JPWO2004074483A1 - TSG gene knockout animal - Google Patents

Abstract

A TSG gene knockout mouse was prepared and analyzed. As a result, TSG gene knockout mice are dwarfism, dwarfism with complex immunodeficiency, osteogenesis imperfecta, hypochondrogenesis, lymphopenia, complex immunodeficiency and renal hypoplasia showed that. It has also been found that TSG deficiency causes various degrees of developmental damage to tissues derived from many mesoderm, particularly the thymus, spleen, cartilage and bone. It has also been found that TSG is essential for mammalian immune-bone development.

Description

  The present invention relates to a TSG gene-modified non-human mammal. The present invention also relates to a drug for the treatment or prevention of a disease associated with developmental failure of tissue derived from mesoderm, a screening method for the candidate compound, and a method for examining a disease associated with developmental failure of tissue derived from mesoderm. And test drugs.

BMPs, members of the transforming growth factor (TGF) -β superfamily, are important developmental regulators. Mutations in signal transducing agents such as TGF-β family ligands, receptors, and SMADs are associated with many human diseases. TSG was identified in Drosophila as one of the seven zygotic genes that govern the dorsal cell fate in the Drosophila embryo (Non-patent Document 1). TSG encodes a secreted cysteine-rich protein that regulates the activity of Decapentaplegic corresponding to vertebrate BMP-4, and when TSG is mutated, the dorsal center called amnion membrane in Drosophila Structural loss occurs (Non-Patent Document 2). On the other hand, in searching for an essential soluble factor produced from the AGM (Non-Patent Document 3) region where final hematopoiesis occurs, the present inventors have obtained the AGM region of the mouse embryo 10.5 days after intercourse (dpc). A mouse homologue of Drosophila TSG was isolated using a retrovirus-mediated signal sequence capture method (Non-patent Document 4).
In 2000, it was found that Xenopus TSGs directly bind to BMP-4 and promote BMP-4 signaling by regulating the extracellular utilization of BMP-4 (Non-Patent Document 5, 6). The ventral-dorsal axis is reversed in Drosophila and vertebrates, and the ventral morphogenetic factor BMP-4 is mesoderm formation (7) and hematopoietic stem cell (HSC) survival (3). Since it is essential for 8), we speculated that TSG may also be involved in mesoderm-derived organogenesis, including ventral formation and mammalian hematopoiesis. Meanwhile, four groups have formed by fine molecular analysis, TSG forming a ternary complex of chodins that are TSG, BMP-4, and BMP-4 antagonists in flies, fish and frogs, or It has been reported that it acts as a BMP-4 antagonist in cooperation with the toroid which is a protease (Non-Patent Documents 9 to 12). Thus, there is a debate over whether TSG acts as a BMP-4 agonist or antagonist.
Prior art document information related to the invention of the present application is described below.
[Patent Document 1] WO 01/18200
[Non-Patent Document 1] Zusman, S .; B. , And E.M. F. Wieschaus. 1985. Dev. Biol. 111: 359-371
[Non-Patent Document 2] Mason, E .; D. et al. 1994. Genes & Dev. 8: 1489-1501
[Non-Patent Document 3] Medvinsky, A. et al. , And E.M. Dzierzak. 1996. Cell 86: 897-906
[Non-Patent Document 4] Kojima, T .; , And T. Kitamura. 1999. Nat. Biotechnol. 17: 487-490
[Non-patent Document 5] Oelgeschlager, M .; et al. 2000. Nature 405: 757-763
[Non Patent Literature 6] Larain J et al. 2001. Development 128 (22): 4439-4447
[Non-Patent Document 7] Winnier, G. et al. et al. , 1995. Genes & Dev. 9: 2105-2116
[Non-Patent Document 8] Bhatia, M. et al. et al. 1999. J. et al. Exp. Med. 189: 1139-1147
[Non-Patent Document 9] Chang, C .; et al. 2001. Nature 410: 483-487
[Non-Patent Document 10] Ross, J. et al. J. et al. et al. 2001. Nature 410: 479-483
[Non-patent Document 11] Scott, I. et al. C. et al. 2001. Nature 410: 475-478
[Non-patent Document 12] Yu K et al. 2000. Development 127 (10): 2143-2154

This invention is made | formed in view of such a condition, The objective is to clarify the function in vivo of TSG, and to find the relationship with a disease. Furthermore, the present invention provides a model animal of the disease, a drug for treatment or prevention of the disease, a screening method for the candidate compound, a test method for the disease, and a test based on the relationship with the found disease The purpose is to provide medicine.
The inventors of the present invention have intensively studied to solve the above problems. In order to elucidate the function of TSG in vivo, TSG gene knockout mice were prepared and analyzed. As a result, the TSG gene knockout mice are dwarfism, dwarfism with complex immunodeficiency, osteogenesis imperfecta, hypochondrosis, lymphopenia, complex immunodeficiency and renal hypoplasia showed that. It has also been found that TSG deficiency causes various degrees of developmental damage to tissues derived from many mesoderm, particularly the thymus, spleen, cartilage and bone. Interestingly, it has recently been reported that thymic proliferation and differentiation is inhibited by BMP-4, which generally functions as a mesoderm morphogen (Graf, D. et al., 2002. J. Exp. Med. 196: 163-171). These findings indicate that TSG acts as both an agonist and antagonist for BMP-4 signaling and is essential for mammalian immune-bone development.
That is, the present invention relates to a model animal of a disease associated with developmental failure of tissue derived from mesoderm, a drug for treatment or prevention of the disease, a screening method for the candidate compound, a test method for the disease, and a test drug The following [1] to [18] are provided.
[1] A genetically modified non-human mammal, wherein the expression of the TSG gene is artificially suppressed.
[2] A genetically modified non-human mammal, wherein a foreign gene is inserted into one or both of the TSG gene pairs.
[3] The genetically modified non-human mammal according to [1] or [2], which is a model animal of a disease accompanied by developmental failure of a tissue derived from mesoderm.
[4] Diseases accompanied by developmental failure of tissues derived from mesoderm are dwarfism, dwarfism with complex immunodeficiency, osteogenesis imperfecta, hypochondrosis, lymphopenia, complex immunity The genetically modified non-human mammal according to [3], which is deficiency or renal hypoplasia.
[5] A genetically modified mammalian cell, wherein the expression of the TSG gene is artificially suppressed.
[6] A genetically modified mammalian cell, wherein a foreign gene is inserted into one or both of the TSG gene pairs.
[7] Treatment of diseases associated with developmental failure of tissue derived from mesoderm, bone loss caused by drugs, osteoporosis, fractures, or growth inhibition by drugs, containing TSG or TSG-encoding DNA as an active ingredient Or drugs for prevention.
[8] Diseases accompanied by developmental failure of tissues derived from mesoderm are dwarfism, dwarfism with complex immunodeficiency, osteogenesis imperfecta, cartilage hypoplasia, lymphopenia, complex immunity [7] The therapeutic or preventive drug according to [7], which is deficiency or renal hypoplasia.
[9] Treatment of diseases accompanied by developmental failure of tissue derived from mesoderm, bone loss by drugs, osteoporosis, fractures, or growth inhibition by drugs, including the following steps (a) to (c) Alternatively, a screening method for drug candidate compounds for prevention.
(A) A step of bringing a test compound into contact with TSG
(B) detecting the binding between the TSG and the test compound
(C) a step of selecting a test compound that binds to the TSG
[10] Treatment of diseases accompanied by developmental failure of tissue derived from mesoderm, bone loss by drugs, osteoporosis, fractures, or growth inhibition by drugs, including the following steps (a) to (c) Alternatively, a screening method for drug candidate compounds for prevention.
(A) A step of administering a test compound to the genetically modified non-human mammal according to any one of [1] to [4]
(B) A step of determining whether or not the test compound substitutes the function of TSG
(C) a step of selecting a compound that substitutes for the function of TSG as compared to the case where no test compound is administered.
[11] Treatment of diseases accompanied by developmental failure of tissue derived from mesoderm, bone loss by drugs, osteoporosis, fractures, or growth inhibition by drugs, including the following steps (a) to (c) Alternatively, a screening method for drug candidate compounds for prevention.
(A) A step of bringing a test compound into contact with TSG
(B) a step of measuring the activity of the TSG
(C) a step of selecting a compound that increases the activity of the TSG compared to the case where no test compound is administered.
[12] Treatment of diseases accompanied by developmental failure of tissue derived from mesoderm, bone loss by drugs, osteoporosis, fractures, or growth inhibition by drugs, including the following steps (a) to (d) Alternatively, a screening method for drug candidate compounds for prevention.
(A) A step of providing a cell or a cell extract having DNA to which a reporter gene is operably linked downstream of the promoter region of the TSG gene
(B) A step of bringing a test compound into contact with the cells or the cell extract
(C) measuring the expression level of the reporter gene in the cell or the cell extract
(D) a step of selecting a compound that increases the expression level of the reporter gene compared to the case where no test compound is administered.
[13] A method for examining a disease accompanied by developmental failure of a tissue derived from mesoderm, comprising a step of measuring the expression level of a TSG gene.
[14] A method for examining a disease accompanied by developmental failure of a tissue derived from mesoderm, comprising a step of detecting a mutation in a TSG gene region.
[15] Diseases associated with developmental failure of tissues derived from mesoderm are dwarfism, dwarfism with complex immunodeficiency, osteogenesis imperfecta, hypochondrosis, lymphopenia, complex immunity The test method according to any one of [9] to [14], which is insufficiency or renal hypoplasia.
[16] A diagnostic agent for diseases associated with developmental failure of tissue derived from mesoderm, comprising an oligonucleotide that hybridizes to a TSG gene region and has a chain length of at least 15 nucleotides.
[17] A test agent for a disease associated with developmental failure of a tissue derived from mesoderm, comprising an antibody that binds to TSG.
[18] Diseases accompanied by developmental failure of tissues derived from mesoderm are dwarfism, dwarfism with complex immunodeficiency, osteogenesis imperfecta, hypochondrosis, lymphopenia, complex immunity The test agent according to [16] or [17], which is deficiency or renal hypoplasia.
The present inventors created a TSG gene knockout mouse in order to elucidate the function of TSG in vivo. As a result, the relationship between TSG and diseases accompanied by developmental failure of tissues derived from mesoderm was clarified. Based on this finding, the present invention provides a genetically modified non-human mammal characterized in that TSG gene expression is artificially suppressed.
The genetically modified non-human mammal of the present invention is useful as a model animal for diseases associated with developmental failure of tissues derived from mesoderm. In addition, the genetically modified non-human mammal of the present invention is used for the treatment and prevention of diseases associated with developmental failure of tissues derived from mesoderm, bone loss caused by drugs, osteoporosis, fractures, or growth inhibition by drugs. It can be used in a screening method for candidate compounds of these drugs.
The TSG (twisted gastrulation) gene of the present invention (the protein encoded by the gene is described as “TSG”) is known in humans, mice, Drosophila, Xenopus, zebrafish and the like. The accession number of the TSG sequence in each sequence is shown below.
Human NCBI, Entree Nucleotide, NM 020648
Mouse NCBI, Entree Nucleotide, NM 023053
Drosophila NCBI, Entre Nucleotide, NM 078580
Xenopus NCBI, Entre Nucleotide, AF 279246
Zebrafish NCBI, Enterprise Nucleotide, NM 131797
Further, the TSG of the present invention is not limited to the above example, and includes a protein functionally equivalent to the above TSG. Functionally equivalent proteins include proteins consisting of amino acid sequences in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of natural TSG. In order to prepare a DNA encoding a mutant protein functionally equivalent to the TSG of the present invention, other methods well known to those skilled in the art include hybridization under stringent conditions (Southern EM: J Mol Biol 98: 503, 1975) and polymerase chain reaction (PCR) technology (Saiki RK, et al: Science 230: 1350, 1985, Saiki RK, et al: Science 239: 487, 1988). .
In the present invention, stringent hybridization conditions refer to hybridization conditions of 6M urea, 0.4% SDS, 0.5 × SSC, or equivalent stringency. It is expected that DNA with higher homology can be isolated under conditions with higher stringency, for example, 6M urea, 0.4% SDS, 0.1 × SSC. High homology refers to sequence identity of at least 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 95% or more of the entire amino acid sequence. Further, the number of amino acids to be mutated in the mutant is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids, still more preferably within 3 amino acids, and even more preferably 2 amino acids. Is within.
The identity of amino acid sequences and base sequences is determined using the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993) by Carlin and Arthur. can do.
In the present invention, examples of tissues derived from mesoderm include, but are not limited to, thymus, spleen, cartilage, bone, and kidney.
In the present invention, examples of diseases accompanied by developmental failure of tissues derived from mesoderm include dwarf growth, dwarf growth with combined immunodeficiency (SCID), osteogenesis imperfecta, hypochondrosis, lymph Examples include cytopenia, combined immunodeficiency, and renal hypoplasia.
In the present invention, “TSG gene expression is artificially suppressed” usually means that one or both of the TSG gene pairs have a gene mutation such as nucleotide insertion, deletion or substitution. It refers to the state where gene expression is suppressed. The expression of a mutant TSG protein whose function as a normal TSG protein is reduced or lost is also included in the “suppression of TSG gene expression”. The “suppression” includes not only the case where the expression of the TSG gene is completely suppressed, but also the case where the expression of only one gene of the gene pair of the gene is suppressed. The site where the gene mutation is present in the present invention is not particularly limited as long as the expression of the gene is suppressed, and examples thereof include an exon site and a promoter site.
In the present invention, the biological species from which the animal subject to modification of the TSG gene is usually a mammal other than a human, preferably a rodent such as a mouse, a rat, a hamster, a rabbit, or a pig. A mouse is particularly preferable.
The present invention also provides a genetically modified mammalian cell characterized in that the expression of the TSG gene is artificially suppressed. Such cells are useful as model cells for diseases associated with developmental failure of tissues derived from mesoderm. In the present invention, the biological species from which the cells to be modified with the TSG gene are not particularly limited, and are cells derived from various biological species including humans. In addition, examples of the types of cells to be modified in the TSG gene in the present invention include somatic cells, fertilized eggs, ES cells, and cells established from the genetically modified non-human mammal of the present invention. It is not limited to. As a method for establishing a cell line derived from the genetically modified non-human mammal, a known method can be used. For example, in rodents, a method of primary culture of fetal cells can be used. (New Chemistry Laboratory, Vol. 18, pp. 125-129, Tokyo Kagaku Dojin, and mouse embryo operation manual, pages 262-264, Modern Publishing).
Means for artificially suppressing the expression of the TSG gene in the genetically modified non-human mammal and the genetically modified mammalian cell of the present invention include a method of deleting the entire TSG gene or a part thereof, a TSG gene expression control region Examples thereof include a method of deleting all or a part thereof, and a method of inactivating the TSG gene by inserting a foreign gene into one or both of the gene pairs of the TSG gene is preferable. That is, in a preferred embodiment of the present invention, the genetically modified non-human mammal and the genetically modified mammalian cell are characterized in that a foreign gene is inserted into one or both of the TSG gene pairs.
The genetically modified non-human mammal of the present invention can be prepared by those skilled in the art by generally known genetic engineering techniques. For example, a genetically modified mouse can be prepared as follows. First, DNA containing the exon portion of the TSG gene is isolated from a mouse, and an appropriate marker gene is inserted into this DNA fragment to construct a targeting vector. This targeting vector is introduced into a mouse ES cell line by electroporation or the like, and a cell line in which homologous recombination has occurred is selected. The marker gene to be inserted is preferably an antibiotic resistance gene such as a neomycin resistance gene. When an antibiotic resistance gene is inserted, a cell line that has undergone homologous recombination can be selected simply by culturing in an antibiotic-containing medium. In addition, in order to perform more efficient selection, it is possible to bind a thymidine kinase gene or the like to a targeting vector. Thereby, the cell line which caused non-homologous recombination can be excluded. Alternatively, homologous recombinants can be assayed by PCR and Southern blotting to efficiently obtain a cell line in which one of the TSG gene pair is inactivated.
When selecting a cell line in which homologous recombination has occurred, it is preferable to produce a chimera using a plurality of clones because there is a risk of unknown gene disruption due to gene insertion in addition to the homologous recombination site. The obtained ES cell line can be injected into a mouse blastoderm to obtain a chimeric mouse. By crossing this chimeric mouse, a mouse in which one of the gene pairs of the TSG gene is inactivated can be obtained. Furthermore, by mating this mouse, a mouse in which both TSG gene pairs are inactivated can be obtained. In animals where ES cells other than mice are established, genetic modification can be performed by the same technique.
An ES cell line in which both TSG gene pairs are inactivated can also be obtained by the following method. That is, by culturing an ES cell line in which one of the gene pair is inactivated in a medium containing a high concentration of antibiotics, the cell line in which the other gene pair is also inactivated, that is, the gene pair of the TSG gene ES cell lines in which both of these are inactivated can be obtained. It can also be produced by selecting an ES cell line in which one of the gene pairs is inactivated, introducing a targeting vector again into this cell line, and selecting a cell line that has undergone homologous recombination. The marker gene to be inserted into the targeting vector is preferably different from the marker gene described above.
Further, the present invention includes TSG or TSG-encoding DNA as an active ingredient, a disease accompanied by developmental failure of tissue derived from mesoderm, bone loss due to drugs such as corticosteroids, osteoporosis, fracture, Alternatively, a drug for the treatment or prevention of growth inhibition by a drug such as a corticosteroid is provided.
There is no restriction | limiting in particular as a form of "DNA which codes TSG" in the chemical | medical agent of this invention, Genomic DNA, cDNA, synthetic DNA, or the vector containing those DNA may be sufficient.
Furthermore, “TSG” in the drug of the present invention can be prepared as a recombinant protein using a known genetic recombination technique in addition to a natural protein. Further, “TSG” in the drug of the present invention is not particularly limited to the organism from which it is derived. When used for treatment or prevention of human diseases, it is preferably derived from mammals, and most preferably derived from humans. The natural protein can be prepared, for example, by a method using affinity chromatography using an antibody against TSG to an extract of a tissue such as heart, brain, lung, liver, kidney or the like that is considered to express TSG. Is possible.
On the other hand, a recombinant protein can be prepared by a person skilled in the art by a known method, for example, as a recombinant polypeptide. The recombinant polypeptide can be obtained by, for example, incorporating a TSG-encoding DNA into an appropriate expression vector, introducing the DNA into an appropriate host cell, collecting the transformant, obtaining an extract, It can be purified and prepared by subjecting to chromatography such as reverse phase, gel filtration, or affinity chromatography in which an antibody against TSG is immobilized on the column, or by further combining a plurality of these columns.
In addition, when TSG was expressed in a host cell (eg, animal cell or Escherichia coli) as a fusion polypeptide with glutathione S-transferase protein or as a recombinant polypeptide to which a plurality of histidines were added, it was expressed. The recombinant polypeptide can be purified using a glutathione column or a nickel column.
As the vector, for example, when Escherichia coli is used as a host, the vector is amplified in Escherichia coli in order to amplify it in large quantities in Escherichia coli (for example, JM109, DH5α, HB101, XL1Blue) and the like. There is no particular limitation as long as it has a selected gene of E. coli that has “ori” and is transformed (for example, a drug resistance gene that can be distinguished by ampicillin, tetracycline, kanamycin, or chloramphenicol). Examples include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, etc. For the purpose of cDNA subcloning and excision, in addition to the above vectors, for example, pGEM-T, pDIRECT , PT7 etc. An expression vector is particularly useful when a vector is used for the purpose of producing TSG, for example, when the expression vector is intended for expression in E. coli, the vector is amplified in E. coli. In addition to the above characteristics, when the host is E. coli such as JM109, DH5α, HB101, XL1-Blue, a promoter that can be expressed efficiently in E. coli, such as the lacZ promoter (Ward et al., Nature ( 1989) 341, 544-546: FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), or T7 promoter is essential. There are such vectors and Examples of the vector include pGEX-5X-1 (Pharmacia), “QIA express system” (Qiagen), pEGFP, and pET.
The vector may contain a signal sequence for polypeptide secretion. As a signal sequence for polypeptide secretion, a pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379) may be used when the polypeptide is produced in the periplasm of E. coli. Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.
In addition to E. coli, for example, vectors for producing TSG include mammalian-derived expression vectors (for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17)). , P5322), pEF, pCDM8), insect cell-derived expression vectors (eg, “Bac-to-BAC baculovirus expression system” (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2), animals Expression vectors derived from viruses (eg, pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (eg, pZIPneo), expression vectors derived from yeast (eg, “Pichia Expression”) it "(Invitrogen), pNV11, SP-Q01), expression vectors derived from Bacillus subtilis (for example, pPL608, pKTH50) or the like.
For the purpose of expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, etc., promoters necessary for expression in cells such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have an MMLV-LTR promoter, an EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), a CMV promoter, etc., and a gene for selecting transformation into cells (for example, More preferably, it has a drug resistance (drug resistance gene that can be discriminated by a drug such as neomycin or G418). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
Examples of the system for producing a polypeptide in vivo include a production system using animals and a production system using plants. DNA encoding TSG is introduced into this animal or plant, and TSG is produced in the body of the animal or plant and recovered.
When animals are used, there are production systems using mammals and insects. As mammals, goats, pigs, sheep, mice, and cattle can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Moreover, when using a mammal, a transgenic animal can be used.
For example, DNA encoding TSG is prepared as a fusion gene with a gene encoding a polypeptide inherently produced in milk such as goat β casein. Next, a DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transferred to a female goat. TSG can be obtained from the milk produced by the transgenic goat born from the goat that received the embryo or its progeny. In order to increase the amount of milk containing the polypeptide produced from the transgenic goat, hormones may be used in the transgenic goat as appropriate (Ebert, KM et al., Bio / Technology (1994) 12, 699-702).
Moreover, as an insect, a silkworm can be used, for example. When a silkworm is used, TSG can be obtained from the body fluid of the silkworm by infecting the silkworm with a baculovirus inserted with DNA encoding TSG (Susumu, M. et al., Nature (1985) 315, 592). -594).
Furthermore, when using plants, for example, tobacco can be used. When tobacco is used, DNA encoding TSG is inserted into a plant expression vector such as pMON 530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens. This bacterium can be infected with tobacco, for example Nicotiana tabacum, and TSG can be obtained from the leaves of this tobacco (Julian K.-C. Ma et al., Eur. J. Immunol. (1994). 24, 131-138).
The TSG thus obtained can be isolated from the inside of the host cell or outside the cell (medium etc.) and purified as a substantially pure and homogeneous polypeptide. Separation and purification of polypeptides may be carried out using separation and purification methods used in usual polypeptide purification, and are not limited in any way. For example, a chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected. When combined, polypeptides can be separated and purified.
The TSG of the present invention can be optionally modified or partially removed by allowing a suitable protein modifying enzyme to act before or after purification. Examples of protein modifying enzymes include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase and the like.
The anti-TSG antibody is not particularly limited, and includes monoclonal antibodies as well as polyclonal antibodies. Also included are antisera obtained by immunizing animals such as rabbits with TSG, polyclonal antibodies and monoclonal antibodies of all classes, humanized antibodies and human antibodies by genetic recombination. The above antibody can be prepared by the following method. In the case of a polyclonal antibody, for example, a small animal such as a rabbit is immunized with TSG to obtain serum, and a fraction that recognizes only TSG is obtained from an affinity column coupled with TSG. Globulin G or M can be prepared by purifying with protein A or protein G column. In the case of a monoclonal antibody, TSG can be used to immunize small animals such as mice, the spleen is removed from the mice, ground into cells, and fused with mouse myeloma cells using a reagent such as polyethylene glycol. A clone producing an antibody against TSG is selected from the fused cells (hybridoma). Next, the obtained hybridoma was transplanted into the abdominal cavity of the mouse, and ascites was collected from the mouse, and the obtained monoclonal antibody was cupped with, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, TSG. It can be prepared by purification using a ringed affinity column or the like. In addition to being used for purification and detection of TSG, the antibody can be used as a drug for controlling the function of TSG. When using an antibody as a drug for humans, a human antibody or a humanized antibody is effective in terms of immunogenicity. Human antibodies or humanized antibodies can be prepared by methods known to those skilled in the art. For example, human antibodies can be prepared, for example, by immunizing mice with the immune system replaced with humans. In addition, a humanized antibody can be prepared by, for example, a CDR graft method in which an antibody gene is cloned from a monoclonal antibody-producing cell and the antigen determination site is transplanted to an existing human antibody.
The drug can be administered either orally or parenterally, but is preferably administered parenterally, specifically, injection form, nasal form, pulmonary form, transdermal form Etc. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.
When DNA encoding TSG is administered in vivo, viral vectors such as retroviruses, adenoviruses, and Sendai viruses, and non-viral vectors such as liposomes can be used. As an administration method, an in vivo method and an ex vivo method can be exemplified.
In addition to directly administering the drug of the present invention to a patient, it can be administered as a drug formulated by a known pharmaceutical method. For example, it can be used in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension. In addition, for example, a pharmacologically acceptable carrier or medium, specifically sterilized water, physiological saline, emulsifier, suspending agent, surfactant, stabilizer, vehicle, preservative, etc. It is envisaged to formulate by blending in the unit dosage form required for recognized pharmaceutical practice. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.
Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection. Examples of the aqueous solution for injection include isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 ^TM , HCO-50 may be used in combination.
Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.
In addition, the dose can be appropriately selected depending on the age and symptoms of the patient. For example, it is possible to select from a range of 0.0001 mg to 1000 mg per kg of body weight at a time. Alternatively, for example, the dose can be selected within the range of 0.001 to 100,000 mg / body per patient. However, the drug of the present invention is not limited to these doses.
The present invention relates to treatment or prevention of diseases associated with developmental failure of tissues derived from mesoderm, bone loss caused by drugs such as corticosteroids, osteoporosis, fractures, or growth inhibition by drugs such as corticosteroids. Methods for screening drug candidate compounds are provided. Although the aspect of the screening method of this invention is illustrated below, the screening method of this invention is not limited to these.
The first aspect of the screening method of the present invention relates to screening for compounds that bind to TSG. In the first embodiment, first, a test compound is brought into contact with TSG. Examples of the test compound used in the screening method of the present invention include single compounds such as natural compounds, organic compounds, inorganic compounds, proteins, peptides, compound libraries, gene library expression products, cell extracts, Examples include cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts and the like.
In the first embodiment, the binding between the TSG and the test compound is then detected. Next, a test compound that binds to the TSG is selected. By this method, the isolated compound can be a candidate compound for a drug for the treatment or prevention of diseases associated with developmental failure of tissues derived from mesoderm. It can also be used as a test compound in the screening method described below.
Many methods known to those skilled in the art can be used as a method of screening for polypeptides that bind to TSG. Such screening can be performed, for example, by immunoprecipitation. Specifically, it can be performed as follows. A gene encoding TSG is inserted into a vector for expressing a foreign gene such as pSV2neo, pcDNA I, or pCD8, thereby expressing the gene in animal cells or the like. As promoters used for expression, SV40 early promoter (Rigby In Williamson (ed.), Genetic Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982)), EF-1 α promoter et al. (91) , P. 217-223 (1990)), CAG promoter (Niwa et al. Gene 108, p. 193-200 (1991)), RSV LTR promoter (Cullen Methods in Enzymology 152, p. 684-704 (1987)). SR α promoter (Takebe et al. Mol. Cell. Biol. 8, p. 466 (1988)), CMV imme date early promoter (Seed and Aruffo Proc. Natl. Acad. Sci. USA 84, p. 3365-3369 (1987)), SV40 late promoter (Gheysen and Fiers J. Mol. (1982)), Adenovirus late promoter (Kaufman et al. Mol. Cell. Biol. 9, p. 946 (1989)), HSV TK promoter, etc., any promoter can be used.
In order to express a foreign gene by introducing a gene into an animal cell, electroporation (Chu, G. et al. Nucl. Acid Res. 15, 1311-1326 (1987)), calcium phosphate method (Chen, C and Okayama, H. Mol. Cell. Biol. 7, 2745-2752 (1987)), DEAE dextran method (Lopata, MA et al. Nucl. Acids Res. 12, 5707-5717 (1984); , DJ and Milman, G. Mol. Cell. Biol. 4, 1644-143 (1985)), Lipofectin method (Derijard, B. Cell 7, 1025-1037 (1994); Lamb, BT et. al.Natur Genetics 5,22-30 (1993); Rabindran, there are S.K.et al.Science 259,230-234 (1993)) method or the like, or by any method.
By introducing a recognition site (epitope) of a monoclonal antibody whose specificity has been clarified into the N-terminus or C-terminus of TSG, TSG can be expressed as a fusion polypeptide having the recognition site of the monoclonal antibody. A commercially available epitope-antibody system can be used (Experimental Medicine 13, 85-90 (1995)). A vector capable of expressing a fusion polypeptide with β-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP) and the like via a multicloning site is commercially available. In addition, a method for preparing a fusion polypeptide by introducing only a small epitope part consisting of several to a dozen amino acids in order to prevent the property of TSG from changing as much as possible by making it into a fusion polypeptide has also been reported. ing. For example, polyhistidine (His-tag), influenza agglutinin HA, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV) -Tag), E-tag (epitope on monoclonal phage), and monoclonal antibodies that recognize them can be used as an epitope-antibody system for screening for polypeptides that bind to TSG (Experimental Medicine 13, 85). -90 (1995)).
In immunoprecipitation, an immune complex is formed by adding these antibodies to a cell lysate prepared using an appropriate surfactant. This immune complex consists of TSG, a polypeptide capable of binding to it, and an antibody. In addition to using an antibody against the epitope, immunoprecipitation can also be performed using an antibody against TSG. An antibody against TSG is, for example, by introducing DNA encoding TSG into an appropriate E. coli expression vector and expressing it in E. coli, purifying the expressed polypeptide, and applying it to rabbits, mice, rats, goats, chickens, etc. It can be prepared by immunization. It can also be prepared by immunizing the above animal with the synthesized partial peptide of TSG.
For example, if the antibody is a mouse IgG antibody, the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose. In addition, when TSG is prepared as a fusion polypeptide with an epitope such as GST, for example, a TSG antibody is utilized using a substance that specifically binds to these epitopes such as glutathione-Sepharose 4B. In the same manner, immune complexes can be formed.
For general methods of immunoprecipitation, for example, according to the method described in the literature (Harlow, E. and Lane, D .: Antibodies, pp. 511-552, Cold Spring Harbor Laboratory publications, New York (1988)), or It may be done according to this.
SDS-PAGE is generally used for analysis of immunoprecipitated polypeptides, and by using a gel with an appropriate concentration, it is possible to analyze a polypeptide that has been bound based on the molecular weight of the polypeptide. At this time, generally, a polypeptide bound to TSG is a radioisotope because it is difficult to detect by a normal staining method such as Coomassie staining or silver staining. ³⁵ S-methionine or ³⁵ Detection sensitivity can be improved by culturing cells in a culture solution containing S-cysteine, labeling the polypeptide in the cells, and detecting this. Once the molecular weight of the polypeptide is known, the polypeptide of interest can be purified directly from an SDS-polyacrylamide gel and its sequence determined.
Moreover, as a method for isolating a polypeptide that binds to TSG using TSG, for example, the method of Skolnik et al. (Skolnik, EY et al., Cell (1991) 65, 83-90) is used. Can be used. That is, a cDNA library using a phage vector (λgt11, ZAP, etc.) is prepared from cells and tissues expected to express a polypeptide that binds to TSG, and this is expressed on LB-agarose and filtered. The TSG labeled with purified and labeled TSG and the above-described filter are reacted, and plaques expressing the polypeptide bound to TSG may be detected with the label. As a method for labeling TSG, a method using the binding property of biotin and avidin, a method using an antibody that specifically binds to TSG or a polypeptide fused to TSG (eg, GST), a method using a radioisotope Or the method of utilizing fluorescence, etc. are mentioned.
In addition, as a first aspect of the screening method of the present invention, a two-hybrid system using cells (Fields, S., and Sternglanz, R., Trends. Genet. (1994) 10, 286-292, Dalton S , And Triisman R (1992) Characteristic of SAP-1, a protein replicated by serum response factor to the c-fos serum response element. Cell 68, 597-612. "Assay Kit", "MATCHMAKER One-Hy rid System "(both manufactured by Clontech), method using the" HybriZAP Two-Hybrid Vector System "(Stratagene)) and the like.
In the 2-hybrid system, it is expected that TSG or a partial peptide thereof is fused with the SRF DNA binding region or GAL4 DNA binding region and expressed in yeast cells to express a polypeptide that binds to TSG. From the cells, a cDNA library that is expressed in a form fused with the VP16 or GAL4 transcriptional activation region is prepared, introduced into the yeast cell, and the library-derived cDNA is isolated from the detected positive clone ( When a polypeptide that binds to TSG is expressed in yeast cells, the reporter gene is activated by the binding of both, and a positive clone can be confirmed). By introducing the isolated cDNA into E. coli and expressing it, a polypeptide encoded by the cDNA can be obtained. This makes it possible to prepare a polypeptide that binds to TSG or its gene.
Examples of the reporter gene used in the 2-hybrid system include, but are not limited to, the HIS3 gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (plasminogen activator inhibitor type 1) gene, and the like. Not. Screening by the two-hybrid method can be performed using yeast, mammalian cells, and the like.
Screening for a compound that binds to TSG can also be performed using affinity chromatography. For example, TSG is immobilized on an affinity column carrier, and a test compound that is expected to express a polypeptide that binds to TSG is applied thereto. Examples of test compounds in this case include cell extracts and cell lysates. After applying the test compound, the column can be washed and a polypeptide bound to TSG can be prepared.
The resulting polypeptide can be analyzed for its amino acid sequence, synthesized oligo DNA based on it, and screened for a cDNA library using the DNA as a probe to obtain DNA encoding the polypeptide. .
As a method for isolating a compound that binds to TSG as well as a polypeptide, for example, a synthetic compound, a natural product bank, or a random phage peptide display library is allowed to act on immobilized TSG to bind to TSG. Molecular screening methods and screening methods using high-throughput combinatorial chemistry technology (Wrighton NC; Farrell FX; Chang R; Kashhap AK; Barbone FP; Mulcahy LS; Johnson DL; Barlet RW; Small peptides as potential mimics of the protein hormone erythropoietin, Scie nce (UNITED STATES) Jul 26 1996, 273 p458-64, Verdine GL., The combinatorial chemistry of nature, Nature (ENGLAND) Nov 7 1996, 384 p11-13, Hog. Nov 7 1996, 384 p17-9) is known to those skilled in the art.
In the present invention, a biosensor using the surface plasmon resonance phenomenon can also be used as a means for detecting or measuring a bound compound. The biosensor using the surface plasmon resonance phenomenon can observe the interaction between the TSG and the test compound in real time as a surface plasmon resonance signal without labeling with a trace amount of polypeptide. (For example, BIAcore, Pharmacia).
The second embodiment of the screening method in the present invention relates to screening for compounds that substitute for the function of TSG. In the second embodiment, first, a test compound is administered to the genetically modified non-human mammal of the present invention. Administration of the test compound to the genetically modified non-human mammal of the present invention can be performed, for example, orally or parenterally, but is not limited thereto. When the test compound is a protein, for example, a viral vector having a gene encoding the protein is constructed, and the gene is introduced into the genetically modified non-human mammal of the present invention using its infectivity. It is also possible. As a second aspect, it is then determined whether or not the test compound substitutes for the function of TSG. Next, a compound that substitutes for the function of TSG is selected as compared with the case where the test compound is not administered.
For example, in the following cases, it is determined that the test compound substitutes the function of TSG. Measure nasal anal length (distance between nose and anus) with calipers, etc. If the length of the genetically modified non-human animal to which the test compound is administered is longer than that to which it is not administered, the number of white blood cells in peripheral blood is measured by Sysmex Observe a separately prepared smear of peripheral blood smeared with May-Giemsa under a microscope to calculate the percentage of leukocytes, multiply the two to calculate the number of lymphocytes, and then apply the test compound If the lymphocytes in human peripheral blood are higher than those not administered, the non-human animal is euthanized, the thymus or spleen is removed, weighed with a balance, and the test compound is administered. This is the case when the animal's thymus or spleen weight is heavier than that of the non-administered animal.
The third aspect of the screening method in the present invention relates to screening for compounds that increase the activity of TSG. As a third aspect, first, a test compound is brought into contact with TSG. There is no restriction | limiting in particular as a state of TSG used for a 3rd aspect, For example, the state refine | purified, the state expressed in the cell, the state expressed in the cell extract etc. may be sufficient.
Purification of TSG can be performed by a well-known method (Yochisha, Protein Experiment Note, page 10-13 until determination of primary structure). Examples of cells expressing TSG include cells expressing endogenous TSG and cells expressing exogenous TSG. Examples of the cells expressing the endogenous TSG include, but are not limited to, cultured cells.
The cells expressing the exogenous TSG can be prepared, for example, by introducing a vector containing DNA encoding TSG into the cells. Introduction of a vector into a cell can be performed by a method common to those skilled in the art. Moreover, the cell which has the said exogenous TSG can be produced by inserting DNA which codes TSG into a chromosome by the gene transfer method using homologous recombination, for example. The biological species from which the cell into which such exogenous TSG is introduced is not particularly limited, and may be any biological species for which a technique for expressing a foreign protein in the cell has been established.
In addition, examples of the cell extract in which TSG is expressed include those obtained by adding a vector containing DNA encoding TSG to a cell extract contained in an in vitro transcription / translation system. The in vitro transcription / translation system is not particularly limited, and a commercially available in vitro transcription / translation kit can be used.
In the present invention, “contact” is performed according to the state of the TSG. For example, if TSG is in a purified state, it can be carried out by adding a test compound to the purified sample. Moreover, if it is the state expressed in the cell or the state expressed in the cell extract, it can be carried out by adding a test compound to the cell culture solution or the cell extract, respectively. When the test compound is a protein, for example, a vector containing DNA encoding the protein is introduced into a cell expressing TSG, or the vector is added to a cell extract expressing TSG. It is also possible to do this. In addition, for example, a two-hybrid method using yeast or animal cells can be used.
In the third embodiment, the activity of the TSG is then measured. The activity of TSG can be measured, for example, by using the binding ability with BMP-4 as an index (Oelgeschlager, M. et al., 2000. Nature 405: 757-763).
In the third embodiment, a compound that increases the activity of the TSG is then selected as compared to the case where no test compound is administered.
The fourth aspect of the screening method in the present invention relates to screening for compounds that increase the expression level of the TSG gene. As a fourth aspect, first, a cell or cell extract having DNA to which a reporter gene is operably linked downstream of the promoter region of the TSG gene is provided. Here, “functionally linked” means that the promoter region of the TSG gene and the reporter gene are bound so that expression of the reporter gene is induced by binding of a transcription factor to the promoter region of the TSG gene. It means that Therefore, even when the reporter gene is bound to another gene and forms a fusion protein with another gene product, expression of the fusion protein is caused by binding of a transcription factor to the promoter region of the TSG gene. If it is derived, it is included in the meaning of the above “functionally coupled”.
The reporter gene is not particularly limited as long as its expression can be detected. For example, a CAT gene, a lacZ gene, a luciferase gene, a β-glucuronidase gene (GUS) and a GFP that are commonly used by those skilled in the art. Examples include genes. The reporter gene also includes DNA encoding TSG.
In the fourth embodiment, the test compound is then brought into contact with the cells or the cell extract. Next, the expression level of the reporter gene in the cell or the cell extract is measured. The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of reporter gene used. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of β-glucuronidase gene (GUS), luminescence of Glucuron (ICN) or 5-bromo-4-chloro-3-indolyl- By detecting the color development of β-glucuronide (X-Gluc), and in the case of the GFP gene, the expression level of the reporter gene can be measured by detecting the fluorescence of the GFP protein.
When the TSG gene is used as a reporter, the expression level of the gene can be measured by methods known to those skilled in the art. For example, the transcription level of the gene can be measured by extracting mRNA of the gene according to a standard method and performing Northern hybridization or RT-PCR using the mRNA as a template. Furthermore, the expression level of the gene can be measured using DNA array technology.
Moreover, the translation level of a gene can also be measured by collect | recovering the fraction containing TSG according to a conventional method, and detecting the expression of this TSG by electrophoresis methods, such as SDS-PAGE. Moreover, it is also possible to measure the translation level of a gene by performing Western blotting using an antibody against TSG and detecting the expression of TSG.
In the fourth embodiment, a compound that increases the expression level of the reporter gene is then selected compared to when no test compound is administered.
The present invention provides a method for examining a disease accompanied by developmental failure of a tissue derived from mesoderm, comprising a step of measuring the expression level of a TSG gene. Here, “expression of TSG gene” includes not only expression of TSG mRNA but also expression of TSG. When the expression of the TSG gene is reduced by the test method, it is determined that the subject is already suffering from a disease accompanied by developmental failure of tissue derived from mesoderm.
Although the aspect of the inspection method of this invention is illustrated below, the inspection method of this invention is not limited to them. As one embodiment of the test method, first, an RNA sample of a subject is prepared. The RNA sample can be extracted from the blood, skin, oral mucosa, hair, tissue or cells collected or excised by surgery, for example.
In the present method, the amount of RNA encoding TSG contained in the RNA sample is then measured. The amount of RNA measured is then compared to a control. Examples of such a method include Northern blotting, DNA array, or RT-PCR.
Moreover, the said test | inspection method can be implemented as follows by measuring the expression level of TSG. First, a polypeptide sample is prepared from a subject. The polypeptide sample can be prepared from, for example, a subject's blood, skin, oral mucosa, hair, tissue or cells collected or excised by surgery.
In this method, the amount of TSG contained in the polypeptide sample is then measured. The amount of TSG measured is then compared to a control. Such methods include SDS polyacrylamide electrophoresis and Western blotting, dot blotting, immunoprecipitation, enzyme-linked immunoassay (ELISA), and immunofluorescence using antibodies that bind to TSG. It can be illustrated.
The present invention also provides a method for examining a disease associated with developmental failure of a tissue derived from mesoderm, comprising a step of detecting a mutation in the TSG gene region. When a mutation occurs in the TSG gene region by the test method, it is determined that the disease is already suffering from a disease accompanied by developmental failure of tissue derived from mesoderm.
In the present invention, the TSG gene region means a TSG gene and a region that affects the expression of the gene. The region that affects the expression of the gene is not particularly limited, and examples thereof include a promoter region.
The mutation in the present invention is not limited in its type, number, site, etc., as long as it is a mutation that causes abnormal cell growth or differentiation. Examples of the type of mutation include deletion, substitution, or insertion mutation.
Hereinafter, although the preferable aspect of the test | inspection method including the process of detecting the variation | mutation which arose in the TSG gene region is described, the method of this invention is not limited to those methods.
In a preferred embodiment of the above inspection method, first, a DNA sample is prepared from a subject. The DNA sample can be prepared based on, for example, a subject's blood, skin, oral mucosa, hair, chromosomal DNA extracted from tissue or cells collected or excised by surgery, or RNA.
In this method, DNA containing the TSG gene region is then isolated. The gene region can be isolated, for example, by PCR using a primer that hybridizes to DNA containing the gene region and using chromosomal DNA or RNA as a template.
In this method, the base sequence of the isolated DNA is then determined. The base sequence of the isolated DNA can be determined by methods known to those skilled in the art.
In this method, the determined DNA base sequence is then compared with a control. In the present invention, the control refers to DNA containing a normal (wild-type) TSG gene region. In general, since the sequence of DNA containing the TSG gene region of a healthy person is considered to be normal, the above “comparison with the control” usually means comparing with the DNA sequence containing the TSG gene region of a healthy person. Means.
Detection of the mutation in the present invention can also be performed by the following method. First, a DNA sample is prepared from a subject. Next, the prepared DNA sample is cleaved with a restriction enzyme. Next, the DNA fragments are separated according to their sizes. The size of the detected DNA fragment is then compared to a control. In another embodiment, a DNA sample is first prepared from a subject. Next, DNA containing the TSG gene region is amplified. Furthermore, the amplified DNA is cleaved with a restriction enzyme. Next, the DNA fragments are separated according to their sizes. The size of the detected DNA fragment is then compared to a control.
Examples of such a method include a method using a restriction fragment length polymorphism / RFLP, a PCR-RFLP method, and the like. Specifically, if there is a mutation at the restriction enzyme recognition site, or if there is a base insertion or deletion in the DNA fragment produced by the restriction enzyme treatment, the size of the fragment produced after the restriction enzyme treatment is compared with the control. Change. By amplifying a portion containing this mutation by the PCR method and treating with each restriction enzyme, these mutations can be detected as a difference in band mobility after electrophoresis. Alternatively, the presence or absence of a mutation can be detected by treating chromosomal DNA with these restriction enzymes, performing electrophoresis, and then performing Southern blotting using the probe DNA of the present invention. The restriction enzyme used can be appropriately selected according to each mutation. In this method, in addition to genomic DNA, RNA prepared from a subject can be converted to cDNA using reverse transcriptase, and this can be cleaved with a restriction enzyme as it is, followed by Southern blotting. It is also possible to examine the difference in mobility after amplifying DNA containing the TSG gene region by PCR using this cDNA as a template, cleaving it with a restriction enzyme.
In yet another method, a DNA sample is first prepared from a subject. Next, DNA containing the TSG gene region is amplified. Furthermore, the amplified DNA is dissociated into single-stranded DNA. The dissociated single-stranded DNA is then separated on a non-denaturing gel. The mobility of the separated single-stranded DNA on the gel is compared with the control.
As the method, for example, the PCR-SSCP (single-strand conformation polymorphism, single-strand conformation polymorphism, single-strand conformation polymorphism, single-strand conformation polymorphism, single-strand conformation polymorphism, 19-strand conformation polymorphism) 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumours by single-strand conformation analysis of polymerase. chain reaction products. Oncogene.1991 Aug 1; 6 (8): 1313-1318., Multiple fluorescence-based PCR-SSCP analysis with posted labeling. Is mentioned. This method is particularly convenient for screening a large number of DNA samples because it is relatively simple to operate and has the advantage of requiring a small amount of test sample. The principle is as follows. When a double-stranded DNA fragment is dissociated into single strands, each strand forms a unique higher order structure depending on the base sequence. When the dissociated DNA strand is electrophoresed in a polyacrylamide gel not containing a denaturing agent, single-stranded DNA having the same complementary strand length moves to a different position according to the difference in each higher-order structure. The higher-order structure of this single-stranded DNA also changes by substitution of a single base, and shows different mobility in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, it is possible to detect the presence of mutations due to point mutations, deletions or insertions in the DNA fragment.
Specifically, first, DNA containing the TSG gene region is amplified by a PCR method or the like. As a range to be amplified, a length of about 200 to 400 bp is usually preferable. Those skilled in the art can perform PCR by appropriately selecting reaction conditions and the like. During PCR ³² By using a primer labeled with an isotope such as P, a fluorescent dye, or biotin, the amplified DNA product can be labeled. Or in PCR reaction solution ³² It is also possible to label the amplified DNA product by performing PCR by adding a substrate base labeled with an isotope such as P, a fluorescent dye, or biotin. Furthermore, using a Klenow enzyme after the PCR reaction, ³² Labeling can also be performed by adding a substrate base labeled with an isotope such as P, a fluorescent dye, or biotin to the amplified DNA fragment. The labeled DNA fragment thus obtained is denatured by applying heat or the like and electrophoresed on a polyacrylamide gel containing no denaturing agent such as urea. In this case, the conditions for separating DNA fragments can be improved by adding an appropriate amount (about 5 to 10%) of glycerol to the polyacrylamide gel. In addition, although the electrophoresis conditions vary depending on the properties of each DNA fragment, it is usually carried out at room temperature (20 to 25 ° C.). Review. After electrophoresis, the mobility of the DNA fragment is analyzed by autoradiography using an X-ray film, a scanner for detecting fluorescence, or the like. If a band with a difference in mobility is detected, this band can be directly excised from the gel, amplified again by PCR, and directly sequenced to confirm the presence of the mutation. Even when the labeled DNA is not used, the band can be detected by staining the gel after electrophoresis with ethidium bromide or silver staining.
In yet another method, a DNA sample is first prepared from a subject. Next, DNA containing the TSG gene region is amplified. Furthermore, the amplified DNA is separated on a gel where the concentration of the DNA denaturant is gradually increased. The mobility of the separated DNA on the gel is then compared to the control.
Examples of such a method include denaturant gradient gel electrophoresis (DGGE method). The DGGE method is a method in which a mixture of DNA fragments is run in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated according to differences in instability. When a mismatched unstable DNA fragment migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to its instability. The mobility of this partially dissociated DNA fragment becomes very slow and can be separated from the mobility of complete double-stranded DNA without a dissociated portion. Specifically, a DNA containing a TSG gene region is amplified by a PCR method or the like using the primer of the present invention, and this is gradually increased as the concentration of a denaturing agent such as urea moves. Electrophoresis with and compare to control. In the case of a DNA fragment with mutation, the DNA fragment becomes single-stranded at a lower denaturant concentration position, and the movement speed becomes extremely slow. Therefore, the presence or absence of mutation is detected by detecting the difference in mobility. be able to.
In yet another method, first, a DNA containing a TSG gene region prepared from a subject and a substrate on which a nucleotide probe that hybridizes to the DNA is immobilized are provided.
In the present invention, the “substrate” means a plate-like material on which a nucleotide probe can be fixed. In the present invention, the nucleotide includes oligonucleotides and polynucleotides. The substrate of the present invention is not particularly limited as long as the nucleotide probe can be immobilized, but a substrate generally used in DNA array technology can be preferably used. In general, a DNA array is composed of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate. The surface layer of the substrate is generally glass, but a porous film such as a nitrocellulose membrane can be used.
In the present invention, an oligonucleotide-based array developed by Affymetrix can be exemplified as a nucleotide immobilization (array) method. In an oligonucleotide array, oligonucleotides are usually synthesized in situ. For example, in situ synthesis methods of oligonucleotides using photolithographic technology (Affymetrix) and ink-jet (Rosetta Informatics) technology for immobilizing chemical substances are already known. Can be used.
The nucleotide probe immobilized on the substrate is not particularly limited as long as it can detect a mutation in the TSG gene region. That is, the probe is, for example, a probe that hybridizes to DNA containing the TSG gene region. As long as specific hybridization is possible, the nucleotide probe need not be completely complementary to the DNA containing the gene region. In the present invention, the length of the nucleotide probe to be bound to the substrate is usually 10 to 100 bp, preferably 10 to 50 bp, more preferably 15 to 25 bp when the oligonucleotide is immobilized.
Next, in this method, the DNA containing the TSG gene region is brought into contact with the substrate. Through this process, DNA is hybridized to the nucleotide probe. The hybridization reaction solution and reaction conditions may vary depending on various factors such as the length of the nucleotide probe immobilized on the substrate, but can generally be performed by methods well known to those skilled in the art.
In this method, the intensity of hybridization between the DNA containing the TSG gene region and the nucleotide probe immobilized on the substrate is then detected. This detection can be performed, for example, by reading the fluorescence signal with a scanner or the like. In a DNA array, DNA fixed to a slide glass is generally called a probe, while labeled DNA in a solution is called a target. Therefore, the nucleotide immobilized on the substrate is referred to as a nucleotide probe in this specification. In this method, the detected hybridization intensity is further compared with a control.
Examples of such a method include a DNA array method (SNP gene mutation strategy, Kenichi Matsubara, Yoshiyuki Tsuji, Nakayama Shoten, p128-135, Nature Genetics (1999) 22: 164-167) and the like.
In addition to the above method, an allele specific oligonucleotide (ASO) hybridization method can be used for the purpose of detecting only a mutation at a specific position. When an oligonucleotide containing a nucleotide sequence that is considered to have a mutation is prepared and hybridized with DNA, the efficiency of hybridization is reduced when the mutation exists. It can be detected by Southern blotting or a method using the property of quenching by intercalating a special fluorescent reagent into the hybrid gap.
In the present invention, MALDI-TOF / MS method (SNP gene polymorphism strategy, Kenichi Matsubara, Yoshiyuki Tsuji, Nakayama Shoten, p106-117, Trends Biotechnol (2000): 18: 77-84), TaqMan PCR method (Strategy for SNP gene polymorphism, Kenichi Matsubara, Yoshiyuki Tsuji, Nakayama Shoten, p94-105, Genet Anal. (1999) 14: 143-149), Invader method (strategy for SNP gene polymorphism, Kenichi Matsubara, Yoshiyuki Tsuji, Nakayama Shoten, p94-105, Genome Research (2000) 10: 330-343), Pyrosequencing method (Anal. Biochem. (2000) 10: 103-110), AcycloPrime method (Genome Research (1999) 9: 492-498). , SNuPE method (Rapid Vommun Mass Spectrom (2000) 14:. 950-959), or the like can also be used.
Furthermore, the present invention provides a test agent for use in the test method of the present invention. One embodiment thereof is a test agent comprising an oligonucleotide that hybridizes to the TSG gene region and has a chain length of at least 15 nucleotides. Here, the oligonucleotide includes a polynucleotide.
The oligonucleotide specifically hybridizes to DNA (normal DNA or mutant DNA) containing the TSG gene region. Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, The condition described in the second edition 1989) means that cross-hybridization does not occur significantly with DNA encoding other proteins. If specific hybridization is possible, the oligonucleotide need not be perfectly complementary to the DNA containing the TSG gene region.
Oligonucleotides that hybridize to DNA containing the TSG gene region and have a chain length of at least 15 nucleotides can be used as probes (including the substrate on which the probe is immobilized) and primers in the above-described inspection method of the present invention. When using this oligonucleotide as a primer, the length is 15 bp-100 bp normally, Preferably it is 17 bp-30 bp. The primer is not particularly limited as long as it can amplify at least a part of the TSG gene region including the mutated portion.
In addition, when the oligonucleotide is used as a probe, the probe is not particularly limited as long as it specifically hybridizes to DNA containing the TSG gene region. The probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp.
The oligonucleotide of the present invention can be prepared, for example, by a commercially available oligonucleotide synthesizer. The probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like.
When the oligonucleotide of the present invention is used as a probe, it is preferably used after appropriately labeling. As a labeling method, T4 polynucleotide kinase is used to remove the 5 ′ end of the oligonucleotide. ³² A method of labeling by phosphorylation with P, and a DNA polymerase such as Klenow enzyme, and using random hexamer oligonucleotides as primers ³² Examples thereof include a method (such as a random prime method) of incorporating a substrate base labeled with an isotope such as P, a fluorescent dye, or biotin.
Another aspect of the test agent of the present invention is a test reagent containing an antibody that binds to TSG. The antibody is not particularly limited as long as it can be used for testing. The antibody is labeled as necessary.
In the above test agents, in addition to the active ingredients such as oligonucleotides and antibodies, for example, sterilized water, physiological saline, vegetable oil, surfactants, lipids, solubilizers, buffers, protein stabilizers (such as BSA and gelatin) ), Preservatives and the like may be mixed as necessary.

FIG. 1 is a photograph showing TSG expression patterns in embryos and adult mice. A to D show sagittal cross sections of 11.5 dpc mouse embryos by in situ hybridization. White dots represent hybridization signals. A, Probing with the sense strand RNA of TSG. Probing with antisense strands of BD, TSG. C, D, Partial enlarged view of B by hematoxylin-eosin (HE) staining. V, area where spine is appearing; A, aorta; GM, gonadal-medium kidney. E, Northern blot analysis of multiple tissues from adult mice with TSG cDNA probe.
FIG. 2 is a photograph showing TSG expression in lung, thymus and kidney of 17.5 dpc wild-type mouse embryo. Each tissue section was subjected to in situ hybridization using TSG antisense or sense riboprobe and stained with HE. The scale bar indicates 100 μm, and all panels are shown at the same magnification.
FIG. 3 is a diagram and photograph showing targeted destruction of TSG. Map of A, TSG locus (upper), targeting construct (middle), and targeting locus (lower). Restriction enzymes are EcoRI (E), BamHI (B), and NheI (N). The targeting vector consists of a 4.8 and 1.6 kb TSG genomic fragment, a neomycin resistance gene (Neo) cassette derived from pMC1NeoPolyA (Stratagene), a herpes simplex virus thymidine kinase (tk) gene cassette containing the same promoter as pMC1NeoPolyA. . B, Southern blot analysis of tail DNA from mice. Genomic DNA digested with EcoRI was hybridized with 5 ′ flanking probes. The 8.1 and 7.4 kb bands represent wild type and mutant alleles, respectively. When the blot was rehybridized with the Neo probe, only a 7.4 kb band was detected, and the BamHI digest probed with the 3 ′ flanking exon probe further confirmed the suitability of homologous recombination (data is Not shown).
FIG. 4 shows hybridization using 3′TSG cDNA probe to total RNA from liver and kidney of adult TSG ^{+ / +} mice or adult TSG ^{− / −} mice, and human glyceraldehyde triphosphate dehydrogenase ( It is a photograph which shows the Northern blot analysis by rehybridization using a GAPDH probe. The RNA ladder marker (kb) (Promega) is shown on the left of the photo.
FIG. 5 is a diagram and a photograph showing growth retardation in mice. A, Appearance of TSG ^{+ / +} and TSG ^{− / −} littermate mice at 15 days of age. The bar represents 1 cm. Growth profile with age of B, C, TSG ^{+ / +} , TSG ^+/− , and TSG ^{− / −} mice. TSG ^{− / −} mice were divided into two groups; appearance health and disease. All very small mice died within one month of life or were sacrificed when they appeared very ill. D, Survival of TSG ^{+ / +} , TSG ^+/− , and TSG ^{− / −} mice by age. Sick mice sacrificed before death were excluded from the data.
FIG. 6 is a photograph showing a truncated tail of a TSG ^{− / −} mouse. A, Shrunken tail of 26 day old TSG ^{− / −} mice. B, X-ray images of TSG ^{− / −} mice and TSG ^{+ / +} littermate tails shown in A. C and B partially enlarged views. The arrow indicates the partial disappearance of the intervertebral disc with calcification.
FIG. 7 is a photograph showing X-ray images of 22 day old TSG ^{− / −} mice and TSG ^{+ / +} littermates. -/-Mice are thinner than + / + mice.
FIG. 8 is a photograph showing the skeletons of 27-day-old TSG ^{− / −} mice and litters stained with alizarin red and Aleutian blue.
FIG. 9 is a photograph showing dwarfism with delayed endochondral ossification and immature structure of kidney glomeruli in TSG ^{− / −} mice. HE-stained sections of the distal epiphysis of the femurs of A, B, 17-day-old TSG ^+/− and TSG ^{− / −} mice. Bar = 250 μm. C, D, partially enlarged views of A and B showing metaphyseal cartilage growth plates, respectively. The growth plate rest (R), proliferation (P), and hypertrophy (H) layers are shown. Bar = 100 μm. E, F, HE stained sections from kidneys of TSG ^{+ / +} and TSG ^{− / −} mice at 32 days of age. Bar = 40 μm.
FIG. 10 is a diagram showing bone density of TSG ^{+ / +} , TSG ^+/− , and TSG ^{− / −} mice. The bone mineral density of the right femur and lumbar vertebra L2-4 was measured by X-ray photography using DCS-600EX-II (Aloka). + / + Is n = 7, +/− is n = 3, and − / − is n = 9. Three-/-male mice (1 at 189 days and 2 at 224 days) and 16 female mice (others) were analyzed. A shows the femur and B shows the lumbar spine.
FIG. 11 is a diagram showing lymphatic defects in the peripheral blood of TSG ^{− / −} mice. Analysis of peripheral blood in A, TSG ^{− / −} and TSG ^{+ / +} littermates. WBC, leukocytes; RBC, erythrocytes; Hb, hemoglobin; PLT, platelets. Severe dwarfism that typically appears ill at 12-28 days --- 3 mice, 4 seemingly healthy to show mild dwarfism --- 4 mice, and 4 + / + litters Were analyzed for their peripheral blood. The correspondence between each bar pattern and the mouse subgroup is the same as B. B, blood cell image of WBCs. The same sample used in A was analyzed.
FIG. 12 is a diagram and a photograph showing defects in lymphatic system development in TSG ^{− / −} mice. A, B, spleen and thymus freshly isolated from 27 day old TSG ^{− / −} mice and TSG ^{+ / +} littermates. Bar = 5 mm for A and 3 mm for B. HE stained sections of thymus from 15 day old TSG ^{− / −} mice (D) and TSG ^{+ / +} littermates (C). Bar = 30 μm. Comparison of spleen and thymocyte numbers by age in E, F, TSG ^{+ / +} , TSG ^+/− , and TSG ^{− / −} mice.
FIG. 13 is a photograph showing the results of TUNEL analysis using fluorescein-dUTP (green) on cells visualized with propidium iodide (red). A is derived from the same paraffin-embedded specimen as in FIG. 12C. The same applies to B and the intercept in FIG. 12D.
FIG. 14 shows flow cytometric analysis of thymocytes and bone marrow cells from 27 day old TSG ^{− / −} mice and TSG ^{+ / +} littermates. Thymocytes (Thy), bone marrow cells (BM) were analyzed by FACS Calibur flow cytometry (Becton Dickinson). A typical pattern of four independent experiments is shown.
FIG. 15 is a diagram showing the proliferation of TSG-deficient lymphocytes in vitro. Thymocytes (A) and splenocytes (B) from 26-day-old TSG ^{+ / +} and TSG ^{− / −} littermates were stimulated with various polyclonal activators for 24 hours to allow ³ H-thymidine incorporation after pulse labeling. It was measured.
FIG. 16 is a photograph showing promotion of phosphorylation of SMAD1 accompanied by an increase in RUNX1 transcript in TSG ^{− / −} thymocytes. A, Western blot analysis of 10 day old + / + and − / − littermate thymocytes using anti-phosphorylated SMAD1 antibody. Freshly isolated thymocytes (labeled in vivo) were collected from 7.5% knockout serum replacement (KSR) medium (labeled BMP-4), human BMP-4 supplemented with 100 ng / ml human BMP-4 prior to recovery, human BMP-4. Was cultured in 7.5% KSR medium (denoted as BMP-4 + TSG) supplemented with 100 ng / ml and mouse TSG at 1 μg / ml for 45 minutes. Each lane was loaded with cell lysate corresponding to 1.5 × 10 ⁵ cells. The results are shown in two independent photographs. The left picture shows only thymocyte results in vivo. B, RT-PCR analysis of thymocytes from 37 day old + / + and − / − mice. RUNX1 mRNA was detected as a 292 bp PCR product. RNA integrity was confirmed by detection of a 373 bp β ₂ -microglobulin (β ₂ -MG) transcript.

興味深いことに、Ｂ−リンパ球コロニー形成は−／−で減少しなかった（表１）。この結果は図１５と矛盾しない。同様のアッセイを２５〜２８日齢でのマウス（＋／＋，ｎ＝３；−／−，ｎ＝３）でも行ったところ、同様の結果が得られた（データは示していない）。コロニーアッセイに用いたウシ胎仔血清に存在する可能性があるウシＴＳＧがｉｎ ｖｉｔｒｏでＴＳＧ^−／−細胞の増殖および分化を増強するように作用した可能性は除外できないが（ＫＳＲはコロニー形成効率が非常に低いために用いなかった）、アッセイは、ＴＳＧ^−／−骨髄が＋／＋骨髄と同等のレベルでＢ−リンパ球前駆体細胞を含むことを示している。一方、他系列の前駆体細胞は全て減少していることを示す。
［実施例５］ ＴＳＧ欠損胸腺細胞におけるＳＭＡＤ１のリン酸化の促進に伴うＲＵＮＸ１転写産物の増加
ＴＳＧ機能の分子メカニズムを解明するために、ＢＭＰ−４シグナル伝達の下流で起こる現象について調べた。１０日齢の＋／＋および−／−同腹仔の胸腺細胞におけるＳＭＡＤ１のセリンリン酸化について調べた。図１６Ａに示されるように、ＳＭＡＤ１はインビボにおいては、胸腺細胞中で既にリン酸化されており、ｉｎ ｖｉｔｒｏにおいては、組み換えＴＳＧの存在あるいは非存在下でのＢＭＰ−４によるさらなる細胞の刺激は、ＳＭＡＤ１のリン酸化にほとんど影響を及ぼさなかった。しかしながら、インビボにおけるＳＭＡＤ１のリン酸化の程度は、＋／＋細胞より−／−細胞の方が有意に高かった。これらの結果は、インビボにおける胸腺細胞中でのＳＭＡＤ１のリン酸化という面からは、ＴＳＧがＢＭＰ−４アンタゴニストとして機能することを示す。これと一致して、インビボにおいては、ＲＵＮＸ１（その遺伝子産物はＳＭＡＤ１と機能的に結合し（Ｈａｎａｉ，Ｊ．ｅｔ ａｌ．，１９９９．Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２７４：３１５７７−３１５８２）、ＲＵＮＸ１自身の転写を刺激すると予測される）の発現は、＋／＋胸腺細胞と比較して−／−胸腺細胞では上昇していた（図１６Ｂ）。
［実施例６］ ＴＳＧ欠損マウスの表現型の重症度の変動
ＴＳＧ欠損マウスの表現型は、個体によって変化する；生存して子孫を生じるマウスもあるが（雄性および雌性ＴＳＧ^−／−マウスはいずれも念性である）、ＴＳＧ欠損マウスの半数以上が、骨化の遅延による重度の矮小発育症、リンパ系前駆細胞の枯渇を伴うリンパ系欠損、腎発達の遅延、ならびに衰弱および／または免疫不全によっておそらく引き起こされた肺繊維症のようなさらなる疾患により出生後１ヶ月以内に死亡する。ＴＳＧ^−／−マウスの表現型が多様である理由は、ＴＳＧの非存在下での微小環境におけるＢＭＰｓおよびコーディンを含む可溶性因子の確率論的分布の異常、および遺伝的背景の差によるＢＭＰシグナル伝達の差に由来する可能性がある。母親からのＴＳＧが子宮内で異なる局所利用率で−／−胚を救出するとの推測がある。しかし、軽度の表現型を示す生存ＴＳＧ^−／−マウス同士を交配させると、＋／−の親に由来する−／−の仔と出生時に表現型を区別することができない−／−の仔を生じることから、この可能性は除外された（表２）。

軽度の表現型を示す−／−の親は、軽度の表現型を示す−／−マウスのみならず、生後に重度の表現型を示す−／−マウスを生じた（データは示していない）。
ＴＳＧはＡＧＭ領域において発現され、ＴＳＧ欠損マウスは中等度の血小板減少症と軽度の貧血を伴うリンパ系発達欠損を示した。低濃度のＢＭＰ−４は、ＣＤ３４^＋ＣＤ３８⁻Ｌｉｎ⁻ヒトＨＳＣｓの増殖および分化を誘導することが知られているが、より高濃度のＢＭＰ−４はＨＳＣｓの生存を促進する（Ｂｈａｔｉａ，Ｍ．ｅｔ ａｌ．，１９９９．Ｊ．Ｅｘｐ．Ｍｅｄ．１８９：１１３９−１１４７）。したがって、ＴＳＧは、造血の初期段階で中胚葉誘発物質ＢＭＰ−４のアゴニストとして機能すると推測される。しかし、造血のＢＭＰ−４媒介調節は、より複雑である。ＢＭＰ−４の上流の調節物質であるＳｈｈが、胸腺間質によって産生され、その受容体であるパッチド＆スムースンドが、二重陰性（ＤＮ）胸腺細胞において発現され、Ｓｈｈが胸腺細胞の分化をＤＮ段階で停止させることが示された（Ｏｕｔｒａｍ，Ｓ．Ｖ．ｅｔ ａｌ．，２０００．Ｉｍｍｕｎｉｔｙ １３：１８７−１９７）。最近、胸腺間質によって産生されたＢＭＰ−４も同様に、胸腺細胞の増殖および分化を阻害することが示され、その胸腺細胞における発現がＴ細胞受容体シグナル伝達によって誘導されるＴＳＧが、ＢＭＰ−４媒介胸腺発達阻害効果を遮断するようにコーディンと相乗作用を示すことが証明された（Ｇｒａｆ，Ｄ．ｅｔ ａｌ．，２００２．Ｊ．Ｅｘｐ．Ｍｅｄ．１９６：１６３−１７１）。したがって、ＢＭＰ−４アンタゴニストとしてのＴＳＧは胸腺細胞の発達の正の調節物質であるように思われる。出生前後のＴＳＧ^−／−マウスにおける胸腺の発達障害に関する本発明者らの知見は、ｉｎ ｖｉｔｒｏでのこれらの結果と一致する。それにもかかわらず、ＴＳＧは、繊維芽細胞に対するＴＧＦ−β作用の下流の媒介物質である細胞分裂促進性ペプチドである結合組織増殖因子（ＣＴＧＦ）（Ｂｒａｄｈａｍ，Ｄ．Ｍ．．ｅｔ ａｌ，１９９１．Ｊ．Ｃｅｌｌ Ｂｉｏｌ．１１４：１２８５−１２９４）に対して弱い相同性を有することから（Ｇｒｏｔｅｎｄｏｒｓｔ，Ｇ．Ｒ．１９９７．Ｃｙｔｏｋｉｎｅ ＆ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ Ｒｅｖｉｅｗｓ ８：１７１−１７９）、ＴＳＧはＢＭＰ−４シグナル伝達を調節することによって機能するのみならず、間質機能に影響を及ぼす特定されていない標的にとっての増殖／分化因子として機能する可能性もある。
ＴＳＧは最近、ＢＭＰシグナル伝達に対して時期に応じてアゴニストおよびアンタゴニスト機能の双方を発揮することが提唱されている；第一に、ＴＳＧはＢＭＰおよび完全長のコーディンと三元複合体を形成し、ＢＭＰのその受容体への結合を妨害する、そして第二に、全てのコーディンがゾロイド（ヒトＢＭＰ−１、ショウジョウバエトロイド）によって切断されると、ＴＳＧはＢＭＰとの結合に関して、なおも抗ＢＭＰ活性を保持しているコーディン断片と競合することによってＢＭＰシグナル伝達を促進する。このように、ＢＭＰシグナル伝達のスイッチのオン・オフが、ＴＳＧによって鋭敏に行われる（Ｌａｒｒａｉｎ，Ｊ．ｅｔ ａｌ．，２００１．Ｄｅｖｅｌｏｐｍｅｎｔ １２８：４４３９−４４４７）。ＴＳＧ^−／−マウスにおける本発明者らの知見は、分子スイッチとしてのＴＳＧによるＢＭＰ活性の微細な制御が多数の臓器の適切な発達にとって必須であることを示唆している。双機能ＴＳＧが、ＢＭＰ、コーディン、ＴＳＧ、ゾロイド、および他の関連分子の局所濃度に応じて、異なる動物種の発達の際に異なるＢＭＰシグナル伝達を誘発する可能性がある。ＴＳＧ欠損マウスは、ヘッジホッグ−ＢＭＰ−ＳＭＡＤシグナル伝達、および／または推定のＢＭＰ−無関係シグナル伝達によって調節される、初期造血前駆細胞、胸腺、脾臓、軟骨、骨、および腎臓の発達の分子メカニズムを解明するために用いることができる。
ヒトＴＳＧ遺伝子は、染色体バンド１８ｐ１１にマッピングされ、これには免疫−骨疾患はこれまで連鎖していない（Ｓｃｏｔｔ，Ｉ．Ｃ．ｅｔ ａｌ．，２００１．Ｎａｔｕｒｅ ４１０：４７５−４７８）。病気のＴＳＧ^−／−マウスの表現型は、ＳＣＩＤを伴うヒト早期致死性短肢骨格異常の表現型と類似である（Ｇａｔｔｉ，Ｒ．Ａ．ｅｔ ａｌ．，１９６９．Ｊ．Ｐｅｄｉａｔｒ．７５：６７５−６８４）。ほとんどのヒト免疫−骨疾患の病因は、まだ分子的に明らかにされていないが、ＴＳＧ欠損マウスはまた、これらの疾患の発病の基礎となるメカニズムを解明するために有用となるであろう。
要約すると、ＴＳＧ欠損マウスにおける構造的に未成熟な腎臓を有する矮小発育症とリンパ系欠損の表現型は、インビボでのＴＳＧの双機能（骨格−腎形成に関するＢＭＰ−４アゴニストと、胸腺発達に関するＢＭＰ−４アンタゴニスト）を示唆している。同様に、ＣＴＧＦファミリーとしてのＴＳＧのもう一つの機能は、今後の研究において解明されなければならない。いずれにせよ、哺乳類のＴＳＧは、中胚葉臓器の適切な発達にとって必須である。EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
(1) TSG gene targeting The targeting vector is a subgroup of TSG genomic clone (derived from ES129 / SvJ strain DNA (Stratagene) cloned into λ phage (Stratagene)) to pBluescript II (Stratagene). After cloning, the NheI site in exon 20 bp downstream of the first ATG was converted into a SalI site, and then a 1.1 kb XhoI-SalI fragment of pMC1NeoPolyA (Stratagene) was inserted in the same direction into the antisense direction. The HSV tk cassette was also inserted into the NotI site of pBluescript II for negative selection. The vector was linearized at the 5 'end of the left arm and electroporated into E14-1 embryonic stem (ES) cells. Screening of Southern blot analysis for homologous recombination was performed as described in the literature (Nosaka, T. et al., 1995. Science 270: 800-802). Targeting efficiency was 5-10%. All mice were maintained in specific pathogen free conditions.
(2) In situ hybridization A 0.6 kb mouse TSG cDNA fragment from the cDNA start site to the EcoRV site, containing most of the protein coding sequence, was subcloned into the BamHI-EcoRV site of pBluescript II SK. The sense RNA probe was prepared from EcoRV digested DNA template with T3 RNA polymerase, and the antisense RNA probe was prepared from BamHI digested template with T7 RNA polymerase using ³⁵ S-UTP. Hybridization was performed as described (Sugiyama, T. et al., 2000. Biochemistry 39: 15817-15825).
(3) Northern blots Multiple tissue blots of mice were purchased from Clontech and hybridized with a 4.0 kb mouse TSG full-length cDNA probe, as previously described (Nosaka, T. et al. 1999. EMBO J. 18: 4754-4765) hybridized with mouse β-actin probe.
(4) Thymocyte and Spleen Cell Proliferation Assay Three thymocytes (5 × 10 ⁴ ) per sample were prepared using 10% knockout serum replacement medium (KSR) (Invitrogen) instead of fetal bovine serum (FBS). Concanavalin A (Con A, 20 μg / ml), Con A + IL-2 (10 units / ml), Con A + IL-7 (5 ng / ml), anti-CD3ε antibody (clone 145-2C11, 10 μg / ml) in the presence + phorbol 12-myristate 13-acetate (PMA, 10ng / ml), or after stimulation for 24 hours with either anti--CD3ε antibody + PMA + ^IL-7, 3 H- thymidine 0.5μCi a and 14 hours ^{pulse, 3} H-thymidine incorporation was measured. Splenocytes were similarly analyzed by KSR or KSR + lipopolysaccharide (LPS, 10 μg / ml).
[Example 1] Targeted destruction of TSG Mouse TSG cDNA (accession number: NCBI NO. BD013035) isolated from the mouse aorta-gonad-medium kidney (AGM) region by the present inventors has a length of 4. It encodes 222 amino acids of the TSG protein (accession number: NCBI NO. AAG00605) at 0 kb, which is another group (Graf, D. et al., 2001. Mamm. Genome 124: 554-560, Ross, J J. et al., 2001. Nature 410: 479-483, Scott, I. C. et al., 2001. Nature 410: 475-478). In situ hybridization of 11.5-dpc mouse embryos revealed that TSG mRNA was present in the spine developing region, aortic wall, gonadal-medium kidney region, and stromal tissue in general (FIGS. 1A-D). ). Northern blot analysis of adult mouse tissue revealed that a 4.1 kb TSG transcript (FIG. 1E) was present in the heart, lung, liver, and kidney. The expression of TSG mRNA in 17.5 dpc sections of lung, thymus, and kidney was also examined (Figure 2). TSG mRNA was expressed in lung alveoli and bronchial epithelial cells, thymic stroma, especially the medulla, and cells around the renal tubule. To investigate the physiological role of TSG in mammalian development, TSG-deficient mice were generated by gene targeting. The present inventors constructed a TSG targeting vector that disrupts the first coding exon to completely suppress TSG gene expression (FIG. 3A). The construct was electroporated into E14-1 ES cells to obtain three independent clones that were injected into C57BL / 6 blastula to create chimeric mice. Chimeric mice were backcrossed with C57BL / 6 mice to produce heterozygous (+/−) mice, and +/− mice were bred to produce homozygous mutant (− / −) mice. Genotyping of 100 offspring (FIG. 3B) resulted in 27 wild-type (+ / +) mice, 47 +/− mice and 26 − / − mice; this expected ratio (1: 2: 1) showed no embryo lethality. The absence of wild type TSG mRNA was confirmed by Northern blot analysis (FIG. 4).
Example 2 Growth retardation in TSG-deficient mice 12.5% of mice died on the day of birth and the rest of the mice appeared healthy at birth, but they were + / + and + / 10-20% smaller than the litters of − (described later), this difference increased with age (FIG. 5A). More than half-/-mice had severe growth retardation, followed by pathological appearance and subsequent death, or in some cases sudden death (FIGS. 5B-D). Some of the sick-/-mice died of 3 weeks of age with open eyes, jerky gait, and general tremor. The cause of death of TSG ^{− / −} mice could not be unified except for weakness. All neonates − / − mice showed milky spots similar in size to + / + or +/− littermates, indicating that breastfeeding was appropriate. At necropsy, several sick mice were found to have pulmonary fibrosis, intra-abdominal or subarachnoid hemorrhage in addition to thymic and splenic developmental failure (see below). Regardless of the presence of pathological appearance, almost half of the mice showed a clearly shrunken tail (FIG. 6). BMP-7 null mice (Dudley, AT et al., 1995. Genes Dev 9: 2795-2807, Jena, N. et al., 1997. Exp Cell Res. 230: 28-37, Luo, G. et. al., 1995. Genes Dev 9: 2808-2820). The shrunken tail reported in several cases of gene-disrupted mice was observed from day 4 after birth of TSG ^{− / −} mice. Radiographs revealed that the disc was partially lost and ossified in the shrunken tail of the mice.
Example 3 Delayed Bone Formation and Renal Formation in TSG-Deficient Mice + / +, +/−, and − / − Littermate X-rays (typical in FIG. 7) and autopsy along with their aleutians Skeletal specimen (Figure 8) stained with blue (for cartilage) and alizarin red (for mineral-rich bone) reveals that the leg bone is short and thin in TSG ^-/- mice and the skull is also very thin. However, at 27 days of age, there was no difference in their mineralized areas. Histological analysis of the distal metaphyseal cartilage growth plate in the femur revealed a thin proliferative layer, a hypertrophic layer and an enlarged resting layer of chondrocytes, and epiphyseal (secondary) ossification was +/- littermates -/-Mice were significantly delayed (Figs. 9A-D), indicating that the endochondral ossification of mice was reduced. Furthermore, the growth of femoral cord-like cortical bone in − / − mice was delayed compared to +/− littermates at 17-32 days of age (data not shown). With severe dwarfism at 32 days of age-/-The bone density of the femur and lumbar spine of mice is approximately half that of + / + littermates and is long alive-/- It was also shown that the decreasing trend is moderate (FIG. 10). These phenotypes are similar to the phenotype of transgenic mice expressing dominant negative IB type BMP receptors driven by osteoblast lineage specific promoters (Zhao, M. et al., 2002.J. Cell Biol.157: 1049-1060). These findings indicate that TSG deficiency is based on the attenuation of BMP signaling and decreases intramembranous ossification as well as dwarfism due to delayed endochondral ossification by blocking the transition from resting state to proliferative state of chondrocytes And shows that osteopenia occurs. In addition to bone formation disorders, − / − mice also showed small and immature, histologically undeveloped kidneys compared to + / + littermates (FIGS. 9E, F). This finding is reminiscent of impaired renal development in BMP-7-deficient mice, but the activity of BMP-7 as a mesoderm inducer is consistent with higher heterodimers with BMP-4 than with homodimers (Suzuki, A Et al., 1997. Biochem. Biophys. Res. Commun. 232: 153-156). Thus, TSG is likely to act as a BMP-4 agonist during skeletal system and kidney development.
[Example 4] Lymphoid cell depletion in TSG-deficient mice We isolated mouse TSG cDNA from the AGM region. BMP-4 was also found to be expressed in the human AGM region (Marshall, CJ et al., 2000. Blood 96: 1591-1593). BMP-4 proliferates pluripotent hematopoietic cells, unlike Sonic hedgehog (shh) (Bhardwaj, G. et al., 2001. Nat. Immunol. 2: 172-180), which is an upstream regulator. Can not be allowed to remain, but they can be maintained (Bhatia, M. et al., 1999. J. Exp. Med. 189: 1139-1147). Therefore, the inventors were interested in the effect of TSG deficiency on hematopoiesis. -/-Peripheral blood counts and smears from mice showed a severe decrease in the number of lymphoid cells, a moderate decrease in platelets, and a moderate decrease in red blood cells. On the other hand, the relative proportion of granulocytes and monocytes increased (FIG. 11). Consistent with this, thymus and spleen development was very poor in more than half of the mice. These organs contain very few cells, such as a 3000-fold decrease in some-/-mice whose body weights differ only by 2-5 fold from the weight of + / + or +/- litters. Not found (Figures 12A, B, E, F). There was a positive correlation between growth retardation and lymphatic deficiency level, suggesting that lymphatic deficiency may be a secondary event. However, the thymus and spleen cells at birth of − / − mice are already reduced by 2 to 4 times and 2 to 8 times compared to + / + or +/− littermates, respectively (FIGS. 12E, F). This suggests that the lymphatic deficiency is not simply due to postnatal weakness. Histologically, in the spleen of − / −, the white pulp area was reduced (data not shown) and most cells of the thymus had an apoptotic appearance, whereas the + / + thymus was apoptotic The appearance was not shown (FIGS. 12C and D). TUNEL analysis revealed that many thymocytes in diseased − / − mice caused DNA fragmentation to varying degrees, but thymocytes in + / + mice did not (FIG. 13). Flow cytometric analysis of thymus revealed that the percentage of CD4- and CD8- single positive cells increased and double positive cells decreased in-/-mice compared to + / + littermates (Fig. 14, top panel). The total number of bone marrow cells did not decrease significantly in the − / − mice when normalized by physique with the exception of a few cases. By CD43 / B220 and B220 / IgM double staining of bone marrow cells, pro-B (CD43 ⁺ B220 ^low IgM ⁻ ), pre-B (CD43 ⁻ B220 ^low IgM ⁻ ) compared to + / + littermates. ), And immature B (CD43 ⁺ B220 ^low IgM ⁺ ) cells were dramatically reduced, whereas mature B cells (CD43 ⁻ B220 ^high IgM ⁺ ) were demonstrated to be retained (FIG. 14, middle). 4 panels). These findings suggest that TSG ^{− / −} mice impair precursor B and T cell proliferation, but do not block lymphoid differentiation itself. However, spleen cells and thymocytes from − / − mice show normal dividing activity in vitro, and when cultured in KSR medium (ie, in the absence of fetal bovine TSG), lipopolysaccharide ( Compared to splenocytes and thymocytes from + / + mice by stimulation with polyclonal activators such as LPS), concanavalin A (ConA), and anti-CD3 antibody plus phorbol myristyl acetate (PMA), or IL-7 Thus, they grew at an equivalent or rather enhanced level (FIG. 15). Thus, lymphoid defects in TSG ^{− / −} mice do not appear to be due to endogenous defects in B- and T-cells, microenvironmental abnormalities such as stromal cells and cytokine production or distribution. It was thought that this was caused by an abnormality. It should also be noted that TSG has a weak homology with connective tissue growth factor. The proportion of CD11b ⁺ Gr-1 ⁺ bone marrow cells is increased in − / − bone marrow cells, indicating that they are rich in mature granulocytes (FIG. 14, lower panel). Granulocyte-macrophage (GM) colony, megakaryocyte (Meg) colony, erythroid lineage (7), by clonal culture assay of bone marrow cells of mice (+ / +, n = 3; − / −, n = 3) E) It was shown that the formation of burst, erythrocyte-megakaryocyte colonies, and mixed hematopoietic colonies (GM / Meg, GM / E, or GM / E / Meg) were all reduced in TSG-deficient mice (Table 1). ).

Interestingly, B-lymphocyte colony formation did not decrease at − / − (Table 1). This result is consistent with FIG. Similar assays were performed in mice at 25-28 days of age (+ / +, n = 3; − / −, n = 3), and similar results were obtained (data not shown). Although it is not possible to exclude the possibility that bovine TSG, which may be present in the fetal bovine serum used in the colony assay, acted to enhance TSG ^{− / −} cell proliferation and differentiation in vitro (KSR is effective for colony formation) The assay has shown that TSG ^{− / −} bone marrow contains B-lymphocyte progenitor cells at levels comparable to + / + bone marrow. On the other hand, all the other series of precursor cells are decreased.
[Example 5] Increase in RUNX1 transcript accompanying promotion of phosphorylation of SMAD1 in TSG-deficient thymocytes In order to elucidate the molecular mechanism of TSG function, the phenomenon occurring downstream of BMP-4 signaling was examined. SMAD1 serine phosphorylation in 10 day old + / + and − / − littermate thymocytes was examined. As shown in FIG. 16A, SMAD1 is already phosphorylated in thymocytes in vivo, and in vitro further cell stimulation by BMP-4 in the presence or absence of recombinant TSG is There was little effect on SMAD1 phosphorylation. However, the degree of phosphorylation of SMAD1 in vivo was significantly higher in − / − cells than in + / + cells. These results indicate that TSG functions as a BMP-4 antagonist in terms of phosphorylation of SMAD1 in thymocytes in vivo. Consistent with this, in vivo, RUNX1 (its gene product functionally binds to SMAD1 (Hanai, J. et al., 1999. J. Biol. Chem. 274: 31577-31582) and RUNX1's own The expression of (predicted to stimulate transcription) was increased in − / − thymocytes compared to + / + thymocytes (FIG. 16B).
[Example 6] Variation in severity of phenotype of TSG-deficient mice The phenotype of TSG-deficient mice varies from individual to individual; some mice survive to produce offspring (male and female TSG ^-/- mice are either More than half of TSG-deficient mice have severe dwarfism due to delayed ossification, lymphoid deficiency with depletion of lymphoid progenitors, delayed kidney development, and weakness and / or immunodeficiency Death within one month after birth due to additional diseases such as possibly caused by pulmonary fibrosis. The phenotypes of TSG ^{− / −} mice are diverse because of abnormal probabilistic distribution of soluble factors including BMPs and chodin in the microenvironment in the absence of TSG, and BMP signaling due to genetic background differences May come from the difference. There is speculation that TSGs from mothers will rescue embryos with different local utilization in the womb. However, survival TSG shows a mild phenotype - when the mating mice with each other, + / - ^- / from the parent - / - can not be distinguished phenotypically upon the pups born - / - of pups This possibility was ruled out because it occurred (Table 2).

Parents with a mild phenotype-/-gave rise to not only mice with a mild phenotype-/-mice but also mice with a severe phenotype after birth (data not shown).
TSG was expressed in the AGM region, and TSG-deficient mice showed a lymphoid developmental defect with moderate thrombocytopenia and mild anemia. Low concentrations of BMP-4 are known to induce the proliferation and differentiation of CD34 ⁺ CD38 ^- Lin ^- human HSCs, but higher concentrations of BMP-4 promote the survival of HSCs (Bhatia, M. et al. et al., 1999. J. Exp. Med. 189: 1139-1147). Therefore, it is speculated that TSG functions as an agonist of the mesoderm inducer BMP-4 at an early stage of hematopoiesis. However, BMP-4-mediated regulation of hematopoiesis is more complex. The upstream regulator of BMP-4, Shh, is produced by the thymic stroma, and its receptor, patched and smoothened, is expressed in double negative (DN) thymocytes, and Shh induces thymocyte differentiation. It was shown to stop at the DN stage (Outram, SV et al., 2000. Immunity 13: 187-197). Recently, BMP-4 produced by thymic stroma has also been shown to inhibit thymocyte proliferation and differentiation, and TSG whose expression in thymocytes is induced by T cell receptor signaling is -4 proved to be synergistic with chodin to block the mediated thymic development inhibitory effect (Graf, D. et al., 2002. J. Exp. Med. 196: 163-171). Thus, TSG as a BMP-4 antagonist appears to be a positive regulator of thymocyte development. Our findings regarding thymic developmental disorders in pre- and post-natal TSG ^{− / −} mice are consistent with these results in vitro. Nevertheless, TSG is a connective tissue growth factor (CTGF), a mitogenic peptide that is a downstream mediator of TGF-β action on fibroblasts (Bradham, DM, et al, 1991. J. Cell Biol. 114: 1285-1294) (Grotendorst, G.R. 1997. Cytokine & Growth Factor Reviews 8: 171-179), TSG does not transmit BMP-4 signaling. In addition to functioning by modulating, it may also function as a growth / differentiation factor for unspecified targets that affect stromal function.
TSG has recently been proposed to exert both agonist and antagonist functions on BMP signaling in a timely manner; first, TSG forms a ternary complex with BMP and full-length chodin. Interfere with the binding of BMP to its receptor, and, secondly, if all chodins are cleaved by zoloids (human BMP-1, Drosophila toroid), TSG is still anti-BMP for binding to BMP. Promotes BMP signaling by competing with codein fragments that retain activity. In this way, the switch of BMP signaling is turned on and off sharply by TSG (Larrain, J. et al., 2001. Development 128: 4439-4447). Our findings in TSG ^{− / −} mice suggest that fine control of BMP activity by TSG as a molecular switch is essential for the proper development of many organs. Bifunctional TSG can induce different BMP signaling during development of different animal species, depending on the local concentration of BMP, chodin, TSG, zoloid, and other related molecules. TSG-deficient mice display molecular mechanisms of early hematopoietic progenitor cell, thymus, spleen, cartilage, bone, and kidney development that are regulated by hedgehog-BMP-SMAD signaling and / or putative BMP-independent signaling Can be used to elucidate.
The human TSG gene maps to chromosomal band 18p11, to which no immune-bone disease has previously been linked (Scott, IC et al., 2001. Nature 410: 475-478). The phenotype of diseased TSG ^{− / −} mice is similar to the phenotype of human early lethal short limb skeletal abnormalities with SCID (Gatti, RA et al., 1969. J. Pediatr. 75: 675). -684). Although the pathogenesis of most human immune-bone diseases has not yet been revealed molecularly, TSG-deficient mice will also be useful to elucidate the mechanisms underlying the pathogenesis of these diseases.
In summary, the phenotype of dwarfism with structurally immature kidney and lymphoid deficiency in TSG-deficient mice is related to the dual function of TSG in vivo (BMP-4 agonists for skeletal-nephrogenesis and thymic development). BMP-4 antagonist). Similarly, another function of TSG as a CTGF family must be elucidated in future studies. In any case, mammalian TSG is essential for the proper development of mesoderm organs.

Industrial applicability

  The first industrial usefulness of the present invention is that TSG protein, TSG gene and TSG are used for various diseases that exhibit traits such as dwarf growth and lymphocyte hematopoiesis observed in the animals of the present invention. A compound having a similar biological activity is applicable as a therapeutic or prophylactic agent. The second point is to provide a method for screening a compound having the same biological activity as TSG and a compound that modifies the activity of endogenous TSG using the animal and TSG gene-modified mammalian cells. The third point is that it can provide a test method and a test drug for the above diseases by measuring the expression level of the TSG gene or protein.

Claims (18)

A genetically modified non-human mammal, wherein the expression of TSG gene is artificially suppressed. A genetically modified non-human mammal, wherein a foreign gene is inserted into one or both of the TSG gene pairs. The genetically modified non-human mammal according to claim 1 or 2, which is a model animal of a disease accompanied by developmental failure of a tissue derived from mesoderm. Diseases with developmental failure of tissue derived from mesoderm include dwarfism, dwarfism with complex immunodeficiency, osteogenesis imperfecta, hypochondrosis, lymphopenia, complex immunodeficiency, Alternatively, the genetically modified non-human mammal according to claim 3, which is hypoplastic kidney. A genetically modified mammalian cell, characterized in that the expression of the TSG gene is artificially suppressed. A genetically modified mammalian cell, wherein a foreign gene is inserted into one or both of the TSG gene pairs. TSG or TSG or TSG-containing DNA as an active ingredient for treatment or prevention of diseases associated with developmental failure of tissue derived from mesoderm, bone loss by drugs, osteoporosis, fractures, or growth inhibition by drugs Drug for. Diseases with developmental failure of tissue derived from mesoderm include dwarfism, dwarfism with complex immunodeficiency, osteogenesis imperfecta, hypochondrosis, lymphopenia, complex immunodeficiency, Alternatively, the agent for treatment or prevention according to claim 7, wherein the agent is renal hypoplasia. Treatment or prevention of diseases accompanied by developmental failure of tissue derived from mesoderm, bone loss due to drugs, osteoporosis, fractures, or growth inhibition by drugs, including the following steps (a) to (c): A screening method for drug candidate compounds.
(A) contacting TSG with a test compound (b) detecting the binding between the TSG and the test compound (c) selecting a test compound that binds to the TSG Treatment or prevention of diseases accompanied by developmental failure of tissue derived from mesoderm, bone loss due to drugs, osteoporosis, fractures, or growth inhibition by drugs, including the following steps (a) to (c): A screening method for drug candidate compounds.
(A) a step of administering a test compound to the genetically modified non-human mammal according to any one of claims 1 to 4 (b) a step of determining whether or not the test compound substitutes a function of TSG ( c) A step of selecting a compound that substitutes for the function of TSG as compared to the case where no test compound is administered. Treatment or prevention of diseases accompanied by developmental failure of tissue derived from mesoderm, bone loss due to drugs, osteoporosis, fractures, or growth inhibition by drugs, including the following steps (a) to (c): A screening method for drug candidate compounds.
(A) contacting the test compound with TSG (b) measuring the activity of the TSG (c) selecting a compound that increases the activity of the TSG compared to the case where the test compound is not administered Process Treatment or prevention of diseases accompanied by developmental failure of tissue derived from mesoderm, bone loss by drugs, osteoporosis, fractures, or growth inhibition by drugs, comprising the following steps (a) to (d): A screening method for drug candidate compounds.
(A) a step of providing a cell or a cell extract having DNA to which a reporter gene is operably linked downstream of the promoter region of the TSG gene (b) a step of bringing a test compound into contact with the cell or the cell extract ( c) a step of measuring the expression level of the reporter gene in the cell or the cell extract (d) selecting a compound that increases the expression level of the reporter gene compared to the case where no test compound is administered Process A method for examining a disease accompanied by developmental failure of a tissue derived from mesoderm, comprising a step of measuring the expression level of a TSG gene. A method for examining a disease associated with developmental failure of a tissue derived from mesoderm, comprising a step of detecting a mutation in a TSG gene region. Diseases with developmental failure of tissue derived from mesoderm include dwarfism, dwarfism with complex immunodeficiency, osteogenesis imperfecta, hypochondrosis, lymphopenia, complex immunodeficiency, Or the test | inspection method in any one of Claims 9-14 which is renal hypoplasia. A diagnostic agent for diseases associated with developmental failure of tissue derived from mesoderm, comprising an oligonucleotide that hybridizes to a TSG gene region and has a chain length of at least 15 nucleotides. A test agent for a disease associated with developmental failure of a tissue derived from mesoderm, comprising an antibody that binds to TSG. Diseases with developmental failure of tissue derived from mesoderm include dwarfism, dwarfism with complex immunodeficiency, osteogenesis imperfecta, hypochondrosis, lymphopenia, complex immunodeficiency, Alternatively, the test agent according to claim 16 or 17, which is renal hypoplasia.

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