19.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC- IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the relevant field, and examples thereof are as follows.
  • DNA Deoxylipo nucleic acid
  • RNA Liponucleic acid A adenine
  • Example 1 Cloning of rat type I collagen gene promoter and enhancer
  • the region of the promoter of the rat type II collagen gene is a primer (5'-GTGGTGGTGGAC AACTAGGAAACTCTGG-3 ': SEQ ID NO: 1) designed based on the nucleotide sequence of Kohno et al. (LBiol. Chem. 260: 4441, 1985). And (5′-CGAGGCGAAKATGGCTCACCGCG-3 ′: SEQ ID NO: 2) by PCR.
  • the obtained fragment of about 1.2 Kb was cloned into pCRII-TOPII using TOPO TA Cloning Kit (manufactured by Invitrogen) according to the attached protocol (referred to as pCRI I-promoter 2).
  • nucleotide sequence of the inserted DNA fragment was confirmed by a conventional method using a DNA sequencer manufactured by ABI, the nucleotide sequence was consistent with the nucleotide sequence of the promoter region in the aforementioned literature (SEQ ID NO: 9).
  • pCRI I Not I site (5 'side) of multiple-cloning site of pCRI I plasmid in promoter 2
  • pCRI I p The fragment consisting of the SmaI site in the type II collagen gene promoter sequence in the robot 2 was used in the following experiments.
  • the rat type II enhancer region was prepared using a primer (5′-TCCACGCGTTTGGGAAACTTCTTGGCTGCG-3) designed based on the nucleotide sequence of Krebsbach et al. (J. Biol. Chem. ': SEQ ID NO: 3) and (5'-GCTTCGTCGC CGCTACGCGTGGGGCCGGA-3': SEQ ID NO: 4).
  • An EcoRI linker was added to the obtained 0.35 Kb MluI fragment by a conventional method, and inserted into the EcoRI site of pBluescript KSII + (hereinafter, referred to as pKS_enhancer1-4).
  • An expression vector for producing a transgenic rat, pKS-MMPBcon- 19, was constructed according to a conventional method.
  • Co 12 A 1 pr omo ter promoter region of rat type II collagen gene, pCR II described in Example 1—from Not I site in the multiple cloning site of pCR II in pr omoter 2 CR II—a 1120 bp fragment to the SmaI site in the type II collagen gene promoter in promoter 2 (the SmaI site was converted to a Sal1 site by conventional linker ligation).
  • SV40 sp1icing a DNA fragment containing a splicing site derived from pTB399 (R. Sasada et al., Cell Structure and Function 12: 205, 1987)
  • MMP-19 Approximately 1600 b from the SacI site to the XhoI site in the MMP-19 cDNA of pTB1921 (Japanese Patent Laid-Open No. 10-080283). P gene fragment. (Sac I site was converted to CI a I site and Xho I site was converted to Bg 1 II site by linker ligation).
  • Co 12 A 1 enh ancer rat type II collagen described in Example 1—enrichment of pKS containing enhancer region of gene—from Hind III site to Not I site of enhancer 1_4 fragment.
  • the rat SD strain was purchased at 8 weeks of age for egg collection and reared for 1 week at 7:00 to 19:00 12 hours under light conditions.
  • First day at 11:00, follicle stimulating hormone (pregnant horse serum gonad stimulation) Hormone, PMSG) (301 U / individual) was injected intraperitoneally, and at 11:00 on day 3, male rats were injected intraperitoneally with luteinizing hormone (human chorionic gonadotropin, hCG) (5 IUZ individuals).
  • the SD line lived and bred with individuals after 10 weeks of age at 1: 1 at 15: 0.
  • the female rats bred at 9:00 were checked for vaginal plugs, and at 13:30, the individuals whose vaginal plugs were confirmed were sacrificed and egg collection was started.
  • a pronucleus-forming egg was selected from fertilized eggs, and the plasmid pKS—MMPBc 011-19 obtained in Example 2 was cut from 1:30 at 1:30 with 1 ⁇ 0-se I and adjusted to a concentration of 10 g / m1.
  • DNA fragments 1-21 containing the MMP-19 gene were injected into the male pronucleus of fertilized eggs of the SD strain rat at the single cell stage while observing under a microscope.
  • the egg cells were cultured in a known HER medium (HAM-F12 powder medium (Dainippon Pharmaceutical) 3.180 g, PMI-1640 powder medium (Dainippon Pharmaceutical) 1.040 g, MEM Eag1e powder medium Ground (Dainippon Pharmaceutical) 0.95 g, NaHCO s (Wako Pure Chemical) 0.780 g, Penicillin-G (GIBCO BRL) 50000 U and Streptomycin (GIBCO BRL) 5000 0U were dissolved in 500 ml of distilled water ) And confirmed the two-cell stage embryos at 13:30 on day 5, and then reported to Wagner et al. (Proc. Natl. Acad. Sci. USA 78: 5016, 1981). In accordance with the method described above, it was transplanted into the oviduct of a pseudopregnant female Wistar rat and implanted.
  • Genomic DNA of these two PCR-positive individuals was analyzed by Southern hybridization. That is, 10 X g of DNA was completely digested with BamHI, transferred to a nylon filter after 2.0% agarose gel electrophoresis. This filter was combined with a probe obtained by labeling a DNA fragment at the Bg1 II site from the NheI site of MMP-19 (Example 2) with a DIG RNA labeling kit (Roche Diagnostics). Hybridized overnight, washed twice with 2 ⁇ SSC, 0.1% SDS at room temperature, and then twice with 0.1 ⁇ SSC, 0.1% SDS at 68 ° C. For detection, a DIG fluorescence detection kit (manufactured by Roche Diagnostics) was used.
  • Example 5 Acquisition of transgenic rats in heterozygote
  • the B6 OF (first generation (F 0 )) obtained in Example 4 reached the age of 12 weeks, it was bred with the SD strain rat to obtain the second generation (F). The second generation was 4 weeks old.
  • PCR was performed by the method described in Example 4 to select a heterozygote and used in Example 6.
  • Example 6 Confirmation of human mRNA in cartilage of transgenic rat
  • the transgenic rat obtained in Example 5 was homogenized in ISOGEN (manufactured by Futabajin) to extract total RNA by a conventional method.
  • ISOGEN manufactured by Futabajin
  • cDNA was synthesized according to the protocol using First strand cDNA synthesis kit (Amersham Pharmacia Biotech) and used as a template for RT-PCR.
  • rat and human MMP- Simultaneously amplified and detected 19 mRNA, and used as a negative control was an individual determined to be wild-type by PCR performed in Example 5.
  • RT PCR amplified 538 bp fragment was completely digested with ApaLI, which is only present in human fragments. At this time, fragments of 347 bp and 191 bp could be detected, confirming that human MMP-19 mRNA was expressed in the cartilage of the transgenic rat.
  • 19 mRNA was found in heart, lung, liver, spleen, kidney, and cartilage ( Figure 2)
  • Example 7 Obtaining homozygous transgenic rats
  • Example 7 The body weight of each of the wild-type, heterozygous and homozygous individuals (B6OF strain, 12-week-old, male) obtained in Example 7 was measured and compared with the wild-type. However, they also found that the weight of homozygotes was smaller than that of heterozygotes.
  • the individual used for weight measurement was anesthetized with Nembutal, and the lengths of forelimbs, hindlimbs and skull were measured with calipers. Heterozygotes did not differ in forelimb, hindlimb, and skull lengths compared to wild-type. Homozygote was found to have shorter forelimbs and hindlimbs than wild-type and heterozygote. It was also found that the skull was flattened in the long axis direction and expanded in the direction perpendicular to the long axis. Table 1 shows the results.
  • the animals were port-fixed from the heart with 4% paraformaldehyde under ether anesthesia, and the right knee joint was removed. After resection of the muscle tissue and connective tissue attached to the periphery, the joint was divided into two parts in the median direction using a diamond cutter. Each joint was further fixed in 4% paraformaldehyde for 14 hours at room temperature. After the fixation, the tissue pieces were washed with distilled water, and the knee joint was decalcified with 20% EDTA (pH 7.4) for 8 days. Decalcification liquid exchange was performed every day.
  • the prepared section was left overnight in a drier heated at 37 to dry completely.
  • Deparaffinization The following references were used for in- and general staining (hematoxylin eosin double staining method). (Histology Research Method, Yutaka Sano, Nanzando, 1985).
  • staining of sulfated glycosaminoglycan the sections were deparaffinized, dehydrated, washed with hematoxylin for 2 minutes and running water, then washed with a 2% aqueous solution of 1% dextrin for 5 minutes, and immersed in a 1% aqueous solution of acetic acid. Performed in a 1% aqueous solution of safranin for 10 minutes.
  • the sections were dehydrated by taking in and out 95% ethanol and 100% ethanol three times in each step, and finally immersing the sections in 100% ethanol for 5 minutes.
  • the transparency and encapsulation were performed with reference to the following literature. (Histology Research Method, Yutaka Sano, Nanzando, 1985).
  • the sections after staining were examined microscopically in a bright field using an optical microscope. No abnormality was found in the articular cartilage and growth plate of the femur and tibia of the heterozygous compared to the wild type.
  • the articular cartilage of the femur and tibia of the homozygote had sites where the articular cartilage layer was thinner and thicker than in the wild type, and where the surface layer was replaced by fibrous articular cartilage tissue.
  • the growth plate was found to be ruptured by homozygote.
  • the surface layer of the articular cartilage layer was replaced with fibrous articular cartilage tissue
  • lack of staining at the surface layer indicated the lack of sulfated glycosaminodalican.
  • the direction of the original chondrocyte growth direction was not constant, indicating that normal cartilage differentiation did not occur.
  • New transgenic rat (strain name is MMP—B60F)
  • heterozygous body weight is smaller than wild type
  • homozygous body weight is smaller than heterozygous body
  • homozygous body is wild type.
  • Watashita To determine whether this abnormality was acquired postnatally or innately during fetal life, we examined whether there were abnormalities in fetal skeletal formation.
  • a homozygote of a new transgenic rat (strain name: MMP—B60F) The male of the bird and the female of the homozygote lived together and were mated. After confirmation of mating, pregnant animals were sacrificed on the 18th day of pregnancy, cesarean section was performed, and fetuses were removed. Since all of the removed fetuses were homozygous, we examined whether fetal skeletal abnormalities were seen in all cases.
  • the fetuses were subjected to double staining of osteochondral cartilage with reference to the following (Histology research method, Yutaka Sano, Nanzando, 1985).
  • New transgenic rats (strain name: MMP-B60F) homozygote, shortening of limbs, cranial deformity and caudal vertebra deformity observed in adult searches may have occurred from embryonic stage. all right. Delayed calcification was observed in the posterior lumbar spine and hind limbs. The incidence of abnormalities in the stomach was about 50% in Adalto, but any abnormalities were observed in all cases during the embryonic period. From these results, it was inferred that the skeletal abnormalities occurring in the homozygote of the new transgenic rat (strain name: MMP-B60F) were congenital in the fetal period.
  • New transgenic rats (strain name: MMP-B60F) homozygote, skeletal abnormalities are known to occur in adults. There is a thinned part in the articular cartilage of this rat. Whether or not cartilage differentiation was normally occurring at this site was analyzed by examining the expression of various transcription factors by immunostaining.
  • a novel transgenic rat (strain name: MMP-B60F) homozygote, 12-week-old male, section of articular cartilage at the distal end of the femur was prepared in the same manner as in Example 8.
  • the prepared sections were subjected to immunostaining using the following antibodies.
  • FGFR3 Fibroblast Growth Factor Receptor 3
  • IHH Indian hedgehog
  • PTC Patched
  • SMO Smoothend
  • FGFR3 expression was observed in the proliferating chondrocyte layer at the site where the cartilage layer of the homozygote was thinned, and there was no abnormality.
  • IHH is not The expression of its receptors, PTC and SMO, was not found at this site. Since normal IHH expression is found in pre-hypertrophic chondrocytes, abnormalities occur in the stage where pre-hypertrophic chondrocytes differentiate into hypertrophic chondrocytes at sites where the cartilage layer is thinning Power ⁇ guessed.
  • the transgenic non-human mammal of the present invention includes various diseases characterized by cartilage destruction, for example, prophylactic or therapeutic agents for osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis. It can be used for evaluation, experiments for gene therapy of patients with MMP-19 gene abnormalities, and the like. Cartilage fragments or chondrocytes collected from the transgenic non-human mammal of the present invention are cultured, and MMP-1 9 Can be used to evaluate inhibitors.
  • cartilage destruction for example, prophylactic or therapeutic agents for osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis. It can be used for evaluation, experiments for gene therapy of patients with MMP-19 gene abnormalities, and the like.
  • Cartilage fragments or chondrocytes collected from the transgenic non-human mammal of the present invention are cultured, and MMP-1 9 Can be used to evaluate inhibitors.

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Abstract

It is intended to a transgenic nonhuman mammal which is usable as a disease model animal for preventives or remedies for chondrogenic failure, osteogenetic failure, osteoporosis, arthritis deformans, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, eye diseases, malignant tumor and complications thereof and, therefore, makes it possible to clarify the pathological mechanisms of these diseases, examine methods for treating these diseases and screen remedies therefor.

Description

遺伝子導入動物 技術分野  Transgenic animal technology
本発明は MMP— 19遺伝子導入非ヒト哺乳動物に関するものである。 背景技術  The present invention relates to an MMP-19 transgenic non-human mammal. Background art
細胞外マトリックスは、 組織の細胞を取り巻く細胞支持組織であり、 コラーゲ ンゃエラスチンなどの繊維性タンパク質、 プロテオダリカン、 フイブロネクチン 、 ラミニンなどの糖タンパク質及びヒアルロン酸などの糖質からなる。 細胞外マ トリックスは細胞の形態 ·代謝 ·移動 ·増殖 ·分化など細胞の活動に重大な影響 を与え、 生体の発生 ·加齢 ·炎症 ·創傷治癒 ·免疫 ·腫瘍など多くの生体現象と 関連することが知られている。 さらに、 慢性関節リウマチ、 変形性関節症、 骨粗 鬆症、 ガンの転移，浸潤、 動脈硬化、 角膜潰瘍など種々の疾病においては細胞外 マトリックスの異常な分解が起こることも知られている。 この細胞外マトリック スを分解に関与する酵素として、 MMP (matrix metalloproteinase) と名付け られた一群の金属プロテアーゼファミリーが知られている。 MMPの活性を調節 する化合物はこれらの疾患の治療薬となる可能性が示唆されている。 MM Pはヒ トにおいては約 20種類がクローン化され、 その塩基配列が報告されている。 M MPは基質特異性やその構造上の類似性から、 コラゲナ一ゼ群、 ゼラチナーゼ群 、 ストロメリシン群および MT— MMP群などのサブファミリ一に分類されてい る （I· Massova et al., FASEB J. 12:1075-1095, 1998) 。 ヒト MMP— 19 ( MMP— 18や r a s i— 1とも呼ばれるが同一のタンパク質である） は 508 アミノ酸からなる夕ンパク質でその構造から上記のサブフアミリーに属さない新 たなサブファミリーを形成する MMPとして注目されている （J. Cossins et al ., Bioc em. Biophys. Res. Co腿 un. 228: 494-498, 1996, (MMP— 18と記 載されている） 、 BA. M. Pendas et al. , J. Biol. Cheni. 272:4281-4286, 1997 、 I. Massova et al., FASEB J. 12:1075-1095, 1998) 。 しかし、 MMP— 19 の生理機能については***との関係が指摘されているにすぎない （A. Hagglund et al. , Endocrinology 140 :4351-4358, 1999) 。 また疾病との関連では、 慢性 関節リウマチ患者の 2 6 %以上に MM P— 1 9の自己抗体の上昇が観察され、 慢 性関節リウマチ患者の炎症を起こした関節滑膜血管において強く発現しているこ とが知られている (C. Kolb et al. , Immunology Let ters 57 : 83-88, 1997、 ( r a s i一 1と記載されている） ) 。 このように、 関節疾患等の疾患と MM P— 1 9の関係が注目されている。 The extracellular matrix is a cell-supporting tissue surrounding cells of the tissue, and is composed of fibrous proteins such as collagen-elastin, glycoproteins such as proteodarican, fibronectin, laminin, and carbohydrates such as hyaluronic acid. Extracellular matrices have a significant effect on cell activities such as cell morphology, metabolism, migration, proliferation, and differentiation, and are associated with many biological phenomena such as development, aging, inflammation, wound healing, immunity, and tumors. It is known. It is also known that abnormal degradation of extracellular matrix occurs in various diseases such as rheumatoid arthritis, osteoarthritis, osteoporosis, cancer metastasis, invasion, arteriosclerosis, and corneal ulcer. A group of metal protease families named MMP (matrix metalloproteinase) is known as an enzyme involved in the degradation of this extracellular matrix. Compounds that modulate the activity of MMPs have been suggested as potential therapeutics for these diseases. About 20 types of MMPs have been cloned in humans, and their nucleotide sequences have been reported. MMPs are classified into subfamilies, such as the collagenase group, the gelatinase group, the stromelysin group, and the MT-MMP group, based on their substrate specificity and their structural similarity (I. Massova et al., FASEB J 12: 1075-1095, 1998). Human MMP-19 (also called MMP-18 or rasi-1 but the same protein) is a 508 amino acid protein that forms a new subfamily that does not belong to the above subfamily because of its structure. Attention has been drawn (J. Cossins et al., Biochem. Biophys. Res. Co. thigh un. 228: 494-498, 1996, (denoted as MMP-18), BA. M. Pendas et al. , J. Biol. Cheni. 272: 4281-4286, 1997, I. Massova et al., FASEB J. 12: 1075-1095, 1998). However, only the relationship between ovulation and the physiological function of MMP-19 has been pointed out (A. Hagglund et al., Endocrinology 140: 4351-4358, 1999). In relation to the disease, an increase in autoantibodies of MMP-19 was observed in more than 26% of patients with rheumatoid arthritis, and it was strongly expressed in inflamed joint synovial vessels in patients with chronic rheumatoid arthritis. (C. Kolb et al., Immunology Letters 57: 83-88, 1997, (described as rasi-11)). As described above, attention has been paid to the relationship between MMP-19 and diseases such as joint diseases.
新たな遺伝子導入 （トランスジエニック） 動物は、 例えば慢性関節リウマチや 変形性関節症などの関節疾患の予防や治療に役立つ新たな医薬品の開発を可能に すると考えられてきた。  New transgenic animals have been thought to enable the development of new medicines that can help prevent and treat joint diseases such as rheumatoid arthritis and osteoarthritis.
したがって、 本発明の分野では、 慢性関節リウマチや変形性関節症などの関節 疾患などで観察される軟骨基質の分解が認められる遺伝子導入非ヒト動物 （以下 、 卜ランスジエニック動物と称することもある） を見出し、 関節疾患モデル動物 を大量に生産する方法の開発が望まれていた。 発明の開示  Therefore, in the field of the present invention, transgenic non-human animals in which degradation of cartilage matrix observed in joint diseases such as rheumatoid arthritis and osteoarthritis are observed (hereinafter sometimes referred to as transgenic animals) ), And the development of a method for mass-producing animal models of joint disease was desired. Disclosure of the invention
本発明者らは、 上記の課題を解決するために鋭意研究を重ねた結果、 I I型コ ラーゲンプロモ一夕一の制御下に MM P— 1 9を発現させた新規なトランスジェ ニックラットを作製したところ、 軟骨の細胞外マトリックスが分解され、 さらに 四肢の短縮などの表現型を示すことを見出した。  The present inventors have conducted intensive studies to solve the above problems, and as a result, have produced a novel transgenic rat expressing MMP-19 under the control of type II collagen promoter overnight. As a result, they found that the extracellular matrix of cartilage was degraded and exhibited a phenotype such as shortening of limbs.
本発明者は、 これらの知見を基づいて、 さらに検討を重ねた結果、 本発明を完 成するに至った。  The present inventors have conducted further studies based on these findings, and as a result, have completed the present invention.
すなわち、 本発明は  That is, the present invention
( 1 ) 外来性 MM P— 1 9遺伝子またはその変異遺伝子を組み込んだ D N Aを 有する非ヒト哺乳動物またはその生体の一部、  (1) a non-human mammal having a DNA incorporating an exogenous MMP-19 gene or a mutant gene thereof or a part of a living body thereof,
( 2 ) 非ヒト哺乳動物が、 ゥサギ、 ィヌ、 ネコ、 モルモット、 ハムスター、 マ ウスまたはラットである上記 （1 ) 記載の動物またはその生体の一部、  (2) The animal or a part of the living body thereof according to (1), wherein the non-human mammal is a heron, a dog, a cat, a guinea pig, a hamster, a mouse, or a rat.
( 3 ) 非ヒト哺乳動物がラットである上記 （1 ) 記載の動物またはその生体の 一部、  (3) The animal according to the above (1), wherein the non-human mammal is a rat, or a part of a living body thereof,
( 4) 外来性 MMP— 1 9遺伝子が配列番号： 1 2で表される塩基配列を有す る上記 （1) 記載の動物またはその生体の一部、 (4) The exogenous MMP-19 gene has the nucleotide sequence represented by SEQ ID NO: 12. The animal or part of the living body thereof described in (1) above,
(5) 関節疾患を発症している上記 （1) から （4) 記載の動物またはその生 体の一部、  (5) The animal or a part of the animal according to (1) to (4) above, which has developed joint disease,
(6) ①四肢の短縮と変形、 ②頭蓋の変形、 ③咬合不全、 ④上顎及び下顎切歯 の過長、 ⑤腰椎の石灰化不全、 ⑥尾椎の変形および⑦尾椎椎間板の欠損から選ば れる症状を発症している上記 （1) から （4) 記載の動物またはその生体の一部  (6) Select from (1) shortening and deformation of limbs, (2) deformity of the skull, (3) malocclusion, (4) excessive maxillary and mandibular incisors, (4) inadequate calcification of the lumbar spine, (4) deformity of the tail vertebra, and (4) loss of the caudal disc The animal or a part of the living body thereof according to the above (1) to (4), which has a symptom of
(7) 外来性 MMP— 19遺伝子またはその変異遺伝子を含有し、 非ヒト哺乳 動物において該遺伝子を発現し得るベクター、 (7) a vector containing the exogenous MMP-19 gene or a mutant gene thereof and capable of expressing the gene in a non-human mammal,
(8) 外来性 MMP— 19遺伝子が配列番号： 12で表される塩基配列を有す る上記 （7) 記載のベクター、  (8) The vector according to (7) above, wherein the exogenous MMP-19 gene has the nucleotide sequence represented by SEQ ID NO: 12.
(9) さらにラット I I型コラーゲン遺伝子のプロモ一夕一領域およびラット I I型コラーゲン遺伝子のェンハンサ一領域を含有する上記 （7) 記載のベクタ (10) pKS— MMPB c o n— 19で表示される上記 （7) 記載のベクタ  (9) The vector according to the above (7), which further contains a promoter overnight region of the rat type II collagen gene and an enhancer region of the rat type II collagen gene. (10) The above described (pKS-MMPB con-19) 7) described vector
(11) 上記 （7) 記載のベクターで形質転換された形質転換体、 (11) a transformant transformed with the vector of (7) above,
(12) 形質転換体が大腸菌 JM109/pKS-MMPB c on— 19 (F ERM BP— 7647) である上記 （11) 記載の形質転換体、  (12) the transformant according to the above (11), wherein the transformant is Escherichia coli JM109 / pKS-MMPB con—19 (FERM BP-7647);
(13) 上記 （1から 6のいずれかに） 記載の動物またはその生体の一部に被 験物質を適用し、 細胞外マトリックスの異常な分解に起因する疾患の改善効果を 検定することを特徴とする、 細胞外マトリックスの異常な分解に起因する疾患の 予防 ·治療のために用いられる物質のスクリーニング方法、  (13) A test substance is applied to the animal or a part of the organism described in any of (1) to (6) above, and the effect of ameliorating a disease caused by abnormal degradation of extracellular matrix is assayed. Screening methods for substances used for the prevention and treatment of diseases caused by abnormal degradation of extracellular matrix,
(14) 細胞外マトリックスの異常な分解が MMP— 19の機能異常に起因す るものである上記 （13) 記載のスクリーニング方法、  (14) The screening method according to (13), wherein the abnormal degradation of the extracellular matrix is caused by a dysfunction of MMP-19.
(15) 細胞外マトリックスの異常な分解に起因する疾患が、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代 謝性関節症、 眼疾患および悪性腫瘍である上記 （13) 記載のスクリーニング方 法、 (16) 上記 （13) 記載の方法により細胞外マトリックスの異常な分解に起 因する疾患の改善効果を有すると判定される物質を含有してなる細胞外マトリツ クスの異常な分解に起因する疾患の予防 ·治療用医薬、 (15) Diseases caused by abnormal degradation of extracellular matrix include chondrodysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis, eye disease and The screening method according to the above (13), which is a malignant tumor, (16) A disease caused by abnormal degradation of extracellular matrix containing a substance determined to have an improving effect on a disease caused by abnormal degradation of extracellular matrix by the method described in (13) above Prevention and treatment of medicines,
(17) 細胞外マトリックスの異常な分解に起因する疾患が、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代 謝性関節症、 眼疾患および悪性腫瘍である上記 （16) 記載の医薬、  (17) Diseases caused by abnormal degradation of extracellular matrix include cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis, eye disease and The medicament according to the above (16), which is a malignant tumor,
(18) 哺乳動物に対して、 上記 （13) 記載の方法により細胞外マトリック スの異常な分解に起因する疾患の改善効果を有すると判定される物質の有効量を 投与することを特徴とする細胞外マトリックスの異常な分解に起因する疾患の予 防 ·治療法、  (18) To a mammal, an effective amount of a substance determined to have a ameliorating effect on a disease caused by abnormal degradation of the extracellular matrix by the method described in (13) above, is administered. Prevention and treatment of diseases caused by abnormal degradation of extracellular matrix,
(19) 細胞外マトリックスの異常な分解に起因する疾患が、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代 謝性関節症、 眼疾患および悪性腫瘍である上記 （18) 記載の予防，治療法、 (19) Diseases resulting from abnormal degradation of extracellular matrix include chondrodysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthritis, eye disease and The method for preventing or treating malignant tumor according to the above (18),
(20) 細胞外マトリックスの異常な分解に起因する疾患の予防 ·治療のため に用いられる物質をスクリーニングするための上記 （1) から （6) のいずれか に記載の動物またはその生体の一部の用途、 (20) The animal or a part of the living body thereof according to any one of (1) to (6) above, for screening a substance used for prevention and treatment of a disease caused by abnormal degradation of the extracellular matrix. Use of
(21) 卵胞刺激ホルモン約 20ないし 50 I U/個体を投与した後に黄体形 成ホルモン約 0ないし 10 I UZ個体を投与した雌ラットを雄ラッ卜と交配させ て得られる受精卵に、 外来性 MMP— 19遺伝子またはその変異遺伝子を組み込 んだ DNAを導入し、 該受精卵を雌ラッ卜に着床させることを特徴とする上記 （ 3) 記載のラットまたはその生体の一部の作製方法、  (21) Fertilized eggs obtained by mating female rats to which follicle stimulating hormone was administered at about 0 to 10 IUZ after administering follicle stimulating hormone at about 20 to 50 IU / individual, and exogenous MMP The method for producing a part of a rat or a living body thereof according to the above (3), wherein a DNA incorporating the 19 gene or its mutant gene is introduced, and the fertilized egg is implanted in a female rat.
(22) 黄体形成ホルモン放出ホルモンまたはその類縁体を投与した後、 雄ラ ットと交配させた雌の偽妊娠ラットに、 外来性 MMP— 19遺伝子またはその変 異遺伝子を組み込んだ DNAを導入した受精卵を着床させることを特徴とする上 記 （3) 記載のラットまたはその生体の一部の作製方法、  (22) DNA containing the exogenous MMP-19 gene or its mutant gene was introduced into female pseudopregnant rats mated with male rats after administration of luteinizing hormone-releasing hormone or its analogs. The method for producing a part of the rat or the living body thereof according to the above (3), wherein the fertilized egg is implanted.
(23) 外来性 MMP— 19遺伝子またはその変異遺伝子を組み込んだ DNA を導入した受精卵などを提供するものである。 図面の簡単な説明 図 1は実施例 2で構築したプラスミド p K S— MM P B c o n _ 1 9の概略図 を示す。 (23) The present invention provides fertilized eggs into which DNA having the exogenous MMP-19 gene or its mutant gene incorporated is introduced. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows a schematic diagram of the plasmid pKS-MMPBcon_19 constructed in Example 2.
図 2は実施例 6で行われた mR N Aの検出結果を示す。 レーン 1〜 6はコント ロールとして用いた野生型ラッ卜の、 レーン 7〜1 2はトランスジエニックラッ 卜の臓器を示す。 また、 レーン 1および 7は心臓を、 レーン 2および 8は肺を、 レーン 3および 9は肝臓を、 レーン 4および 1 0は脾臓を、 レーン 5および 1 1 は腎臓を、 レーン 6および 1 2は軟骨をそれぞれ示す。  FIG. 2 shows the results of mRNA detection performed in Example 6. Lanes 1 to 6 show wild-type rats used as controls, and lanes 7 to 12 show organs of transgenic rats. Lanes 1 and 7 are the heart, lanes 2 and 8 are the lungs, lanes 3 and 9 are the liver, lanes 4 and 10 are the spleen, lanes 5 and 11 are the kidneys, lanes 6 and 12 are the Each shows cartilage.
図 3は実施例 7で行われたサザンハイプリダイゼーション法による解析結果を 示す。 レーン 1、 3〜6、 8〜1 3はへテロザィゴ一ト、 レーン 2および 7はホ モザィゴートを示す。 発明の実施のための最良の形態  FIG. 3 shows the results of analysis by the Southern hybridization method performed in Example 7. Lanes 1, 3 to 6, 8 to 13 indicate heterozygous, and lanes 2 and 7 indicate homozygous. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の遺伝子導入非ヒト動物は、 未受精卵、 受精卵、 ***およびその始原細 胞を含む胚芽細胞などに対して、 好ましくは、 非ヒト哺乳動物の発生における胚 発生の段階 （さらに好ましくは、 単細胞または受精卵細胞の段階でかつ一般に 8 細胞期以前） において、 リン酸カルシウム法、 電気パルス法、 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、 D E A E—デ キストラン法などの遺伝子導入方法によって、 目的とする外来性 MM P— 1 9遺 伝子またはその変異遺伝子を目的とする細胞に導入することにより作出される。 また、 該遺伝子導入方法により、 体細胞、 生体の臓器、 組織細胞などに目的とす る遺伝子を導入し、 細胞培養、 組織培養などに利用することもでき、 さらに、 こ れらの細胞を上述の胚芽細胞と公知の細胞融合法によって融合させることにより 遺伝子導入動物を作出することもできる。  The transgenic non-human animal of the present invention is preferably used for embryo development during non-human mammal development (more preferably, for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their primordial cells). , At the stage of single cells or fertilized egg cells and generally before the 8-cell stage), gene transfer methods such as the calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, particle gun method, DEAE-dextran method, etc. It is created by introducing the desired exogenous MMP-19 gene or its mutant gene into the target cell. In addition, the target gene can be introduced into somatic cells, organs of living organisms, tissue cells, and the like by the gene transfer method, and can be used for cell culture, tissue culture, and the like. A transgenic animal can also be produced by fusing the germ cell with a known cell fusion method.
また、 このようにして作製された遺伝子導入動物の生体の一部 （例えば、 ①外 来性 MM P— 1 9遺伝子またはその変異遺伝子を組み込んだ D N Aを有する細胞 、 組織、 臓器など、 ②これらに由来する細胞または組織を培養し、 必要に応じ、 継代したものなど、 ③該遺伝子導入動物から単離し得る各種タンパク質または D NAなど） も、 本発明の 「外来性 MM P— 1 9遺伝子またはその変異遺伝子を組 み込んだ D NAを有する非ヒト哺乳動物の生体の一部」 として、 本発明の 「外来 性 MM P— 1 9遺伝子またはその変異遺伝子を組み込んだ D NAを有する非ヒト 哺乳動物」 と同様な目的に用いることが出来る。 In addition, a part of the living body of the transgenic animal produced in this manner (for example, 1) cells, tissues, organs, etc. having DNA incorporating the exogenous MMP-19 gene or its mutant gene; The derived cells or tissues are cultured and passaged as necessary. (3) Various proteins or DNA that can be isolated from the transgenic animal) can also be used as the "exogenous MMP-19 gene or DNA" of the present invention. As a part of the living body of a non-human mammal having a DNA incorporating the mutant gene, The non-human mammal having DNA incorporating the sex MMP-19 gene or its mutant gene can be used for the same purpose.
遺伝子導入動物の生体の一部である組織としては、 関節組織などが好ましい。 遺伝子導入動物の生体の一部である細胞としては、 関節組織を構成する細胞な どが好ましい。  The tissue that is a part of the living body of the transgenic animal is preferably a joint tissue or the like. Cells that are part of the living body of the transgenic animal are preferably cells that constitute joint tissues.
本発明で対象とし得る 「非ヒト哺乳動物」 としては、 ゥシ、 ブタ、 ヒッジ、 ャ ギ、 ゥサギ、 ィヌ、 ネコ、 モルモット、 ハムス夕一、 ラット、 マウスなどが挙げ られる。 好ましくは、 ゥサギ、 ィヌ、 ネコ、 モルモット、 ハムスター、 マウスま たはラットであり、 なかでも齧歯目 （R o d e n t i a ) が好ましく、 とりわけ ラッ卜 （W i s t a r、 S Dなど） 、 特に W i s t a r系統のラッ卜が疾患モデ ル動物として最も好ましい対象動物である。 他に鳥類動物として、 ニヮトリなど も本発明で対象する 「非ヒト哺乳動物」 と同様な目的に用いることが出来る。 対象となる非ヒト哺乳動物に導入する外来性 MM P— 1 9遺伝子としては、 例 えば、 ヒト、 ブ夕、 ヒッジ、 ャギ、 ゥサギ、 ィヌ、 ネコ、 モルモット、 ハムス夕 一、 ラット、 マウスなどの哺乳動物由来の MM P— 1 9遺伝子を用いることがで きる。  "Non-human mammals" that can be targeted in the present invention include porcupines, pigs, sheep, sheep, goats, puppies, dogs, cats, guinea pigs, hampus, rats, mice, and the like. Preferred are egrets, dogs, cats, cats, guinea pigs, hamsters, mice or rats, among which rodents (Rodentia) are preferred, especially rats (Wistar, SD, etc.), especially those of the Wistar strain. Rats are the most preferred target animals for disease model animals. In addition, birds and the like as bird animals can be used for the same purpose as the “non-human mammal” targeted in the present invention. Examples of the exogenous MMP-19 gene to be introduced into the target non-human mammal include, for example, human, bush, higgin, goat, rabbit, dog, cat, guinea pig, hamus, rat, mouse For example, a mammalian MMP-19 gene can be used.
外来性 MM P— 1 9遺伝子とは、 遺伝子導入対象動物が有する内在性の遺伝子 とは異なる遺伝子であり、 具体的には前記の哺乳動物から単離 ·精製した MM P 一 1 9遺伝子または合成した MM P— 1 9遺伝子などが用いられる。 なかでも、 外来性 MM P— 1 9遺伝子としては、 イントロンを保持しない MM P— 1 9遺伝 子が好ましい。  The exogenous MMP-19 gene is a gene different from the endogenous gene of the animal into which the gene is to be introduced, and specifically, the MMP-19 gene isolated or purified from the mammal or a synthetic MMP-19 gene. For example, the MM P-19 gene is used. Among them, the exogenous MMP-19 gene is preferably an MMP-19 gene that does not have an intron.
本発明の外来性 MM P— 1 9遺伝子の変異遺伝子としては、 本発明の D NAに 変異 （例えば、 突然変異、 部位特異的突然変異など） が生じたもの、 具体的には 、 塩基の付加、 欠損、 他の塩基への置換などが生じた遺伝子が挙げられる。 より 具体的には、 該塩基の付加、 欠損、 他の塩基への置換の結果、 MM P— 1 9を構 成するアミノ酸配列において、 1ないし 5個 （好ましくは 1または 2個） のアミ ノ酸に置換、 付加または欠損が生じるように変異させることが好ましく、 MM P 一 1 9の機能を失わない変異であれば何れの変異であってもよい。  As the mutant gene of the exogenous MMP-19 gene of the present invention, a mutation (for example, mutation, site-specific mutation, etc.) in the DNA of the present invention, specifically, the addition of a base Or a gene in which a deletion, substitution with another base, or the like has occurred. More specifically, as a result of the addition, deletion, or substitution of another base, 1 to 5 (preferably 1 or 2) amino acids are contained in the amino acid sequence constituting MMP-19. It is preferable to mutate so that substitution, addition or deletion occurs in the acid, and any mutation may be used as long as it does not lose the function of MMP-19.
外来性 MM P— 1 9遺伝子としては、 例えば、 配列番号： 1 3で表されるアミ ノ酸配列を有するヒト MMP—1 9をコードする、 配列番号： 1 2で表される塩 基配列を有する遺伝子などが用いられる。 The exogenous MMP-19 gene includes, for example, the amino acid sequence represented by SEQ ID NO: 13. For example, a gene encoding the human MMP-19 having the amino acid sequence and having the base sequence represented by SEQ ID NO: 12 is used.
本発明における外来性 MM P—1 9遺伝子またはその変異遺伝子 （以下、 単に MM P _ 1 9遺伝子と称することもある） は、 導入または発現の対象とする非ヒ ト哺乳動物と同種あるいは異種のどちらの哺乳動物由来のものであってもよい。 該遺伝子を対象動物に導入させるにあたっては、 当該遺伝子を対象となる動物の 細胞で発現させうるプロモーターの下流に連結した遺伝子コンストラクト （例、 ベクターなど） として用いるのが一般に有利である。 具体的には、 ヒトの MM P 一 1 9遺伝子を導入させる場合、 ヒト MM P— 1 9遺伝子と相同性が高い MM P 一 1 9遺伝子を有する各種哺乳動物 （ゥサギ、 ィヌ、 ネコ、 モルモット、 ハムス 夕一、 ラット、 マウスなど （好ましくはラットなど） ） に由来し、 ヒトの MM P 一 1 9遺伝子を発現させうる各種プロモーターの下流に、 該遺伝子を連結したベ クタ一を、 対象となる非ヒト哺乳動物の受精卵 （例えばラット受精卵） へマイク 口インジェクションすることによって、 目的とするヒト MM P— 1 9遺伝子を高 発現する遺伝子導入非ヒト哺乳動物を作出できる。  The exogenous MMP-19 gene or a mutant gene thereof (hereinafter sometimes simply referred to as the MMP_19 gene) in the present invention may be of the same or different species as the non-human mammal to be introduced or expressed. It may be derived from any mammal. When introducing the gene into a target animal, it is generally advantageous to use the gene as a gene construct (eg, a vector, etc.) linked downstream of a promoter that can be expressed in the cells of the target animal. Specifically, when introducing the human MMP-l9 gene, various mammals having the MMP-l 19 gene having high homology to the human MMP-l9 gene (Egret, dog, cat, guinea pig) Hamsters, rats, mice, etc. (preferably rats, etc.), and a vector obtained by ligating the gene downstream of various promoters capable of expressing the human MMP119 gene. Microinjection into a fertilized egg of a non-human mammal (eg, a rat fertilized egg) can produce a transgenic non-human mammal that highly expresses the desired human MMP-19 gene.
MM P— 1 9遺伝子の発現ベクターとしては、 大腸菌由来のプラスミド、 枯草 菌由来のプラスミド、 酵母由来のプラスミド、 λファージなどのパクテリオファ —ジ、 モロニ一白血病ウィルスなどのレ卜ロウィルス、 ワクシニアウィルスまた はバキュロウィルスなどの動物ウィルスなどが用いられる。 なかでも、 大腸菌由 来のプラスミド、 枯草菌由来のプラスミドまたは酵母由来のプラスミドなどが好 ましく用いられ、 特に大腸菌由来のプラスミドが好ましい。  Examples of the expression vector for the MMP-19 gene include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, pateriophage such as λ phage, retroviruses such as Moroni leukemia virus, vaccinia virus or Animal viruses such as baculovirus are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used, and a plasmid derived from Escherichia coli is particularly preferred.
MM P - 1 9遺伝子の遺伝子発現調節を行うプロモータ一としては、 例えばゥ ィルス （サイトメガロウィルス、 モロニ一白血病ウィルス、 J Cウィルス、 乳癌 ウィルスなど） に由来する遺伝子のプロモーター、 各種哺乳動物 （ヒト、 ゥサギ 、 ィヌ、 ネコ、 モルモット、 ハムスター、 ラット、 マウスなど） および鳥類 （二 ヮトリなど） に由来する遺伝子 （例えば、 アルブミン、 エンドセリン、 ォステオ カルシン、 筋クレアチンキナーゼ、 I型および I I型コラーゲン、 サイクリック AM P依存タンパクキナーゼ ]3 Iサブユニット、 心房ナトリウム利尿性因子、 ド —パミン |3 _水酸化酵素、 ニューロフィラメント軽鎖、 メタ口チォネイン Iおよ び I I A、 メタ口プロティナーゼ 1組織インヒビ夕一、 平滑筋 aァクチン、 ポリ ペプチド鎖延長因子 1ひ （E F 1 —ひ） 、 βァクチン、 ひおよび j8ミオシン重鎖 、 ミオシン軽鎖 1および 2、 ミエリン基礎タンパク、 血清アミロイド Ρコンポ一 ネント、 レニンなど） のプロモ一夕一などが挙げられるが、 好ましくは軟骨組織 で高発現することが可能なモロニ一白血病ウィルスプロモ一夕一、 ヒトおよび二 ヮトリ jQァクチンプロモーターなどを用いることができる。 MM P— 1 9遺伝子 をヒ卜、 ラット、 マウスなどで軟骨特異的に発現させるには、 軟骨で発現するこ との知られている I I型コラーゲン遺伝子のプロモーター領域などが有効である 上記ベクターは、 遺伝子導入哺乳動物において、 目的とするメッセンジャー R N Aの転写を終結する配列 （ポリ A、 一般に夕一ミネタ一と呼ばれる） を有して いることが好ましく、 例えば、 ウィルス由来、 各種哺乳動物および鳥類由来の各 遺伝子の配列を用いて遺伝子発現を操作することが出来る。 好ましくは、 シミア ンウィルスの S V 4 0夕一ミネ夕一などが用いられる。 その他、 目的の遺伝子を さらに高発現させる目的で、 各遺伝子のスプライシングシグナル、 ェンハンサ一 領域、 真核遺伝子のイントロンの一部を、 プロモータ一領域の 5 ' 上流、 プロモ 一夕一領域と翻訳領域間あるいは翻訳領域の 3 ' 下流に連結することも目的によ り可能である。 Examples of the promoter that regulates the gene expression of the MMP-19 gene include, for example, promoters of genes derived from viruses (cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, etc.), various mammals (human, Genes from egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc. and birds (eg, birds) (eg, albumin, endothelin, osteocalcin, muscle creatine kinase, type I and type II collagen, cyclic) AMP-dependent protein kinase] 3 I subunit, atrial natriuretic factor, dopamine | 3_hydroxylase, neurofilament light chain, And IIA, meta-oral proteinase 1 tissue inhibitor, smooth muscle a-actin, polypeptide chain elongation factor 1 (EF1-sp), β-actin, spike and j8 myosin heavy chain, myosin light chain 1 and 2, myelin basic Protein, serum amyloid component, renin, etc.), preferably Moroni leukemia virus promoter, which can be highly expressed in cartilage tissue, human and bird jQa. A Kuching promoter or the like can be used. In order to express the MMP-19 gene specifically in cartilage in humans, rats, mice, etc., the promoter region of the type II collagen gene, which is known to be expressed in cartilage, is effective. It is preferable that the transgenic mammal has a sequence that terminates the transcription of the target messenger RNA (poly A, generally referred to as “Yuichi Mineta”). For example, it is derived from viruses, various mammals and birds Gene expression can be manipulated using the sequence of each gene. Preferably, the simian virus SV40 and the like are used. In addition, in order to further express the target gene, the splicing signal of each gene, the enhancer region, and a part of the intron of the eukaryotic gene are transferred 5 'upstream of the promoter region, between the promoter region and the translation region. Alternatively, it may be linked to the 3 'downstream of the translation region depending on the purpose.
MM P— 1 9の翻訳領域は、 ヒトゃ各種非ヒ卜哺乳動物 （ゥサギ、 ィヌ、 ネコ 、 モルモット、 ハムスター、 ラット、 マウスなど） の肝臓、 腎臓、 繊維芽細胞な どに由来する D N Aおよび市販の各種ゲノム D NAライブラリ一に由来するゲノ ム D NAの全てあるいは一部を原料として用い、 あるいはヒトゃ各種非ヒ卜哺乳 動物の肝臓、 腎臓、 繊維芽細胞に由来する RNAから公知の方法により調製された 相補 D NAを原料として用いて、 取得することが出来る。 また、 上記の細胞ある いは組織などから得られた MM P— 1 9の翻訳領域を用いて、 点突然変異誘発法 などにより変異した翻訳領域を作製することもできる。 これらは何れも遺伝子導 入動物に利用可能な材料である。  The translation region of MMP-19 is derived from DNA derived from the liver, kidney, fibroblasts, etc. of humans and various non-human mammals (eg, herons, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). A known method using all or part of the genomic DNA derived from various commercially available genomic DNA libraries as a raw material, or from RNA derived from liver, kidney, and fibroblasts of human and various non-human mammals Can be obtained using the complementary DNA prepared by the above as a raw material. Further, a translation region mutated by a point mutagenesis method or the like can also be prepared using the translation region of MMP-19 obtained from the above cells or tissues. These are all materials that can be used for transgenic animals.
以上の翻訳領域は、 導入動物において発現しうる遺伝子コンストラクト （例、 ベクタ一など） として前記のプロモーターの下流 （好ましくは、 転写終結部位の 上流） に連結させる通常の遺伝子工学的手法により、 MM P— 1 9遺伝子を組み 込んだ D N Aを作製することができる。 The above-mentioned translation region is located downstream of the above promoter (preferably at the transcription termination site) as a gene construct (eg, a vector, etc.) that can be expressed in an introduced animal. DNA incorporating the MMP-19 gene can be produced by the usual genetic engineering technique of ligation to the upstream.
受精卵細胞段階における MM P— 1 9遺伝子の導入は、 対象非ヒト哺乳動物の 胚芽細胞および体細胞のすべてに存在するように確保される。 遺伝子導入後の作 出動物の胚芽細胞において、 MM P— 1 9遺伝子が存在することは、 作出動物の 後代の動物全てが、 その胚芽細胞および体細胞のすべてに MM P— 1 9遺伝子が 存在することを意味する。 遺伝子を受け継いだこの種の動物の子孫は、 その胚芽 細胞および体細胞のすべてに MM P— 1 9遺伝子を有する。  Introduction of the MMP-19 gene at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target non-human mammal. The presence of the MMP-19 gene in the germ cells of the transgenic animal after gene transfer indicates that the progeny of the transgenic animal has the MMP-19 gene in all of its germ cells and somatic cells Means to do. The offspring of this type of animal that has inherited the gene carry the MMP-19 gene in all of its germinal and somatic cells.
導入遺伝子を相同染色体の両方に持つホモザィゴ一ト動物を取得し、 この雌雄 の動物を交配することにより、 すべての子孫が該遺伝子を安定に保持し、 また、 該遺伝子を過剰に有することを確認して、 通常の飼育環境で繁殖継代することが できる。  By obtaining homozygous animals having the transgene on both homologous chromosomes and crossing the male and female animals, it is confirmed that all offspring stably maintain the gene and that the gene has an excessive amount of the gene. Then, it can be subcultured in a normal breeding environment.
外来性の MM P— 1 9遺伝子を、 対象非ヒト哺乳動物 （好ましくはラットなど 、 特に好ましくは W i s t a r系統のラッ卜など) またはその先祖の受精卵に導 入する際に用いられる受精卵は、 同種の雄非ヒト哺乳動物 （好ましくは雄ラット など、 特に好ましくは W i s t a r系統の雄ラットなど） と雌非ヒト哺乳動物 （ 好ましくは雌ラットなど、 特に好ましくは W i s t a r系統の雌ラットなど） を 交配させることによって得られる。  The fertilized egg used to introduce the exogenous MMP-19 gene into a target non-human mammal (preferably a rat or the like, particularly preferably a Wistar strain rat or the like) or a fertilized egg of its ancestor is Male non-human mammals of the same species (preferably male rats, etc., particularly preferably male rats of the Wistar strain) and female non-human mammals (preferably female rats, particularly preferably female rats of the Wistar strain) Are obtained by crossing.
受精卵は自然交配によっても得られるが、 雌非ヒト哺乳動物 （好ましくは雌ラ ットなど、 特に好ましくは W i s t a r系統の雌ラットなど） の性周期を人工的 に調節した後、 雄非ヒト哺乳動物 （好ましくは雌ラットなど、 特に好ましくは W i s t a r系統の雌ラットなど） と交配させる方法が好ましい。 雌非ヒト哺乳動 物の性周期を人工的に調節する方法としては、 例えば初めに卵胞刺激ホルモン （ 妊馬血清性性腺刺激ホルモン、 一般に P M S Gと略する） 、 次いで黄体形成ホル モン （ヒト絨毛性性腺刺激ホルモン、 一般に h C Gと略する） を、 例えば腹腔注 射などにより投与する方法が好ましいが、 好ましいホルモンの投与量、 投与間隔 は非ヒト哺乳動物の種類によりそれぞれ異なる。 また、 W i s t a r系統のラッ 卜を用いる場合は、 約 1 2時間明期条件 （例えば 7 : 0 0— 1 9 : 0 0 ) で約 1 週間飼育した 8週齢以上のものが好ましい。 非ヒト哺乳動物が雌ラッ卜 （好まし くは W i s t a r系統の雌ラッ卜） の場合、 通常、 卵胞刺激ホルモン投与後、 約 4 8時間後に黄体形成ホルモンを投与し、 雄ラットと交配させることにより受精 卵を得る方法が好ましく、 卵胞刺激ホルモンの投与量は約 2 0〜約 5 0 I U/個 体、 好ましくは約 3 0 I UZ個体、 黄体形成ホルモンの投与量は約 0〜約 1 0 I UZ個体、 好ましくは約 5 I UZ個体である。 Fertilized eggs can also be obtained by natural mating, but after artificially regulating the estrous cycle of female non-human mammals (preferably female rats, particularly preferably female rats of the Wistar strain), A method of crossing with mammals (preferably female rats, particularly preferably female rats of the Wistar strain) is preferred. Methods for artificially regulating the estrous cycle of female non-human mammals include, for example, follicle-stimulating hormone (pregnant horse serum gonadotropin, generally abbreviated as PMSG) first, and then luteinizing hormone (human chorionic). Gonadotropin, generally abbreviated as hCG) is preferably administered by, for example, intraperitoneal injection, but the preferred hormone dose and administration interval vary depending on the type of non-human mammal. When a Wistar strain rat is used, it is preferable that the rat be reared under a light condition of about 12 hours (for example, 7: 0 to 19:00) for about 1 week or older. Non-human mammals are female rats (preferred In the case of female rats of the Wistar strain, it is usually preferable to administer luteinizing hormone about 48 hours after administration of follicle-stimulating hormone and obtain fertilized eggs by mating with male rats. The dose of the hormone is about 20 to about 50 IU / person, preferably about 30 IUZ individuals, and the dose of luteinizing hormone is about 0 to about 10 IUZ individuals, preferably about 5 IUZ individuals. It is.
得られた受精卵に、 前述の方法により外来性 MM P— 1 9遺伝子が導入された 後、 雌非ヒ卜哺乳動物に人工的に移植 ·着床され、 外来性遺伝子を組み込んだ D NAを有する非ヒ卜哺乳動物が得られる。  After the exogenous MMP-19 gene has been introduced into the obtained fertilized eggs by the method described above, DNA is implanted and implanted artificially into a female non-human mammal, and DNA incorporating the exogenous gene is obtained. A non-human mammal having the above is obtained.
また、 黄体形成ホルモン放出ホルモン （一般に L HRHと略する） あるいはそ の類縁体を投与後、 雄ヒト哺乳動物と交配させることにより、 受精能を誘起され た偽妊娠雌非ヒト哺乳動物に、 得られた受精卵を人工的に移植 ·着床させる方法 も好ましい。  In addition, after administration of luteinizing hormone-releasing hormone (generally abbreviated as L HRH) or an analog thereof, it is mated with a male human mammal to obtain a pseudopregnant female non-human mammal in which fertility has been induced. A method of artificially transplanting and implanting the obtained fertilized egg is also preferable.
L H RHあるいはその類縁体の投与量、 ならびにその投与後に雄非ヒト哺乳動 物と交配させる時期は、 非ヒト哺乳動物の種類によりそれぞれ異なる。 非ヒト哺 乳動物が雌ラット （好ましくは W i s t a r系統の雌ラット） の場合は、 通常、 し111^ぁるぃはその類縁体 （例ぇば[3， 5-011 ] - 11-1^、 [Gin8] - LH- RH、 [ D_Ala6] -LH - RH、 [des-Gly1 °] -LH-RH, [D - His (Bzl) 6] - LH - RHおよびそれらの Ettiy lamideなど） を投与した後、 約 4日目に雄ラットと交配させることが好ましく、 L HR Hあるいはその類縁体の投与量は、 通常、 約 1 0〜6 0 個体、 好ま しくは約 4 0 g Z個体である。 The dose of LHRH or an analog thereof and the timing of mating with a male non-human mammal after its administration differ depending on the type of non-human mammal. When the non-human mammal is a female rat (preferably, a female rat of the Wistar strain), usually, 111111 is an analog thereof (eg, [3, 5-011]-11-1 ^). , [Gin 8 ] -LH-RH, [D_Ala 6 ] -LH-RH, [des-Gly 1 °] -LH-RH, [D-His (Bzl) 6 ] -LH-RH and their Ettiy lamide etc. ), It is preferable to breed with male rats about 4 days after administration, and the dose of LHRH or an analog thereof is usually about 10 to 60 individuals, preferably about 40 gZ. It is an individual.
また、 上記した雌非ヒト哺乳動物の性周期を人工的に調節して受精卵を取得す るする方法と受精能を誘起された偽妊娠雌非ヒト哺乳動物に、 得られた受精卵を 人工的に移植 ·着床させる方法とを組み合わせて用いることが好ましい。  Further, a method for artificially regulating the estrous cycle of a female non-human mammal to obtain a fertilized egg and a method for artificially regulating the sexual cycle of the female non-human mammal, It is preferable to use the method in combination with the method of transplantation and implantation.
本発明の MM P— 1 9遺伝子が導入された非ヒト哺乳動物 （以下、 本発明の非 ヒ卜哺乳動物と略記する場合がある） は、 軟骨破壊を起こし、 関節疾患を発症し ている。 また、 本発明の非ヒ卜哺乳動物は、 細胞外マトリックスの異常な分解を 起こし、 細胞外マトリックスの異常な分解に起因する疾患 （例、 軟骨形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代 謝性関節症、 眼疾患、 悪性腫瘍など） を発症している。 さらに、 本発明の非ヒト哺乳動物は、 ①四肢の短縮と変形、 ②頭蓋の変形、 ③ 咬合不全、 ④上顎及び下顎切歯の過長、 ⑤腰椎の石灰化不全、 ⑥尾椎の変形およ び⑦尾椎椎間板の欠損などから選ばれる症状を発症している。 The non-human mammal into which the MMP-19 gene of the present invention has been introduced (hereinafter sometimes abbreviated as the non-human mammal of the present invention) has cartilage destruction and has developed joint disease. In addition, the non-human mammal of the present invention causes abnormal degradation of the extracellular matrix, and a disease caused by abnormal degradation of the extracellular matrix (eg, cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis) , Rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye diseases, malignant tumors, etc.). Furthermore, the non-human mammals of the present invention include: 1) shortening and deformation of the limbs, 2) deformation of the skull, 3) malocclusion, 4) excessive maxillary and mandibular incisors, 4) insufficient calcification of the lumbar spine, 4) deformation of the tail vertebra, and And symptoms selected from caudal intervertebral disc defects.
本発明の非ヒト哺乳動物は、 上記のような極めてユニークな特徴を有している ので、 以下に示す有用な用途を有している。  Since the non-human mammal of the present invention has the above-mentioned very unique features, it has the following useful uses.
以下に、 本発明の非ヒ卜哺乳動物の用途を概説する。  The use of the non-human mammal of the present invention will be outlined below.
( 1 ) 本発明のヒト哺乳動物は、 外来性 MM P— 1 9遺伝子が高発現させられ ており、 この MM P— 1 9の酵素活性により、 軟骨破壊を起こしているので、 軟 骨破壊を特徴とする各種の疾患、 例えば、 軟骨形成異常、 骨形成異常、 骨粗鬆症 、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症、 眼疾患、 悪性腫瘍などの細胞外マ卜リックスの異常な分解に起因する疾患の予防薬あるい は治療薬の評価のために用いることができる。  (1) In the human mammal of the present invention, the exogenous MMP-19 gene is highly expressed, and cartilage destruction is caused by the enzymatic activity of this MMP-19 gene. Characteristic various diseases, for example, extracellular matrices such as cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye diseases, malignant tumors It can be used to evaluate prophylactic or therapeutic agents for diseases caused by abnormal degradation of Rix.
すなわち、 本発明は、 本発明の非ヒト哺乳動物またはその生体の一部に被験物 質を適用し、 細胞外マトリックスの異常な分解に起因する疾患の改善効果を検定 することを特徴とする、 細胞外マトリックスの異常な分解に起因する疾患の予防 •治療のために用いられる物質のスクリーニング方法を提供する。  That is, the present invention is characterized in that a test substance is applied to the non-human mammal of the present invention or a part of the living body thereof, and the effect of ameliorating a disease caused by abnormal degradation of the extracellular matrix is assayed. Prevention of diseases caused by abnormal degradation of extracellular matrix • Provide a method for screening substances used for treatment.
細胞外マトリックスの異常な分解としては、 MM P— 1 9の機能異常に起因す るものが挙げられる。  Abnormal degradation of the extracellular matrix includes that caused by an abnormal function of MMP-19.
具体的には、 本発明のスクリーニング方法では、 本発明の非ヒト哺乳動物に被 検物質を投与する。 被験物質としては、 公知の合成化合物、 ペプチド、 タンパク 質、 D N Aライブラリーなどの他に、 例えば温血哺乳動物 （例えば、 マウス、 ラ ット、 ブ夕、 ゥシ、 ヒッジ、 サル、 ヒトなど） の組織抽出物、 細胞培養上清など が用いられる。  Specifically, in the screening method of the present invention, a test substance is administered to the non-human mammal of the present invention. The test substance may be a known synthetic compound, peptide, protein, DNA library, or the like, or, for example, a warm-blooded mammal (eg, mouse, rat, bush, horsetail, hidge, monkey, human, etc.) Tissue extract, cell culture supernatant, etc. are used.
次に尿中 ·血中ピリジノリン量などを指標にその薬物評価あるいは治療効果を 調べることにより軟骨分解抑制薬の評価を行うことができる。  Next, the cartilage degradation inhibitor can be evaluated by examining the drug evaluation or therapeutic effect using urinary and blood pyridinoline levels as indicators.
指標として、 ピリジノリン量のほかに、 処理個体の体重、 体長、 四肢長測定、 X線撮影による骨格の観察、 MM P各種、 T I M P (Tissue Inihibi tor of Met al loproteinase) 、 プロテオダリカン、 コラーゲン、 ケラタン硫酸、 ヒアルロン 酸、 ォステオカルシン、 カルシウム、 リン、 サイト力イン、 成長因子などの血中 あるいは尿中軟骨、 骨代謝生化学マーカーを指標にその薬物評価あるいは治療効 果を調べることができる。 実験の必要に応じて、 病理標本の作製、 サフラニン O 染色などの方法により軟骨破壊程度などを調べることも有用である。 As indicators, in addition to the amount of pyridinoline, measurement of body weight, body length, and limb length of the treated animal, observation of the skeleton by X-ray photography, various types of MMP, TIMP (Tissue Inhibitor of Met alloproteinase), proteodalican, collagen, keratan Sulfuric acid, hyaluronic acid, osteocalcin, calcium, phosphorus, cytodynamics, blood growth factors, etc. Alternatively, drug evaluation or therapeutic effect can be examined using urinary cartilage and bone metabolism biochemical markers as indices. It is also useful to examine the degree of cartilage destruction by methods such as preparation of pathological specimens and staining with safranin O if necessary for experiments.
( 2 ) 上記各疾患の予防薬あるいは治療薬の評価のために本発明の非ヒト哺乳 動物に各種抗原 （例、 I I型コラーゲンやァグリカンなどの基質など） 、 細菌 （ 例、 結核菌死菌など） あるいはウィルス （例、 Epstein- Barr virusなど） を全身 あるいは関節などの局所、 一回、 複数回または連続投与により上記疾患の発症促 進を行う。 これらのものはモデル動物で慢性関節リウマチを発症させることが知 られている。 また、 MM Pの活性化を行うことが知られている、 トリプシンなど のセリンプロテア一ゼ、 ダルタチオン、 活性酸素、 N〇などの窒素酸化物、 水銀 化合物 （例、 4- aminophenylmercuric acetateなど） 、 炎症を惹起しプロテア一 ゼ活性を発現させる I L— 1や TN F aなどのサイトカインを関節に投与するこ とにより発症を促進できる。 発症の促進した非ヒ卜哺乳動物は遺伝子を導入して いない非ヒト哺乳動物はもちろん、 発症の促進していない遺伝子導入非ヒト哺乳 動物と比較して上記指標の絶対値大きいため、 発症促進非ヒト動物を用いて （1 ) に記載した評価を行なうことにより、 より精度良く評価することができる。  (2) Various antigens (eg, substrates such as type II collagen and aggrecan), bacteria (eg, killed Mycobacterium tuberculosis, etc.) may be added to the non-human mammal of the present invention for the evaluation of prophylactic or therapeutic agents for the above diseases. ) Or the virus (eg, Epstein-Barr virus, etc.) is promoted by local, single, multiple or continuous administration of the virus systemically or jointly. These are known to cause rheumatoid arthritis in model animals. It is also known to activate MMPs, serine proteases such as trypsin, daltathione, active oxygen, nitrogen oxides such as N〇, mercury compounds (eg, 4-aminophenylmercuric acetate, etc.), inflammation The onset can be promoted by administering to the joints cytokines such as IL-1 and TN Fa that induce protease and express protease activity. Non-human mammals in which the onset is promoted are not only non-human mammals in which the gene has not been introduced, but also have a greater absolute value of the above index than non-human mammals in which the onset has not been promoted. By performing the evaluation described in (1) using human animals, the evaluation can be performed with higher accuracy.
( 3 ) また、 本発明の非ヒ卜哺乳動物から採取した軟骨片または軟骨細胞など を培養し、 MM P— 1 9阻害薬の評価に用いることができる。 具体的には、 これ らの軟骨片あるいは軟骨細胞を MM P— 1 9酵素の材料として、 基質となる合成 ペプチドを加えることで阻害剤が評価できる。 必要に応じて、 水銀化合物やトリ プシンなどのセリンプロテアーゼで活性化しても良い。 MM P— 1 9は軟骨破壊 を促進することから、 これらの評価系を用いることにより、 軟骨形成異常、 骨形 成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性 関節症、 眼疾患および悪性腫瘍などの予防 ·治療剤の評価に用いることができる  (3) Cartilage fragments or chondrocytes collected from the non-human mammal of the present invention can be cultured and used for evaluation of MMP-19 inhibitors. Specifically, inhibitors can be evaluated by using these cartilage fragments or chondrocytes as a material for the MMP-19 enzyme and adding a synthetic peptide as a substrate. If necessary, it may be activated with a serine protease such as a mercury compound or trypsin. Since MMP-19 promotes cartilage destruction, using these evaluation systems, it is possible to use these evaluation systems to determine cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolism Can be used to evaluate prophylactic and therapeutic agents for osteoarthritis, eye diseases and malignant tumors
( 4 ) さらに、 本発明の非ヒト哺乳動物を妊娠させ、 該妊娠中の動物を、 軟骨 形成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症、 眼疾患および悪性腫瘍、 それらの合併症の予防薬あるい は治療薬の評価のために用いることができる。 具体的には、 まず最初、 妊娠中の本発明の非ヒト哺乳動物に被検物質を投与す る。 次に出産後の産仔の体重、 体長、 四肢長測定、 X線撮影による形態計測、 M MP各種、 T I M P、 プロテオダリカン、 コラーゲン、 ケラタン硫酸、 ピリジノ リン、 ヒアルロン酸、 ォステオカルシン、 カルシウム、 リン、 サイト力イン、 成 長因子などの血中あるいは尿中軟骨、 骨代謝生化学マーカーを指標にその薬物評 価あるいは治療効果を調べる。 実験の必要に応じて、 病理標本を作製したり、 続 いてサフラニン O染色などの方法により軟骨破壌程度などを調べることも有用で ある。 (4) Further, the non-human mammal of the present invention is pregnant, and the pregnant animal is treated with cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic It can be used to evaluate prophylactic or therapeutic agents for arthropathy, eye diseases and malignancies, and their complications. Specifically, first, a test substance is administered to a pregnant non-human mammal of the present invention. Next, the weight, body length and limb length of the offspring after childbirth, morphometry by radiography, various types of MMP, TIMP, proteodalican, collagen, keratan sulfate, pyridinoline, hyaluronic acid, osteocalcin, calcium, phosphorus, Investigate drug evaluation or therapeutic effects using biomarkers of cartilage and bone metabolism in blood or urine such as cytokins and growth factors. It is also useful to prepare a pathological specimen if necessary for the experiment, and to subsequently check the degree of cartilage rupture by a method such as safranin O staining.
( 5 ) 本発明の非ヒ卜哺乳動物を軟骨コラーゲンを分解する事が知られている MM P s (例、 MM P—1、 8、 1 3等） やァグリカンを分解する aggrecanase などの遺伝子の導入された非ヒト哺乳動物交配し、 取得された産仔は MM P— 1 9のみならずこれらの遺伝子の産物 （タンパク質） に起因する疾患 （例、 軟骨形 成異常、 骨形成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑 膜炎、 代謝性関節症、 眼疾患および悪性腫瘍） を発症することが期待される。 し たがって、 該産仔は上記 （1 ) から （4) と同様またはそれに準じた方法により かかる疾患の予防 ·治療剤の評価に用いることができる。  (5) The non-human mammal of the present invention can be transformed with genes such as MMPs (eg, MMP-1, 8, 13 etc.), which are known to degrade cartilage collagen, and aggrecanase, which degrades aggrecan. Introduced non-human mammals are mated and the offspring obtained are diseases caused by the products (proteins) of these genes as well as MMP-19 (eg, cartilage dysplasia, bone dysplasia, osteoporosis, It is expected to develop osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye diseases and malignant tumors. Therefore, the offspring can be used for the evaluation of a prophylactic / therapeutic agent for such a disease by a method similar to (1) to (4) or a method analogous thereto.
以上のように、 本発明のスクリーニング方法において、 被検物質の投与により 、 細胞外マトリックスの異常な分解に起因する疾患の改善効果があると判定され た場合、 その被検物質は前記した細胞外マ卜リックスの異常な分解に起因する疾 患の予防 ·治療用医薬として選択することができる。  As described above, in the screening method of the present invention, when it is determined that administration of a test substance has an effect of ameliorating a disease caused by abnormal degradation of the extracellular matrix, the test substance is extracellular as described above. It can be selected as a drug for prevention and treatment of diseases caused by abnormal degradation of matrix.
上記の予防 ·治療用医薬として選択された物質は、 例えば、 必要に応じて糖衣 を施した錠剤、 力プセル剤、 エリキシル剤、 マイクロ力プセル剤などとして経口 的に、 あるいは水もしくはそれ以外の薬学的に許容し得る液との無菌性溶液、 ま たは懸濁液剤などの注射剤の形で非経口的に使用できる。 例えば、 予防 ·治療用 医薬として選択された物質を生理学的に認められる担体、 香味剤、 賦形剤、 べヒ クル、 防腐剤、 安定剤、 結合剤などとともに一般に認められた製剤実施に要求さ れる単位用量形態で混和することによって製造することができる。 これら製剤に おける有効成分量は指示された範囲の適当な用量が得られるようにするものであ る。 錠剤、 カプセル剤などに混和することができる添加剤としては、 例えば、 ゼラ チン、 コーンスターチ、 トラガント、 アラビアゴムのような結合剤、 結晶性セル ロースのような賦形剤、 コーンスターチ、 ゼラチン、 アルギン酸などのような膨 化剤、 ステアリン酸マグネシウムのような潤滑剤、 ショ糖、 乳糖またはサッカリ ンのような甘味剤、 ペパーミント、 ァカモノ油またはチェリ一のような香味剤な どが用いられる。 調剤単位形態がカプセルである場合には、 前記タイプの材料に さらに油脂のような液状担体を含有することができる。 注射のための無菌組成物 は注射用水のようなべヒクル中の活性物質、 胡麻油、 椰子油などのような天然産 出植物油などを溶解または懸濁させるなどの通常の製剤実施に従って処方するこ とができる。 Substances selected as the above preventive and therapeutic drugs include, for example, sugar-coated tablets, forcepsels, elixirs, microforces, etc., orally, or water or other drugs. It can be used parenterally in the form of an injectable preparation such as a sterile solution with a liquid that is acceptable or a suspension. For example, a substance selected as a prophylactic / therapeutic drug may be required to implement a generally accepted formulation with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. Can be prepared by mixing them in unit dosage form. The amount of the active ingredient in such preparations is to be adjusted so that an appropriate dose in the specified range can be obtained. Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
注射用の水性液としては、 例えば、 生理食塩水、 ブドウ糖やその他の補助薬を 含む等張液 （例えば、 D—ソルビト一ル、 D—マンニトール、 塩化ナトリウムな ど） などが挙げられ、 適当な溶解補助剤、 例えば、 アルコール （例えば、 ェタノ ールなど） 、 ポリアルコール (例えば、 プロピレングリコール、 ポリエチレング リコールなど） 、 非イオン性界面活性剤 （例えば、 ポリソルベート 8 0™、 H C 0— 5 0など） などと併用してもよい。 油性液としては、 例えば、 ゴマ油、 大豆 油などが挙げられ、 溶解補助剤として安息香酸ベンジル、 ベンジルアルコールな どと併用してもよい。 また、 緩衝剤 （例えば、 リン酸塩緩衝液、 酢酸ナトリウム 緩衝液など） 、 無痛化剤 （例えば、 塩化ベンザルコニゥム、 塩酸プロ力インなど ) 、 安定剤 （例えば、 ヒト血清アルブミン、 ポリエチレングリコ一ルなど） 、 保 存剤 （例えば、 ベンジルアルコール、 フエノールなど） 、 酸化防止剤などと配合 してもよい。 調製された注射液は、 通常、 適当なアンプルに充填される。  Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). Solubilizers, for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 80 ™, HC 0-50, etc.) ) May be used together. Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, proforce hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.) ), A preservative (eg, benzyl alcohol, phenol, etc.), an antioxidant and the like. The prepared injection is usually filled in an appropriate ampoule.
また、 予防 ·治療用医薬として選択された物質が D NAである場合、 当該 D N Aを単独あるいはレトロウイルスベクタ一、 アデノウイルスベクタ一、 アデノウ ィルスァソシエーテツドウィルスベクタ一などの適当なベクターに挿入した後、 常套手段に従って、 ヒトまたは温血動物に投与することができる。 当該 D NAは 、 そのままで、 あるいは摂取促進のための補助剤などの生理学的に認められる担 体とともに製剤化し、 遺伝子銃やハイドロゲルカテーテルのようなカテーテルに よって投与できる。 このようにして得られる製剤は、 安全で低毒性であるので、 例えば、 ヒトまた は温血動物 （例えば、 ラッ卜、 マウス、 モルモット、 ゥサギ、 トリ、 ヒッジ、 ブ 夕、 ゥシ、 ゥマ、 ネコ、 ィヌ、 サル、 など） に対して投与することができる。 予防 ·治療用医薬として選択された物質の投与量は、 対象疾患、 投与対象、 投 与ルートなどにより差異はあるが、 例えば、 関節炎の治療目的で経口投与する場 合、 一般的に成人 （60 kgとして） においては、 一日につき当該物質を約 0. lmg〜； L 0 Omg、 好ましくは約 1. 0〜 5 Omg、 より好ましくは約 1. 0 〜20mg投与する。 非経口的に投与する場合は、 当該物質の 1回投与量は投与 対象、 対象疾患などによっても異なるが、 例えば、 関節炎の治療目的で注射剤の 形で成人 （体重 6 O kgとして） に投与する場合、 一日につき当該物質を約 0. 01〜30mg程度、 好ましくは約 0. l〜20mg程度、 より好ましくは約 0 . 1〜1 Omg程度を患部に注射することにより投与するのが好都合である。 他 の動物の場合も、 60 k g当たりに換算した量を投与することができる。 When the substance selected as a drug for prophylaxis or treatment is DNA, insert the DNA alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. After that, it can be administered to humans or warm-blooded animals according to conventional means. The DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and can be administered using a gene gun or a catheter such as a hydrogel catheter. The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, birds, higgies, bushes, dogs, dogs, Cats, dogs, monkeys, etc.). The dose of a substance selected as a prophylactic or therapeutic drug may vary depending on the target disease, the subject of administration, the route of administration, and the like. ), the substance is administered from about 0.1 mg to about 0.1 mg per day, preferably from about 1.0 to 5 mg, more preferably from about 1.0 to 20 mg. In the case of parenteral administration, the single dose of the substance may vary depending on the target of administration, target disease, etc., for example, administration to adults (with a body weight of 6 O kg) in the form of injections for the treatment of arthritis In this case, it is convenient to administer the substance by injecting about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day into the affected area. It is. In the case of other animals, the dose can be administered in terms of 60 kg.
(6) さらに、 本発明の非ヒト哺乳動物は、 MMP— 19遺伝子異常患者の遺 伝子治療用実験に用いることができる。  (6) Further, the non-human mammal of the present invention can be used for an experiment for gene therapy of a patient with an MMP-19 gene abnormality.
以上の本発明の遺伝子導入哺乳動物を、 組織培養のための細胞源として使用す ることも可能である。 また例えば、 本発明の遺伝子導入ラットの組織中の DN A もしくは RN Aを直接分析するか、 あるいは遺伝子により発現されたタンパク組 織を分析することにより、 核内レセプターの複雑な作用の転写因子との関連性に ついて解析することもできる。 あるいは、 遺伝子を有する組織の細胞を、 標準組 織培養技術により培養し、 これらを使用して、 例えば軟骨組織を形成する細胞な どの一般に培養が困難な組織に由来する細胞の機能を研究することもできる。 さ らに、 その細胞を用いることにより、 例えば細胞の機能を高めるような薬剤の選 択も可能である。 また、 高発現細胞株があれば、 そこから、 MMP— 19を大量 に単離精製すること、 ならびにその抗体を作製することも可能である。 本発明の配列表の配列番号は、 以下の配列を示す。  The above-described transgenic mammal of the present invention can also be used as a cell source for tissue culture. Further, for example, by directly analyzing DNA or RNA in the tissue of the transgenic rat of the present invention, or by analyzing the protein expressed by the gene, a transcription factor having a complex action of a nuclear receptor can be obtained. It is also possible to analyze the relationship between the two. Alternatively, culturing cells of a gene-bearing tissue by standard tissue culture techniques and using them to study the function of cells derived from tissues that are generally difficult to culture, such as cells forming cartilage tissue. Can also. Further, by using the cells, for example, it is possible to select a drug that enhances the function of the cells. In addition, if there is a high-expressing cell line, it is possible to isolate and purify MMP-19 in a large amount and to produce an antibody thereof. The sequence numbers in the sequence listing of the present invention indicate the following sequences.
〔配列番号： 1〕 後述の実施例 1で行われた PC R (ポリメラ一ゼチェインリ ァクション） 法に用いられたプライマーの塩基配列を示す。 〔配列番号： 2〕 後述の実施例 1で行われた PC R (ポリメラ一ゼチェインリ アクション） 法に用いられたプライマーの塩基配列を示す。 [SEQ ID NO: 1] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 1 described later. [SEQ ID NO: 2] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 1 described later.
〔配列番号： 3〕 後述の実施例 1で行われた PC R (ポリメラ一ゼチェインリ アクション） 法に用いられたプライマーの塩基配列を示す。  [SEQ ID NO: 3] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 1 described later.
〔配列番号： 4〕 後述の実施例 1で行われた PC R (ポリメラ一ゼチェインリ アクション） 法に用いられたプライマ一の塩基配列を示す。  [SEQ ID NO: 4] This shows the base sequence of the primer used in PCR (polymerase chain reaction) method performed in Example 1 described later.
〔配列番号： 5〕 後述の実施例 4で行われた PC R (ポリメラーゼチェインリ アクション) 法に用いられたプライマーの塩基配列を示す。  [SEQ ID NO: 5] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 4 described later.
〔配列番号： 6〕 後述の実施例 4で行われた PC R (ポリメラーゼチェインリ アクション） 法に用いられたプライマーの塩基配列を示す。  [SEQ ID NO: 6] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 4 described later.
〔配列番号： 7〕 後述の実施例 6で行われた PC R (ポリメラーゼチェインリ ァクション） 法に用いられたプライマーの塩基配列を示す。  [SEQ ID NO: 7] This shows the base sequence of the primer used in the PCR (polymerase chain reaction) method performed in Example 6 described later.
〔配列番号： 8〕 後述の実施例 6で行われた P C R (ポリメラ一ゼチエインリ アクション） 法に用いられたプライマーの塩基配列を示す。  [SEQ ID NO: 8] This shows the base sequence of the primer used in the PCR (polymerase zetiein reaction) method performed in Example 6 described later.
〔配列番号： 9〕 後述の実施例 1でクロ一ニングしたラット I I型コラーゲン 遺伝子のプロモーター領域の塩基配列を示す。  [SEQ ID NO: 9] This shows the base sequence of the promoter region of rat type I collagen gene cloned in Example 1 described later.
〔配列番号： 10〕 後述の実施例 1でクローニングしたラット I I型コラーゲ ン遺伝子のェンハンサー領域の塩基配列を示す。  [SEQ ID NO: 10] This shows the base sequence of the enhancer region of rat type I collagen gene cloned in Example 1 described later.
〔配列番号： 1 1〕 後述の実施例 2で pKS—MMPB c on— 19の構築に 用いた pTB 399由来のスプライシングサイトを含む DN A断片の塩基配列を 示す。  [SEQ ID NO: 11] This shows the base sequence of the DNA fragment containing the splicing site derived from pTB399 used for the construction of pKS-MMPB con-19 in Example 2 described later.
〔配列番号： 12〕 ヒ卜 MMP— 19遺伝子の塩基配列を示す。  [SEQ ID NO: 12] This shows the base sequence of human MMP-19 gene.
〔配列番号： 13〕 ヒト MMP— 19のアミノ酸配列を示す。 本願明細書において、 塩基やアミノ酸などを略号で表示する場合、 IUPAC- IUB Commission on Biochemical Nomenclatureによる略号あるいは当該分野における 慣用略号に基づくものであり、 その例を次に挙げる。  [SEQ ID NO: 13] This shows the amino acid sequence of human MMP-19. In the present specification, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC- IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the relevant field, and examples thereof are as follows.
DNA ：デォキシリポ核酸  DNA: Deoxylipo nucleic acid
RNA ： リポ核酸 A アデニン RNA: Liponucleic acid A adenine
T チミン  T thymine
G グァニン  G Guanin
C 後述の実施例 2で得られた形質転換体大腸菌 JMl 09/pKS-MMPB c on— 19は、 2001年 7月 2日から茨城県つくば市東 1丁目 1番地 1 中央 第 6 (郵便番号 305 - 8566) の独立行政法人産業技術総合研究所 特許生 物寄託センタ一に寄託番号 FERM BP— 7647として、 2001年 6月 2 2日から大阪府大阪市淀川区十三本町 2— 17-85 (郵便番号 532 - 868 6 ) の財団法人 ·発酵研究所 （ I F 0) に寄託番号 I FO 16666として寄 託されている。 実施例  C The transformant Escherichia coli JMl 09 / pKS-MMPB con—19 obtained in Example 2 described below has been used since July 2, 2001, 1-1 1-1 Higashi, Tsukuba-shi, Ibaraki Prefecture 1 Central No. 6 (Zip code 305- 8566) at the National Institute of Advanced Industrial Science and Technology (AIST) as the deposit number FERM BP-7647 as a deposit number FERM BP-7647 from June 22, 2001 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka No. 532-868 6) and deposited at the Fermentation Research Institute (IF 0) under the accession number IFO 16666. Example
以下に実施例を挙げて本発明をより具体的に説明するが、 本発明がこれらに限 定されないことは言うまでもない。 実施例 1 ラット I I型コラーゲン遺伝子のプロモーターおよびェンハンサ一の クローニング  Hereinafter, the present invention will be described more specifically with reference to Examples, but it goes without saying that the present invention is not limited thereto. Example 1 Cloning of rat type I collagen gene promoter and enhancer
ラット I I型コラーゲン遺伝子のプロモータ一領域は、 Kohnoら （LBiol. Chem . 260： 4441, 1985) の塩基配列をもとに設計したプライマ一 （5' -GTGGTGGTGGAC AACTAGGAAACTCTGG-3' ：配列番号： 1) および （5' - CGAGGCGAAKATGGCTCACCGCG-3' ：配列番号： 2) を用いて PCR法により得た。 得られた約 1. 2Kbの断片を TOPO TA Cloning Kit (Invitrogen社製） を用い、 添付のプロトコールに従い、 p CR I I一 TOP〇にクロ一ニングした （pCRI I— p r omo t e r 2と 呼ぶ） 。 挿入された DNA断片の塩基配列を AB I社製 DNAシークェンサ一で 常法により確認したところ、 前述文献のプロモーター領域の塩基配列と一致した (配列番号： 9) 。 pCRI I— p romo t e r 2中の pCRI Iプラスミド のマルチプルクロ一ニングサイトの No t Iサイト （5' 側） と pCRI I— p romo t e r 2中の I I型コラーゲン遺伝子プロモーター配列内の Sm a Iサ ィ卜からなる断片を以下の実験に用いた。 The region of the promoter of the rat type II collagen gene is a primer (5'-GTGGTGGTGGAC AACTAGGAAACTCTGG-3 ': SEQ ID NO: 1) designed based on the nucleotide sequence of Kohno et al. (LBiol. Chem. 260: 4441, 1985). And (5′-CGAGGCGAAKATGGCTCACCGCG-3 ′: SEQ ID NO: 2) by PCR. The obtained fragment of about 1.2 Kb was cloned into pCRII-TOPII using TOPO TA Cloning Kit (manufactured by Invitrogen) according to the attached protocol (referred to as pCRI I-promoter 2). When the nucleotide sequence of the inserted DNA fragment was confirmed by a conventional method using a DNA sequencer manufactured by ABI, the nucleotide sequence was consistent with the nucleotide sequence of the promoter region in the aforementioned literature (SEQ ID NO: 9). pCRI I—Not I site (5 'side) of multiple-cloning site of pCRI I plasmid in promoter 2 and pCRI I—p The fragment consisting of the SmaI site in the type II collagen gene promoter sequence in the robot 2 was used in the following experiments.
ラット I I型ェンハンサ一領域は、 Krebsbachら (J. Biol. Chem. 271： 4298, 1 996) の塩基配列をもとに M 1 u Iサイトが生じるように設計したプライマー （5 '-TCCACGCGTTTGGGAAACTTCTTGGCTGCG-3' ：配列番号： 3) および （5' -GCTTCGTCGC CGCTACGCGTGGGGCCGGA-3' ：配列番号： 4) を用いて P C R法により得た。 得られ た 0. 35 Kbの Ml u I断片に常法により Ec oR Iリンカ一を付与し、 pBlu escript KSII+の E c o R Iサイトに揷入した （以下、 pKS_enhanc e r 1—4と呼ぶ） 。 挿入された DNA断片の塩基配列を AB I社製 DNAシーク ェンサ一で常法により確認したところ、 前述文献のェンハンサ一領域の塩基配列 と一致した （配列番号： 10) 。 実施例 2 トランスジエニックラット作出用発現ベクターの構築  The rat type II enhancer region was prepared using a primer (5′-TCCACGCGTTTGGGAAACTTCTTGGCTGCG-3) designed based on the nucleotide sequence of Krebsbach et al. (J. Biol. Chem. ': SEQ ID NO: 3) and (5'-GCTTCGTCGC CGCTACGCGTGGGGCCGGA-3': SEQ ID NO: 4). An EcoRI linker was added to the obtained 0.35 Kb MluI fragment by a conventional method, and inserted into the EcoRI site of pBluescript KSII + (hereinafter, referred to as pKS_enhancer1-4). The nucleotide sequence of the inserted DNA fragment was confirmed by a conventional method using a DNA sequencer manufactured by ABI, and was found to match the nucleotide sequence of the enhancer region in the aforementioned literature (SEQ ID NO: 10). Example 2 Construction of expression vector for producing transgenic rat
トランスジエニックラット作出用発現ベクター、 ； pKS— MMPBc on— 1 9を常法に基づいて構築した。  An expression vector for producing a transgenic rat, pKS-MMPBcon- 19, was constructed according to a conventional method.
本プラスミドは下記の 1) から 5) までの断片が pB 1 u e s c r i p t K S I I +の No t Iサイトに揷入されたものである （図 1) 。  In this plasmid, the following fragments 1) to 5) were inserted into the NotI site of pB1uescripipKSII + (Fig. 1).
1) C o 12 A 1 p r omo t e r ：ラット I I型コラーゲン遺伝子のプロ モーター領域、 実施例 1に記載の pCR I I— p r omo t e r 2中の pCR I Iのマルチプルクローニングサイト内の No t Iサイトから p CR I I— p r o mo t e r 2中の I I型コラーゲン遺伝子プロモーター内にある Sm a Iサイ卜 までの 1120 b p断片 （常法によるリンカー連結により Sm a Iサイトを S a 1 Iサイ卜に変換した） 。  1) Co 12 A 1 pr omo ter: promoter region of rat type II collagen gene, pCR II described in Example 1—from Not I site in the multiple cloning site of pCR II in pr omoter 2 CR II—a 1120 bp fragment to the SmaI site in the type II collagen gene promoter in promoter 2 (the SmaI site was converted to a Sal1 site by conventional linker ligation).
2) SV40 s p 1 i c i n g： p TB 399 (R. Sasadaら、 Cell Structu re and Function 12： 205, 1987) 由来のスプライシングサイトを含む DNA断片 2) SV40 sp1icing: a DNA fragment containing a splicing site derived from pTB399 (R. Sasada et al., Cell Structure and Function 12: 205, 1987)
(配列番号： 11) の 5 ' 側を S a 1 Iサイト、 3 ' 側を C 1 a Iサイトに変換 したもの。 (SEQ ID NO: 11) 5 'side converted to S a1 I site, 3' side converted to C 1 a I site.
3) MMP- 19 ： pTB 1921 (特開平 10— 080283) の MMP— 19 cDNA内にぁるS a c Iサイトから X ho Iサイ卜までの約 1600 b Pの遺伝子断片。 （リンカ一連結により Sac Iサイトを C I a Iサイトに、 X ho Iサイトを Bg 1 I Iサイ卜に変換した） 。 3) MMP-19: Approximately 1600 b from the SacI site to the XhoI site in the MMP-19 cDNA of pTB1921 (Japanese Patent Laid-Open No. 10-080283). P gene fragment. (Sac I site was converted to CI a I site and Xho I site was converted to Bg 1 II site by linker ligation).
4) S V40 ρ o 1 yA : pTB 399 (R. Sasadaら、 Cell Structure and Function 12： 205, 1987) 由来の p o 1 y A付加シグナルを含む B g 1 I Iサイ 卜から H i n d I I Iサイトまでの断片。  4) S V40 ρ o 1 yA: pB 399 (R. Sasada et al., Cell Structure and Function 12: 205, 1987) from the B g 1 II site containing the po 1 y A additional signal to the Hind III site fragment.
5) C o 12 A 1 e nh an c e r ：実施例 1に記載したラット I I型コラ —ゲン遺伝子のェンハンサ一領域を含む pKS— e n h a n c e r 1 _4の H i n d I I Iサイ卜から No t Iサイ卜までの断片。  5) Co 12 A 1 enh ancer: rat type II collagen described in Example 1—enrichment of pKS containing enhancer region of gene—from Hind III site to Not I site of enhancer 1_4 fragment.
pKS— MMPBc on— 19を大腸菌 J M 109に形質転換して大腸菌 J M 109/pKS— MMPBc on— 19を得た。 実施例 3 卜ランスジエニックラッ卜の作出  pKS-MMPBc on-19 was transformed into E. coli J M109 to obtain E. coli J M109 / pKS-MMPBc on-19. Example 3 Production of transgenic rats
採卵用としてラット SD系統を 8週齢で購入し、 7 : 00〜 19 : 00 12 時間明期条件で 1週間飼育し、 まず 1日目 11 ： 00に卵胞刺激ホルモン （妊馬 血清性性腺刺激ホルモン、 PMSG) (301 U/個体） を腹腔内注射し、 3日 目 11 ： 00に黄体形成ホルモン （ヒ卜絨毛性性腺刺激ホルモン、 hCG) (5 IUZ個体） を腹腔内注射して雄ラット SD系統 10週齢以降の個体と 15 ： 0 0に 1 ： 1で同居、 交配させた。 4日目 9 ： 00に交配させた雌ラットの膣栓確 認を行い、 13 : 30から膣栓確認した個体を屠殺後、 採卵を開始した。 受精卵 で前核形成卵を選択し、 14 : 30から実施例 2で得られたプラスミド pKS— MMPBc 011—19を1^0セ Iによって切断し、 10 g/m 1の濃度に調製 したヒト MMP— 19遺伝子を含む DNA断片 1〜2 1を顕微鏡下で観察しな がら単細胞期の SD系統ラット受精卵の雄前核へ注入した。 続いて、 卵細胞を公 知の HER培地 （HAM— F 12粉末培地 （大日本製薬） 3. 180 g、 PM I— 1640粉末培地 （大日本製薬） 1. 040 g、 MEM E a g 1 e粉末培 地 （大日本製薬） 0. 950 g、 NaHCOs (和光純薬） 0. 780 g、 Peni cillin-G (GIBCO BRL) 50000 Uおよび Streptomycin (GIBCO BRL) 5000 0Uを 500m 1の蒸留水に溶かしたもの） で培養し、 5日目 13 : 30に 2細 胞期胚を確認してから、 Wagnerら (Proc. Natl. Acad. Sci. USA 78:5016, 1981) に より記載された方法に従って、 偽妊娠の雌 Wi s t a r系統ラットの卵管に移植 し、 着床させた。 The rat SD strain was purchased at 8 weeks of age for egg collection and reared for 1 week at 7:00 to 19:00 12 hours under light conditions. First day, at 11:00, follicle stimulating hormone (pregnant horse serum gonad stimulation) Hormone, PMSG) (301 U / individual) was injected intraperitoneally, and at 11:00 on day 3, male rats were injected intraperitoneally with luteinizing hormone (human chorionic gonadotropin, hCG) (5 IUZ individuals). The SD line lived and bred with individuals after 10 weeks of age at 1: 1 at 15: 0. On the fourth day, the female rats bred at 9:00 were checked for vaginal plugs, and at 13:30, the individuals whose vaginal plugs were confirmed were sacrificed and egg collection was started. A pronucleus-forming egg was selected from fertilized eggs, and the plasmid pKS—MMPBc 011-19 obtained in Example 2 was cut from 1:30 at 1:30 with 1 ^ 0-se I and adjusted to a concentration of 10 g / m1. DNA fragments 1-21 containing the MMP-19 gene were injected into the male pronucleus of fertilized eggs of the SD strain rat at the single cell stage while observing under a microscope. Subsequently, the egg cells were cultured in a known HER medium (HAM-F12 powder medium (Dainippon Pharmaceutical) 3.180 g, PMI-1640 powder medium (Dainippon Pharmaceutical) 1.040 g, MEM Eag1e powder medium Ground (Dainippon Pharmaceutical) 0.95 g, NaHCO s (Wako Pure Chemical) 0.780 g, Penicillin-G (GIBCO BRL) 50000 U and Streptomycin (GIBCO BRL) 5000 0U were dissolved in 500 ml of distilled water ) And confirmed the two-cell stage embryos at 13:30 on day 5, and then reported to Wagner et al. (Proc. Natl. Acad. Sci. USA 78: 5016, 1981). In accordance with the method described above, it was transplanted into the oviduct of a pseudopregnant female Wistar rat and implanted.
偽妊娠の雌 Wi s t a r系統ラット （11週齢以降） は 0日目 13 ： 00に L HRHを皮下注射 （40 zg/個体） し、 4日目 15 ： 00に雄 W i s t a r系 統 12週齢以降の個体と 1 ： 1で同居、 交配させることにより作成した。 5日目 9 ： 00に交配させた雌ラッ卜の膣栓確認を行い使用した。 実施例 4 トランスジエニックラットの遺伝子解析  Pseudopregnant female Wistar strain rats (11 weeks old and older) were injected subcutaneously with L HRH (40 zg / individual) at 13:00 on day 0, and male Wistar strains were 12 weeks old at 15:00 on day 4 It was created by living and mating with the subsequent individuals at 1: 1. On day 5, female rats mated at 9:00 were checked for vaginal plugs and used. Example 4 Gene analysis of transgenic rats
4週齢に達した出産仔の尾から B. Hoganら （Manupulating The Mouse Embryo, 1986、 Cold Spring Harvor Laboratories) の方法で採取した DNAを用いて、 C 012A1 promoter (実施例 2) の塩基配列を基に設計したプライマ一 （5'- CGCCGC TGGGCTGCCGGGTC-3' ：配列番号： 5) および MMP— 19 (実施例 2) の塩基配 列を基に設計したプライマー （5'-TTTCTCCAGCGGCCCAGCMC- 3' ：配列番号： 6) を用いて PC Rを行った。 合計 104個体の産仔ラットを解析した結果、 630 b pの P C R断片の検出できた P C R陽性個体は 2個体であつた。  Using the DNA collected from the tail of 4-week-old offspring at the age of 4 weeks by B. Hogan et al. (Manupulating The Mouse Embryo, 1986, Cold Spring Harvor Laboratories), the nucleotide sequence of the C012A1 promoter (Example 2) was determined. Primers (5'-CGCCGCTGTGCTGCCGGGTC-3 ': SEQ ID NO: 5) and primers (5'-TTTCTCCAGCGGCCCAGCMC-3': sequence) designed based on the base sequence of MMP-19 (Example 2) PCR was performed using No. 6). As a result of analyzing a total of 104 litter rats, two PCR positive individuals were able to detect the 630 bp PCR fragment.
これら 2個体の P C R陽性個体のゲノム D N Aをサザンハイブリダイゼーショ ン法により解析した。 すなわち、 10 X gの DNAを B amH Iで完全に切断し 、 2. 0%ァガロースゲル電気泳動後、 ナイロンフィルタ一へ移した。 このフィ ルターを、 MMP— 19 (実施例 2) の Nh e Iサイトから B g 1 I Iサイトの DNA断片を D I G RNAラベリングキッ卜 （ロッシュ ·ダイァグノスティッ クス社製） で標識したプローブと一晩ハイブリダィズし、 2 xS SC、 0. 1 % SDSにて室温で 2回洗浄し、 次に 0. l xS SC、 0. 1%SDSにて 68°C で 2回洗浄した。 検出には D I G蛍光検出キット （ロッシュ ·ダイァグノスティ ックス社製） を用いた。 その結果、 これら 2個体は、 全て MMP— 19由来の 8 60 b pの断片が確認され、 MMP— 19遺伝子の導入が確認された。 また、 導 入された遺伝子のコピ一数は B 60 F系統が 40コピー、 B 61 F系統が 10コ ピーであることが確認された。 実施例 5 ヘテロザィゴ一トのトランスジエニックラット取得 実施例 4で得られた B6 O F (第 1世代 （F0) ) が 12週齢に達した時、 S D系統ラットと交配し、 第 2世代 （F を取得した。 第 2世代が 4週齢に達し た時、 実施例 4に記載の方法で PC Rを行い、 ヘテロザィゴー卜を選抜し、 実施 例 6に用いた。 実施例 6 トランスジエニックラッ卜の軟骨でのヒト mRNAの確認 Genomic DNA of these two PCR-positive individuals was analyzed by Southern hybridization. That is, 10 X g of DNA was completely digested with BamHI, transferred to a nylon filter after 2.0% agarose gel electrophoresis. This filter was combined with a probe obtained by labeling a DNA fragment at the Bg1 II site from the NheI site of MMP-19 (Example 2) with a DIG RNA labeling kit (Roche Diagnostics). Hybridized overnight, washed twice with 2 × SSC, 0.1% SDS at room temperature, and then twice with 0.1 × SSC, 0.1% SDS at 68 ° C. For detection, a DIG fluorescence detection kit (manufactured by Roche Diagnostics) was used. As a result, in these two individuals, a 860 bp fragment derived from MMP-19 was confirmed, and introduction of the MMP-19 gene was confirmed. It was also confirmed that the number of copies of the introduced gene was 40 copies for the B60F strain and 10 copies for the B61F strain. Example 5 Acquisition of transgenic rats in heterozygote When the B6 OF (first generation (F 0 )) obtained in Example 4 reached the age of 12 weeks, it was bred with the SD strain rat to obtain the second generation (F). The second generation was 4 weeks old. When reached, PCR was performed by the method described in Example 4 to select a heterozygote and used in Example 6. Example 6 Confirmation of human mRNA in cartilage of transgenic rat
実施例 5で得たトランスジエニックラット （F から摘出した肋軟骨、 約 2 O Omgを I SOGEN (二ツボンジ一ン社製） 中でホモジナイズし、 常法によ り total RNAを抽出した。 total RNA 5 gを使用し、 First strand cDNA synthesis kit (アマシャムフアルマシアバイオテク社製） を用いてプロトコ一 ルに従って c DNA合成を行い、 RT— PCRのテンプレートとした。 プライマ 一はヒトとラッ卜の MMP— 19に共通する配列である （5' -CTGGATGATGCCACAA GGG-3' (配列番号： 7) ) および (5' -GATCCCTGCCACATCATC-3' (配列番号： 8) ) を用い、 ラッ卜とヒト MMP— 19の mRNAを同時に増幅、 検出した。 また、 陰性コン卜ロールとして、 実施例 5で行われた PC Rにより野生型と判断 された個体を用いた。 RT— PCRにより増幅された 538 b pの断片をヒトの 断片中にのみ存在する A p aL Iで完全に切断したところ、 347 bpと 191 b pの断片が検出できたことから、 トランスジエニックラッ卜の軟骨にヒ卜 MM P— 19の mRNAが発現していることが確認できた。 また、 ラット内在性 MM P— 19の mRNAは心臓、 肺、 肝臓、 脾臓、 腎臓、 軟骨において確認された （ 図 2) 。 実施例 7 ホモザィゴ一トのトランスジェニックラット取得  The transgenic rat obtained in Example 5 (costal cartilage extracted from F, about 2 Omg) was homogenized in ISOGEN (manufactured by Futabajin) to extract total RNA by a conventional method. Using 5 g of RNA, cDNA was synthesized according to the protocol using First strand cDNA synthesis kit (Amersham Pharmacia Biotech) and used as a template for RT-PCR. Using the sequences common to MMP-19 (5'-CTGGATGATGCCACAA GGG-3 '(SEQ ID NO: 7)) and (5'-GATCCCTGCCACATCATC-3' (SEQ ID NO: 8)), rat and human MMP- Simultaneously amplified and detected 19 mRNA, and used as a negative control was an individual determined to be wild-type by PCR performed in Example 5. RT—PCR amplified 538 bp fragment Was completely digested with ApaLI, which is only present in human fragments. At this time, fragments of 347 bp and 191 bp could be detected, confirming that human MMP-19 mRNA was expressed in the cartilage of the transgenic rat. — 19 mRNA was found in heart, lung, liver, spleen, kidney, and cartilage (Figure 2) Example 7 Obtaining homozygous transgenic rats
実施例 4で選抜したヘテロザィゴー卜 が 12週齢に達した時、 兄妹交 配を行い第 3世代 （F2) を取得した。 When the heterozygotes selected in Example 4 reached the age of 12 weeks, siblings were bred to obtain the third generation (F 2 ).
第 3世代 （F2) が 4週齢に達した時、 これらの個体の尾から実施例 4に記載 の方法でゲノム DNAを採取し、 PCRで卜ランスジエニック個体を選抜した。 これらのトランスジエニック個体の D N Aについてさらに実施例 4に記載の方法 に従いサザンハイブリダィゼーシヨン法による解析を行い、 検出されたバンドの 濃さを比較することで遺伝子型を判断した。 ヘテロザィゴ一卜のバンドに比較し てホモザィゴ一卜のバンドは約二倍の濃さであった （図 3 ) 。 以上の実験により 、 B 6 0 F系統一腹の F 2の遺伝子型を野生型、 ヘテロザィゴ一トおよびホモザ ィゴートに分離することができた。 実施例 8 トランスジエニックラット （B 6 O F系統） の特徴の観察 When the third generation (F 2 ) reached the age of 4 weeks, genomic DNA was collected from the tails of these individuals by the method described in Example 4, and transgenic individuals were selected by PCR. The DNA of these transgenic individuals was further analyzed by the Southern hybridization method according to the method described in Example 4, and the band of the detected band was analyzed. The genotype was determined by comparing the concentrations. The homozygous band was about twice as dense as the heterozygous band (Fig. 3). The above experiment, B 6 0 F strain wild-type genotype F 2 of litter, can be separated into Heterozaigo one preparative and Homoza Igoto. Example 8 Observation of characteristics of transgenic rats (B6OF strain)
実施例 7で得られた野生型、 ヘテロザィゴートおよびホモザィゴ一トのそれぞ れの個体 （B 6 O F系統、 1 2週齢、 雄） の体重を測定したところ、 野生型に比 較してへテロザィゴートが、 さらにへテロザィゴー卜に比較してホモザィゴー卜 の体重が小さいことを見いだした。  The body weight of each of the wild-type, heterozygous and homozygous individuals (B6OF strain, 12-week-old, male) obtained in Example 7 was measured and compared with the wild-type. However, they also found that the weight of homozygotes was smaller than that of heterozygotes.
次に、 体重測定に用いた個体をネンブタールにて麻酔後、 前肢、後肢及び頭蓋 の長さをノギスにて測定した。 ヘテロザィゴートは野生型と比較して、 前肢、後 肢及び頭蓋の長さに違いは見られなかった。 ホモザィゴートは野生型及びへテロ ザィゴ一トに比較して前肢および後肢の長さが短いことが分かった。 また頭蓋骨 が長軸方向に扁平化し長軸と垂直方向に拡張していることがわかった。 結果を表 1に示す。  Next, the individual used for weight measurement was anesthetized with Nembutal, and the lengths of forelimbs, hindlimbs and skull were measured with calipers. Heterozygotes did not differ in forelimb, hindlimb, and skull lengths compared to wild-type. Homozygote was found to have shorter forelimbs and hindlimbs than wild-type and heterozygote. It was also found that the skull was flattened in the long axis direction and expanded in the direction perpendicular to the long axis. Table 1 shows the results.
[表 1 ] 野生型 ヘテロザィゴ一ト ホモザィゴー卜 体重 （g) 442.26 350.51 266.71 体長 ( c m) 25.0 23.0 20.0 尾長 ( c m) 19.0 18.0 14.0 頭蓋長 （mm) 56.42 55.37 47.38 頭蓋幅 （mm) 30.09 30.29 31.16 右後肢先端〜中足骨近位端 （mm) 46.25 42.57 28.93 右脛骨近位端〜遠位端 (mm) 57.69 56.27 37.28 右前肢先端〜頭骨近位端 （mm) 54.31 51.47 34.10 さらに、 軟 X線写真測定により全身の骨格を野生型、 ヘテロザィゴートおよび ホモザィゴート間で比較した。 動物をネンブ夕一ルにて麻酔後、 軟 X線発生装置 (ソフテックス社）内に入れ、 4 8 K V p、 3 mA、 管球からの距離 6 0 c mの条 件で軟 X線写真を撮影した。 写真は富士レンドールおよびレンフィックスにて使 用説明書に従って現像、 定着した。 フィルムをドライゥヱル水溶液に浸漬後乾燥 させ、 シャ一カステン上にて比較した。 その結果、 前肢、 後肢及び頭蓋の長さの 違いは上記計測結果と一致し、 ホモザィゴ一卜は野生型及びへテロザィゴ一卜に 比較して前肢および後肢の長さが短いことが分かった。 また頭蓋骨が長軸方向に 扁平化し長軸と垂直方向に拡張していることがわかった。 前肢、 後肢の長管骨、 頭蓋骨及び尾椎以外の骨格には異常は見られなかった。 [Table 1] Wild-type heterozygous body homozygous body weight (g) 442.26 350.51 266.71 body length (cm) 25.0 23.0 20.0 tail length (cm) 19.0 18.0 14.0 skull length (mm) 56.42 55.37 47.38 skull width (mm) 30.09 30.29 31.16 right hind limb Tip to metatarsal proximal end (mm) 46.25 42.57 28.93 Right tibia proximal to distal end (mm) 57.69 56.27 37.28 Right forelimb distal to proximal skull (mm) 54.31 51.47 34.10 In addition, whole body skeleton was compared between wild-type, heterozygous and homozygous by soft radiography. After the animals were anesthetized with a nebulizer, they were placed in a soft X-ray generator (Softex), and soft X-rays were taken under the conditions of 48 KV p, 3 mA, and a distance of 60 cm from the tube. Taken. The photographs were developed and fixed at Fuji Rendall and Renfix in accordance with the instruction manual. The film was immersed in an aqueous dry roll solution, dried, and compared on a char caster. As a result, the differences in the lengths of the forelimbs, hindlimbs and skull agreed with the above measurement results, and it was found that the length of the forelimbs and hindlimbs was shorter in the homozygote than in the wild type and heterozygous. It was also found that the skull was flattened in the long axis direction and expanded in the direction perpendicular to the long axis. No abnormalities were found in the skeleton other than the long bones, skull and tail vertebrae of the forelimbs and hind limbs.
組み込んだ遺伝子のプロモ一夕一には I I型コラーゲンのプロモ一夕一を用い ているため、 トランスジーン （組み込まれた遺伝子） による影響は I I型コラ一 ゲンの発現している部位に見られることが予想された。 トランスジエニックラッ ト （B 6 O F系統） の右膝関節の病理検索を行い、 野生型、 ヘテロザィゴ一卜お よびホモザィゴー卜間で比較した。  Since the type II collagen promoter is used for the integrated gene promoter, the effect of the transgene (integrated gene) can be seen at the site where the type II collagen is expressed. Was expected. A pathological search of the right knee joint of the transgenic rat (B6OF strain) was performed and compared between wild-type, heterozygous, and homozygous.
動物をエーテル麻酔下で 4 %パラホルムアルデヒドを用いて心臓より港流固定 後、 右膝関節部を取り出した。 周辺に付着している筋組織や結合組織を切除後、 ダイヤモンドカッターにて正中方向に関節部分を二分割した。 おのおのの関節部 分をさらに 4 %パラホルムアルデヒド中にて 1 4時間室温にて固定した。 固定終 了後、 組織片を蒸留水にて洗浄し、 2 0 % E D T A ( p H 7 . 4 ) にて、 膝関 節を 8日間脱灰した。 脱灰液交換は毎日行った。 脱灰後、 脱イオン水にて膝関節 に付着した 2 0 % E D TAを洗浄し、 7 0 %エタノール、 8 0 %エタノール、 9 0 %エタノール、 1 0 0 %エタノールを 4回、 キシレン 2回の順で各々 1時間 から 2時間浸漬し、 脱水、 透徹を行った。 次に約 6 0 °Cのパラフィンに 2回、 各 々2時間浸漬し、 組織力セットを土台としたパラフィンブロックを作製した。 ブ ロックは一晩以上冷蔵庫にて急速に冷却し、 完全に固めた。 薄切はパラフィンブ ロックを室温に放置し、 室温に戻してから、 ミクロトームにて 5 πιにて行った 。 切片はマスコートを施したスライドグラス上に温浴しながらマウントした。 作 製した切片は 3 7でに加温した乾燥機内で一晩放置し完全に乾かした。 脱パラフ イン及び一般染色 （へマトキシリンェォジン二重染色法） は下記文献を参考に行 つた。 （組織学研究法、 佐野豊、 南山堂、 1985年） 。 また硫酸化グリコサミノグ リカンの染色は切片を脱パラフィン、 脱水後、 へマトキシリン 2分間、 流水にて 水洗後、 0 . 0 2 %ファーストダリ一ン水溶液 5分間、 1 %酢酸水溶液に浸洗、 0 . 1 %サフラニン 0水溶液に 1 0分間行った。 切片の脱水は 9 5 %エタノール 、 1 0 0 %エタノール 3回を各ステップで 5回出し入れすることにより行い、 最 後に 1 0 0 %エタノールに 5分間浸漬した。 透徹、 封入は下記文献を参考に行つ た。 （組織学研究法、 佐野豊、 南山堂、 1985年） 。 光学顕微鏡を用いて明視野に て染色後の切片を鏡検した。 野生型に比較してヘテロザィゴー卜の大腿骨及び脛 骨の関節軟骨及び成長板に異常は見られなかった。 ホモザィゴー卜の大腿骨及び 脛骨の関節軟骨は野生型に比較して関節軟骨層の菲薄化や肥厚が見られる部位や 表層が繊維性の関節軟骨組織に置換している部位が存在した。 また、 成長板につ いてはホモザィゴー卜で断裂している部位が見られた。 関節軟骨層の表層が繊維 性の関節軟骨組織に置換している部位では表層部での染色性がないことから、 硫 酸化グリコサミノダリカンの欠失が見られた。 ホモザィゴー卜の成長板が断裂し ている部位では本来の軟骨細胞の増殖方向への方向性が一定しておらず、 正常な 軟骨分化が起こっていないことがわかった。 これらのことから、 長管骨における 内軟骨性骨化の異常が四肢の短縮を引き起こしていることが示唆された。 実施例 9 胎仔骨格の解析 The animals were port-fixed from the heart with 4% paraformaldehyde under ether anesthesia, and the right knee joint was removed. After resection of the muscle tissue and connective tissue attached to the periphery, the joint was divided into two parts in the median direction using a diamond cutter. Each joint was further fixed in 4% paraformaldehyde for 14 hours at room temperature. After the fixation, the tissue pieces were washed with distilled water, and the knee joint was decalcified with 20% EDTA (pH 7.4) for 8 days. Decalcification liquid exchange was performed every day. After demineralization, wash the 20% EDTA attached to the knee joint with deionized water, and then wash 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol 4 times and xylene 2 times Each was immersed for 1 to 2 hours in order of dehydration and clearing. Next, it was immersed twice in paraffin at about 60 ° C for 2 hours each to produce a paraffin block based on a tissue force set. The block was cooled rapidly in the refrigerator overnight, and was completely hardened. Slicing was performed by leaving the paraffin block at room temperature, returning to room temperature, and then using a microtome at 5πι. The sections were mounted on a slide glass coated with a mass coat while warm bathing. The prepared section was left overnight in a drier heated at 37 to dry completely. Deparaffinization The following references were used for in- and general staining (hematoxylin eosin double staining method). (Histology Research Method, Yutaka Sano, Nanzando, 1985). For staining of sulfated glycosaminoglycan, the sections were deparaffinized, dehydrated, washed with hematoxylin for 2 minutes and running water, then washed with a 2% aqueous solution of 1% dextrin for 5 minutes, and immersed in a 1% aqueous solution of acetic acid. Performed in a 1% aqueous solution of safranin for 10 minutes. The sections were dehydrated by taking in and out 95% ethanol and 100% ethanol three times in each step, and finally immersing the sections in 100% ethanol for 5 minutes. The transparency and encapsulation were performed with reference to the following literature. (Histology Research Method, Yutaka Sano, Nanzando, 1985). The sections after staining were examined microscopically in a bright field using an optical microscope. No abnormality was found in the articular cartilage and growth plate of the femur and tibia of the heterozygous compared to the wild type. The articular cartilage of the femur and tibia of the homozygote had sites where the articular cartilage layer was thinner and thicker than in the wild type, and where the surface layer was replaced by fibrous articular cartilage tissue. In addition, the growth plate was found to be ruptured by homozygote. At sites where the surface layer of the articular cartilage layer was replaced with fibrous articular cartilage tissue, lack of staining at the surface layer indicated the lack of sulfated glycosaminodalican. At the site where the growth plate of the homozygote was torn, the direction of the original chondrocyte growth direction was not constant, indicating that normal cartilage differentiation did not occur. These findings suggest that abnormal endochondral ossification in the long bone causes limb shortening. Example 9 Analysis of fetal skeleton
新規トランスジエニックラッ卜 (系統名は MM P— B 6 0 F ) アダルトではヮ ィルドタイプに比較してヘテロザィゴ一トが、 ヘテロザィゴートに比較してホモ ザィゴートの体重が小さいこと、 ホモザィゴ一卜はワイルドタイプ及びへテロザ ィゴ一卜に比較して前肢および後肢の長さが短いこと及び頭蓋骨が長軸方向に扁 平化し長軸と垂直方向に拡張していることから、 骨格に異常があることがわかつ た。 この異常が生後後天的に起こったものなのか、 それとも胎生期に先天的に起 こったものなのかを検証する目的で、 胎仔期の骨格形成に異常がないかどうかを 調べた。  New transgenic rat (strain name is MMP—B60F) In adults, heterozygous body weight is smaller than wild type, homozygous body weight is smaller than heterozygous body, and homozygous body is wild type. Because the forelimbs and hindlimbs are shorter in length than the heterozygotes and the skull is flattened in the long axis direction and expanded in the direction perpendicular to the long axis, there may be abnormalities in the skeleton. Watashita. To determine whether this abnormality was acquired postnatally or innately during fetal life, we examined whether there were abnormalities in fetal skeletal formation.
新規トランスジエニックラット （系統名は MM P— B 6 0 F ) のホモザィゴー 卜の雄とホモザィゴー卜の雌を同居させ、 交尾させた。 交尾確認後、 妊娠 1 8日 目に妊娠動物を屠殺し、 帝王切開を行い、 胎仔を摘出した。 摘出された胎仔はす ベてホモザィゴートであることから、 胎仔の骨格異常が全例で見られるか否かを 調べた。 A homozygote of a new transgenic rat (strain name: MMP—B60F) The male of the bird and the female of the homozygote lived together and were mated. After confirmation of mating, pregnant animals were sacrificed on the 18th day of pregnancy, cesarean section was performed, and fetuses were removed. Since all of the removed fetuses were homozygous, we examined whether fetal skeletal abnormalities were seen in all cases.
井上法および Kimmei法を用い、 下記を参考に、 胎仔に骨軟骨二重染色を施した (組織学研究法、 佐野豊、 南山堂、 1985年） 。  Using the Inoue method and Kimmei method, the fetuses were subjected to double staining of osteochondral cartilage with reference to the following (Histology research method, Yutaka Sano, Nanzando, 1985).
新規トランスジエニックラット （系統名は MM P— B 6 0 F) ホモザィゴ一卜 、 アダルトの検索で見られた四肢の短縮、 頭蓋の変形及び尾椎の変形が胎生期か ら起こっていることがわかった。 また、 体後部の腰椎から後肢にかけて、 石灰化 の遅延が見られた。 また一腹分の異常発現率はァダルトでは約 5 0 %であったが 、 胎生期では全例にいずれかの異常が観察された。 これらのことから新規トラン スジエニックラット （系統名は MM P— B 6 0 F) ホモザィゴ一卜に起きている 骨格異常は胎生期に先天的に起こったものであることが推察された。 実施例 1 0 軟骨形成シグナル伝達の解析  New transgenic rats (strain name: MMP-B60F) homozygote, shortening of limbs, cranial deformity and caudal vertebra deformity observed in adult searches may have occurred from embryonic stage. all right. Delayed calcification was observed in the posterior lumbar spine and hind limbs. The incidence of abnormalities in the stomach was about 50% in Adalto, but any abnormalities were observed in all cases during the embryonic period. From these results, it was inferred that the skeletal abnormalities occurring in the homozygote of the new transgenic rat (strain name: MMP-B60F) were congenital in the fetal period. Example 10 Analysis of Chondrogenic Signaling
新規トランスジエニックラット （系統名は MM P— B 6 0 F) ホモザィゴート 、 アダルトでは骨格異常が起きることがわかっている。 このラットの関節軟骨で は菲薄化している部位が存在する。 この部位で軟骨分化が正常に起こっているか 否かを免疫染色法によって各種転写因子の発現を調べることで解析した。  New transgenic rats (strain name: MMP-B60F) homozygote, skeletal abnormalities are known to occur in adults. There is a thinned part in the articular cartilage of this rat. Whether or not cartilage differentiation was normally occurring at this site was analyzed by examining the expression of various transcription factors by immunostaining.
新規トランスジエニックラット （系統名は MM P— B 6 0 F) ホモザィゴート 、 1 2週齢雄、 大腿骨遠位端の関節軟骨の切片を実施例 8と同様の方法にて作製 した。 作製した切片について以下の抗体を用いて免疫染色を行った。 F G F R 3 (Fibroblast Growth Factor Receptor 3) 、 I HH (Indian hedgehog) 、 P T C (Patched) 及び S MO (Smoothend) 。 マウントしたスライドガラスを脱パラ フィン後、 免疫染色を行った。 免疫染色はべクタスティン AB Cキット （ペルォ キシダーゼ法、 ベクター社） を用いて行った。 発色基質には AE Cを用いた。 具 体的な実験手順はキッ卜添付のマニュアルに従って行った。  A novel transgenic rat (strain name: MMP-B60F) homozygote, 12-week-old male, section of articular cartilage at the distal end of the femur was prepared in the same manner as in Example 8. The prepared sections were subjected to immunostaining using the following antibodies. FGFR3 (Fibroblast Growth Factor Receptor 3), IHH (Indian hedgehog), PTC (Patched) and SMO (Smoothend). After deparaffinization of the mounted slide glass, immunostaining was performed. Immunostaining was performed using the Vectastine ABC kit (Peroxidase method, Vector). AEC was used as a chromogenic substrate. The specific experimental procedure was performed according to the manual attached to the kit.
ホモザィゴートの軟骨層が菲薄化している部位の F G F R 3の発現は増殖軟骨 細胞層で見られ、 異常はなかった。 しかし、 I HHはこの部位の軟骨層全層にわ たって発現しており、 その受容体である P T C及び S MOの発現はこの部位に見 られなかった。 正常な I HHの発現が前肥大化軟骨細胞に見られることから、 軟 骨層が菲薄化している部位では前肥大化軟骨細胞が肥大化軟骨細胞へと分化する 段階に異常が起こっていること力 ί推察された。 産業上の利用可能性 FGFR3 expression was observed in the proliferating chondrocyte layer at the site where the cartilage layer of the homozygote was thinned, and there was no abnormality. However, IHH is not The expression of its receptors, PTC and SMO, was not found at this site. Since normal IHH expression is found in pre-hypertrophic chondrocytes, abnormalities occur in the stage where pre-hypertrophic chondrocytes differentiate into hypertrophic chondrocytes at sites where the cartilage layer is thinning Power ί guessed. Industrial applicability
本発明の遺伝子導入非ヒト哺乳動物は、 軟骨破壊を特徴とする各種の疾患、 例 えば、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性関節症の予防 薬あるいは治療薬の評価、 MM P— 1 9遺伝子異常患者の遺伝子治療用実験など に用いることができ、 本発明の遺伝子導入非ヒト哺乳動物から採取した軟骨片ま たは軟骨細胞などを培養し、 MM P— 1 9阻害薬の評価に用いることができる。  The transgenic non-human mammal of the present invention includes various diseases characterized by cartilage destruction, for example, prophylactic or therapeutic agents for osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis. It can be used for evaluation, experiments for gene therapy of patients with MMP-19 gene abnormalities, and the like. Cartilage fragments or chondrocytes collected from the transgenic non-human mammal of the present invention are cultured, and MMP-1 9 Can be used to evaluate inhibitors.

Claims

請求の範囲 The scope of the claims
1. 外来性 MMP— 19遺伝子またはその変異遺伝子を組み込んだ DNAを有す る非ヒ卜哺乳動物またはその生体の一部。  1. A non-human mammal or a part of a living body thereof having a DNA into which an exogenous MMP-19 gene or its mutant gene has been incorporated.
2. 非ヒト哺乳動物が、 ゥサギ、 ィヌ、 ネコ、 モルモット、 ハムスター、 マウス またはラッ卜である請求項 1記載の動物またはその生体の一部。  2. The animal or a part of a living body thereof according to claim 1, wherein the non-human mammal is a heron, a dog, a cat, a guinea pig, a hamster, a mouse or a rat.
3. 非ヒ卜哺乳動物がラットである請求項 1記載の動物またはその生体の一部。 3. The animal or a part thereof according to claim 1, wherein the non-human mammal is a rat.
4. 外来性 MMP— 19遺伝子が配列番号： 12で表される塩基配列を有する請 求項 1記載の動物またはその生体の一部。 4. The animal or a part thereof according to claim 1, wherein the exogenous MMP-19 gene has a nucleotide sequence represented by SEQ ID NO: 12.
5. 関節疾患を発症している請求項 1から 4記載の動物またはその生体の一部。  5. The animal or a part of the living body thereof according to claim 1, which has developed a joint disease.
6. ①四肢の短縮と変形、 ②頭蓋の変形、 ③咬合不全、 ④上顎及び下顎切歯の過 長、 ⑤腰椎の石灰化不全、 ⑥尾椎の変形および⑦尾椎椎間板の欠損から選ばれる 症状を発症している請求項 1から 4記載の動物またはその生体の一部。 6. Selected from ① ① limb shortening and deformity, ② cranial deformity, ③ malocclusion, ④ excessive maxillary and mandibular incisors, 不全 inadequate calcification of lumbar vertebra, 変 形 deformity of tail vertebra, and 欠 損 defect of caudate intervertebral disc 5. The animal or a part of the living body thereof according to claim 1, which has developed symptoms.
7. 外来性 MMP— 19遺伝子またはその変異遺伝子を含有し、 非ヒト哺乳動物 において該遺伝子を発現し得るベクタ一。 7. A vector containing an exogenous MMP-19 gene or a mutant gene thereof and capable of expressing the gene in a non-human mammal.
8. 外来性 MMP— 19遺伝子が配列番号： 12で表される塩基配列を有する請 求項 7記載のベクタ一。 8. The vector according to claim 7, wherein the exogenous MMP-19 gene has a nucleotide sequence represented by SEQ ID NO: 12.
9. さらにラット I I型コラーゲン遺伝子のプロモータ一領域およびラット I I 型コラーゲン遺伝子のェンハンサ一領域を含有する請求項 7記載のベクター。 9. The vector according to claim 7, further comprising a promoter region of the rat type I collagen gene and an enhancer region of the rat type I collagen gene.
10. pKS-MMPB c o n- 19で表示される請求項 7記載のベクター。 10. The vector according to claim 7, which is represented by pKS-MMPBcon-19.
11. 請求項 7記載のベクターで形質転換された形質転換体。 11. A transformant transformed with the vector according to claim 7.
12. 形質転換体が大腸菌 JM109ZpKS— MMPB c o n— 19 (FER M BP— 7647) である請求項 11記載の形質転換体。  12. The transformant according to claim 11, wherein the transformant is Escherichia coli JM109ZpKS-MMPBcon-19 (FERMBP-7647).
13. 請求項 1から 6のいずれかに記載の動物またはその生体の一部に被験物質 を適用し、 細胞外マトリックスの異常な分解に起因する疾患の改善効果を検定す ることを特徴とする、 細胞外マトリックスの異常な分解に起因する疾患の予防 ' 治療のために用いられる物質のスクリーニング方法。  13. A test substance is applied to the animal or a part of the living body according to any one of claims 1 to 6, and the effect of ameliorating a disease caused by abnormal degradation of extracellular matrix is assayed. The prevention of diseases caused by abnormal degradation of extracellular matrix, 'screening methods for substances used for treatment.
14. 細胞外マトリックスの異常な分解が MMP— 19の機能異常に起因するも のである請求項 13記載のスクリーニング方法。  14. The screening method according to claim 13, wherein the abnormal degradation of the extracellular matrix is caused by a dysfunction of MMP-19.
15. 細胞外マトリックスの異常な分解に起因する疾患が、 軟骨形成異常、 骨形 成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性 関節症、 眼疾患および悪性腫瘍である請求項 1 3記載のスクリーニング方法。15. Diseases caused by abnormal degradation of extracellular matrix include chondrodysplasia, bone shape 14. The screening method according to claim 13, wherein the screening method is dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye disease and malignant tumor.
1 6 . 請求項 1 3記載の方法により細胞外マトリックスの異常な分解に起因する 疾患の改善効果を有すると判定される物質を含有してなる細胞外マトリックスの 異常な分解に起因する疾患の予防 ·治療用医薬。 16. Prevention of a disease caused by abnormal degradation of extracellular matrix containing a substance determined to have an effect of improving a disease caused by abnormal degradation of extracellular matrix by the method according to claim 13. · Therapeutic medicine.
1 7 . 細胞外マトリックスの異常な分解に起因する疾患が、 軟骨形成異常、 骨形 成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性 関節症、 眼疾患および悪性腫瘍である請求項 1 6記載の医薬。  1 7. Diseases caused by abnormal degradation of extracellular matrix include cartilage dysplasia, bone dysplasia, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthrosis, eye disease and 17. The medicament according to claim 16, which is a malignant tumor.
1 8 . 哺乳動物に対して、 請求項 1 3記載の方法により細胞外マトリックスの異 常な分解に起因する疾患の改善効果を有すると判定される物質の有効量を投与す ることを特徴とする細胞外マトリックスの異常な分解に起因する疾患の予防 ·治 療法。  18. To a mammal, an effective amount of a substance determined to have a ameliorating effect on a disease caused by abnormal degradation of extracellular matrix is administered by the method according to claim 13. Prevention and treatment of diseases caused by abnormal degradation of extracellular matrix.
1 9 . 細胞外マトリックスの異常な分解に起因する疾患が、 軟骨形成異常、 骨形 成異常、 骨粗鬆症、 変形性関節症、 慢性関節リウマチ、 関節炎、 滑膜炎、 代謝性 関節症、 眼疾患および悪性腫瘍である請求項 1 8記載の予防 ·治療法。  1 9. Diseases caused by abnormal degradation of extracellular matrix include chondrodysplasia, bone malformation, osteoporosis, osteoarthritis, rheumatoid arthritis, arthritis, synovitis, metabolic arthropathy, eye disease and 19. The method according to claim 18, wherein the method is a malignant tumor.
2 0 . 細胞外マトリックスの異常な分解に起因する疾患の予防 ·治療のために用 いられる物質をスクリ一ニングするための請求項 1から 6のいずれかに記載の動 物またはその生体の一部の用途。  20. The animal according to any one of claims 1 to 6 for screening a substance used for prevention and treatment of a disease caused by abnormal degradation of an extracellular matrix, or a living body thereof. Use of department.
2 1 . 卵胞刺激ホルモン約 2 0ないし 5 0 I UZ個体を投与した後に黄体形成ホ ルモン約 0ないし 1 0 I U/個体を投与した雌ラッ卜を雄ラッ卜と交配させて得 られる受精卵に、 外来性 MM P— 1 9遺伝子またはその変異遺伝子を組み込んだ D NAを導入し、 該受精卵を雌ラッ卜に着床させることを特徴とする請求項 3記 載のラットまたはその生体の一部の作製方法。  21. After administration of follicle stimulating hormone of about 20 to 50 IUZ, female fertilized egg obtained by mating a female rat to which a luteinizing holmon of about 0 to 10 IU / individual was administered with a male rat. 4. The method according to claim 3, wherein a DNA incorporating the exogenous MMP-19 gene or its mutant gene is introduced, and the fertilized egg is implanted in a female rat. How to make the part.
2 2 . 黄体形成ホルモン放出ホルモンまたはその類縁体を投与した後、 雄ラット と交配させた雌の偽妊娠ラッ卜に、 外来性 MM P— 1 9遺伝子またはその変異遺 伝子を組み込んだ D NAを導入した受精卵を着床させることを特徴とする請求項 3記載のラットまたはその生体の一部の作製方法。  2 2. DNA containing exogenous MMP-19 gene or its mutant gene in a pseudopregnant rat of female bred with male rat after administration of luteinizing hormone-releasing hormone or its analog. 4. The method for producing a rat or a part of a living body thereof according to claim 3, wherein a fertilized egg into which is introduced is implanted.
2 3 . 外来性 MM P— 1 9遺伝子またはその変異遺伝子を組み込んだ D N Aを導 入した受精卵。  23. Fertilized eggs into which DNA incorporating the exogenous MMP-19 gene or its mutant gene has been introduced.
PCT/JP2001/006826 2000-08-09 2001-08-08 Transgenic animal WO2002011530A1 (en)

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