WO2002011530A9

WO2002011530A9 - Transgenic animal

Info

Publication number: WO2002011530A9
Application number: PCT/JP2001/006826
Authority: WO
Inventors: Koji Yoshimura; Atsushi Nishimura; Mayumi Nishida; Kazuhiro Hosono
Original assignee: Takeda Chemical Industries Ltd; Koji Yoshimura; Atsushi Nishimura; Mayumi Nishida; Kazuhiro Hosono
Priority date: 2000-08-09
Filing date: 2001-08-08
Publication date: 2002-11-14
Also published as: AU2001277716A1; WO2002011530A1

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

Transgenic animal technology

The present invention relates to an MMP-19 transgenic non-human mammal. Background art

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.

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.

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

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 present inventors have conducted further studies based on these findings, and as a result, have completed the present invention.

That is, the present invention

(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) 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) The animal according to the above (1), wherein the non-human mammal is a rat, or a part of a living body thereof,

(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) The animal or a part of the animal according to (1) to (4) above, which has developed joint disease,

(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) 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) The vector according to (7) above, wherein the exogenous MMP-19 gene has the nucleotide sequence represented by SEQ ID NO: 12.

(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) a transformant transformed with the vector of (7) above,

(12) the transformant according to the above (11), wherein the transformant is Escherichia coli JM109 / pKS-MMPB con—19 (FERM BP-7647);

(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) The screening method according to (13), wherein the abnormal degradation of the extracellular matrix is caused by a dysfunction of MMP-19.

(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) 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) 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) 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) 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) 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) 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) 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.

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). , 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.

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.

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. 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Since the non-human mammal of the present invention has the above-mentioned very unique features, it has the following useful uses.

The use of the non-human mammal of the present invention will be outlined below.

(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.

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.

Abnormal degradation of the extracellular matrix includes that caused by an abnormal function of MMP-19.

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.

Next, the cartilage degradation inhibitor can be evaluated by examining the drug evaluation or therapeutic effect using urinary and blood pyridinoline levels as indicators.

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) 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) 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) 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) 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.

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.

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.

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.

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) 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.

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: 9] This shows the base sequence of the promoter region of rat type I collagen gene cloned in Example 1 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: 12] This shows the base sequence of human MMP-19 gene.

[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: Deoxylipo nucleic acid

RNA: Liponucleic acid A adenine

T thymine

G Guanin

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

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

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.

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

An expression vector for producing a transgenic rat, pKS-MMPBcon- 19, was constructed according to a conventional method.

In this plasmid, the following fragments 1) to 5) were inserted into the NotI site of pB1uescripipKSII + (Fig. 1).

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 sp1icing: a DNA fragment containing a splicing site derived from pTB399 (R. Sasada et al., Cell Structure and Function 12: 205, 1987)

(SEQ ID NO: 11) 5 'side converted to S a1 I site, 3' side converted to C 1 a I site.

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: 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) 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 was transformed into E. coli J M109 to obtain E. coli J M109 / pKS-MMPBc on-19. Example 3 Production of transgenic rats

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.

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

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.

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

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

When the heterozygotes selected in Example 4 reached the age of 12 weeks, siblings were bred to obtain the third generation (F ₂ ).

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)

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.

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.

[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.

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.

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

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.

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.

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).

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

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) 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.

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

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

The scope of the claims

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. 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. The animal or a part thereof according to claim 1, wherein the non-human mammal is a rat.

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. The animal or a part of the living body thereof according to claim 1, which has developed a joint disease.

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. 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. The vector according to claim 7, wherein the exogenous MMP-19 gene has a nucleotide sequence represented by SEQ ID NO: 12.

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. The vector according to claim 7, which is represented by pKS-MMPBcon-19.

11. A transformant transformed with the vector according to claim 7.

12. The transformant according to claim 11, wherein the transformant is Escherichia coli JM109ZpKS-MMPBcon-19 (FERMBP-7647).

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. The screening method according to claim 13, wherein the abnormal degradation of the extracellular matrix is caused by a dysfunction of MMP-19.

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.

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. 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.

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. 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.

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.

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. 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.

23. Fertilized eggs into which DNA incorporating the exogenous MMP-19 gene or its mutant gene has been introduced.