JP2004242557A - Muscular dystrophy pathological model, and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a muscular dystrophy pathological model which can be used in stead of a conventional spontaneously obtained muscular dystrophy pathological model and can artificially be produced, and to provide a method for producing the same. <P>SOLUTION: The muscular dystrophy pathological model into which a β-defensin gene is transduced is constituted to excessively express the defensin. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】図１は、筋抽出蛋白のウエスタンブロット後、抗マウスβ−ディフェンシン−６血清にて染色した結果を示す。左レーンには野生型マウスの筋抽出蛋白８μｇ、右レーンには本件トランスジェニックマウスの筋抽出蛋白８μｇを含む。本件トランスジェニックマウスにおいて、４ ｋＤのマウスβ−ディフェンシン−６の発現を確認できる。
【図２】図２のＡは本件トランスジェニックマウスの生後２５週の外見を示す。著名な側彎を認め（矢印）、傍脊柱筋の筋力低下の存在を示している。図２のＢは、本件トランスジェニックマウスの生後２５週の下肢筋のＨＥ染色を示す。大部分の筋線維が中心核をもつ再生線維に置き換わり、その中に壊死した筋線維が散在しており（矢印）、まさに筋ジストロフィーの病理所見に合致する。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a disease model mammal of muscular dystrophy and a method for producing the same.
[0002]
[Prior art]
Muscular dystrophy is also referred to as progressive muscular dystrophy and is defined as "a hereditary disease which mainly involves degeneration, necrosis, and regeneration of muscle fibers and is clinically accompanied by progressive muscle weakness". Muscular weakness is progressive and is a type of intractable disease that often results in death from respiratory failure due to weakness of the respiratory muscles or heart failure. Duchenne muscular dystrophy (DMD: Duchenne Muscular Dystrophy) and Becker muscular dystrophy (BMD: Becker Muscular Dystrophy) are typical disease types. DMD is the most frequent genetic muscular disease, affecting one out of every 3,500 male births.
[0003]
Patients with muscular dystrophy often exhibit muscle weakness during early childhood, followed by muscular atrophy that consistently progresses to death around the age of 20. In 1987, the dystrophin gene, which is the causative gene of DMD, was discovered by retrograde genetics, and it was revealed that BMB also develops from the same dystrophin gene abnormality [Koenig, M .; et al. , Cell, 50: 509-517 (1987)].
[0004]
Fundamental treatments for muscular dystrophy have not been found yet, and rehabilitation and respiratory management for delaying the progression of symptoms are considered to be important. At present, there is no generally accepted drug therapy for muscular dystrophy. For this reason, it is presently desired to provide an effective and safe therapeutic agent for muscular dystrophy.
[0005]
To develop therapeutics, the first step after the in vitro test is to test the mammal in vivo. In this therapeutic drug study in vivo, a disease state model is necessary, and it is important how the therapeutic drug is administered to the model mammal as a reference.
[0006]
[Non-patent document 1]
Koenig, M .; et al. , Cell, 50: 509-517 (1987).
[0007]
[Problems to be solved by the invention]
The problem to be solved by the present invention can be used in place of a conventional muscular dystrophy model mammal (Hoffmann EP et al. Cell. 51: 919-28, 1987) obtained spontaneously. Another object of the present invention is to provide a muscular dystrophy model mammal capable of being artificially produced, and a method for producing the same.
[0008]
[Means for Solving the Problems]
The present inventors have been studying defensin, which is a protein component of the host defense system of animals, and have been examining the effect of defensin on mammals.Surprisingly, by introducing the defensin gene, It has been found that a mammal overexpressing skeletal muscle has atrophy of muscles and shows a mode as a muscular dystrophy disease state model, thereby completing the present invention.
[0009]
That is, according to the present invention, a pathological model mammal of muscular dystrophy, which is obtained by overexpressing β-defensin, a progeny thereof, or a part thereof is provided.
Preferably, the pathological model mammal of the present invention is a transgenic mammal into which the β-defensin gene has been introduced.
Preferably, the introduced β-defensin gene is overexpressed in muscle.
[0010]
Preferably, β-defensin is β-defensin-6.
Preferably, the mammal is a rodent.
Preferably, the rodent animal is a mouse.
[0011]
According to another aspect of the present invention, there is provided a method for producing a pathological model mammal of muscular dystrophy, which comprises introducing a β-defensin gene into a mammalian egg cell or stem cell and generating an animal individual using the cell. Provided.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
The pathological model mammal of muscular dystrophy of the present invention is a model mammal overexpressing β-defensin, and more specifically, a transgenic mammal into which the β-defensin gene has been introduced.
[0013]
(1) β-defensin
Defensins are protein components of the host defense system of animals. They have been found in animals in cells involved in destroying invading microorganisms and parasites. A typical mammalian defensin is an amphipathic molecule having a positively charged hydrophilic (basic) domain and a hydrophobic domain with a conserved pattern of six cysteine residues with about 29-40 amino acids. Yes, it acts as the primary defense system on mucosal surfaces. Defensin inhibits bacterial metabolism by binding the hydrophilic domain to negatively charged bacterial membrane surface molecules and then inserting the hydrophobic domain into the bacterial membrane, increasing the permeability of the bacterial membrane and thereby inhibiting bacterial metabolism. Demonstrate.
[0014]
The term defensin as used in the present invention is not limited to the above proteins, and includes proteins isolated from animal cells or synthetically produced, and substantially retains the cytotoxic activity of the parent protein. , Also includes variants in which the sequence has been altered by the insertion or substitution of one or more amino acids.
[0015]
The present inventors have reported in J. Am. Boc. Chem. Vol. 276, No. 34, 31510-31514, 2001, β-defensin-6 (partly expressed in the esophagus, trachea, and skeletal muscle) is partially similar to the amino acid sequence of β-defensin-3 or β-defensin-4 ( The nucleotide sequence is described in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence is described in SEQ ID NO: 2 in the sequence listing). In the present invention, it is particularly preferable to use β-defensin-6 as the defensin.
[0016]
(2) mammal
The mammal that can be used as a pathological model of muscular dystrophy in the present invention is a mammal other than a human, and includes, for example, cows, monkeys, pigs, dogs, cats, guinea pigs, rabbits, rats, mice, and the like. it can. Among these mammals, rodents such as guinea pigs, rats, and mice are preferable because of their easy handling, and mice are particularly preferable.
[0017]
(3) Production of transgenic mammals
The transgenic mammal of the present invention is preferably used at the stage of embryo development in the development of a non-human mammal (more preferably, a single cell, preferably a non-fertilized egg, a fertilized egg, a germ cell containing sperm and its progenitor cells, and the like). Or at the stage of a fertilized egg cell and generally before the 8-cell stage), a β-defensin gene which is a transgene by a calcium phosphate method, an electric pulse method, a lipofection method, an agglutination method, a microinjection method, a particle gun method, a DEAE-dextran method or the like Can be produced by introducing Further, by the gene transfer method, the desired β-defensin gene can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. Transgenic animals can also be prepared by fusing the cells with germ cells by a cell mixing method known per se.
[0018]
When introducing the β-defensin gene into a target animal, it is preferable to introduce the gene as a gene construct linked downstream of a promoter capable of being expressed in cells of the target animal. Specifically, a vector obtained by ligating the β-defensin gene downstream of various promoters capable of expressing the β-defensin gene derived from various mammals having the desired β-defensin gene is used as the target mammal. By microinjecting into a fertilized egg (for example, a mouse fertilized egg), a transgenic mammal that highly expresses the desired β-defensin gene can be produced.
[0019]
Examples of β-defensin gene expression vectors include Escherichia coli-derived plasmids, Bacillus subtilis-derived plasmids, yeast-derived plasmids, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, and animal viruses such as vaccinia virus or baculovirus. Are used. Examples of promoters that regulate gene expression include promoters of genes derived from viruses (cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, etc.), various mammals (human, rabbit, dog, cat, guinea pig, hamster, Rat, mouse, etc.) and birds (chicken, etc.) derived genes [eg, albumin, insulin II, erythropoietin, endothelin, osteocalcin, muscle creatine kinase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen types I and II Atrial natriuretic factor, dopamine β-hydroxylase, endothelial receptor tyrosine kinase, sodium potassium adenosine 3 kinase, neurofilament light chain, metallothionein I and IIA, Metalloproteinase 1 tissue inhibitor, MHC class I antigen, smooth muscle α-actin, polypeptide chain elongation factor 1α (EF-1α), β-actin, α and β myosin heavy chains, myosin light chains 1 and 2, myelin-based protein, serum Genes such as amyloid P component, myoglobin, and renin]. The vector may have a terminator that terminates transcription of the messenger RNA of interest in the transgenic mammal. In addition, in order to further express the β-defensin gene, a splicing signal of each gene, an enhancer region, a part of an intron of a eukaryotic gene, 5 ′ upstream of the promoter region, between the promoter region and the translation region or in the translation region. Connection at the 3 'downstream is also possible if desired.
[0020]
It is preferable to ensure that the β-defensin gene is introduced at the fertilized egg cell stage so that the β-defensin gene is present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the β-defensin gene in the germinal cells of the transgenic animal produced means that the offspring of the produced animal have an excessive amount of the β-defensin gene in all of the germinal and somatic cells. The offspring of such animals that have inherited the gene have an excess of β-defensin protein in all of their germinal and somatic cells. Obtain a homozygous animal having the transgene on both homologous chromosomes, and by mating the male and female animals, all offspring stably retain the gene and confirm that the gene has an excess. It can be subcultured in a normal breeding environment.
[0021]
The fertilized egg used when transferring the exogenous β-defensin gene, which is a gene different from the endogenous gene of the transgenic subject animal, to the non-human mammal (preferably mouse or the like) or its ancestor fertilized egg is Obtained by crossing male mammals and female mammals of the same species. Although a fertilized egg can be obtained by natural mating, a method of artificially regulating the estrous cycle of a female mammal and then mating it with a male mammal is preferred. Methods for artificially regulating the estrous cycle of female mammals include, for example, follicle stimulating hormone (pregnant horse serum gonadotropin (PMSG)) first, and then luteinizing hormone (human chorionic gonadotropin (hCG)). Is preferably administered by intraperitoneal injection, for example.
[0022]
After introducing an exogenous β-defensin gene into the obtained fertilized egg by the method described above, a non-human mammal having a DNA incorporating the exogenous gene can be transplanted and implanted artificially into a female mammal. can get. Preferably, after administration of luteinizing hormone-releasing hormone (LHRH), the fertilized egg obtained in a pseudopregnant female mammal in which fertility has been induced is artificially transplanted and implanted by mating with a male mammal. The method is preferred. In the case of a mouse, a fertilized egg or an early embryo can be used as the totipotent cell into which the gene is introduced. As a method for introducing a gene into a cultured cell, microinjection of DNA is preferable in consideration of the yield rate of an individual transgenic animal and the efficiency of transferring the introduced gene to the next generation.
[0023]
The fertilized eggs into which the gene has been injected are then transplanted into the fallopian tubes of the foster parent, and the animals that have developed and born to the individual are reared in foster parents, and then a part of the body (in the case of mice, for example, the tip of the tail) DNA can be extracted from the DNA and the presence of the introduced gene can be confirmed by Southern analysis or PCR. If the individual in which the presence of the transgene is confirmed is defined as a founder, the transgene is transmitted to 50% of its offspring (F1). Furthermore, by crossing this F1 individual with a wild-type animal or another F1 animal, an individual (F2) having a transgene in one (heterozygous) or both (homozygous) diploid chromosomes can be produced. it can.
[0024]
Alternatively, a transgenic mammal highly expressing β-defensin can also be prepared by introducing the above-mentioned β-defensin gene as a transgene into ES cells. For example, the β-defensin gene is introduced into HPRT negative (lacking the hypoxanthine guanine phosphoribosyltransferase gene) ES cells (embryonic stem cell) derived from normal mouse blastcysts. ES cells in which the β-defensin gene has been integrated on the mouse endogenous gene are selected by the HAT selection method. Next, the selected ES cells are microinjected into a fertilized egg (blastocyst) obtained from another normal mouse. The blastocyst is implanted into the uterus of another normal mouse as a foster mother. Thus, a chimeric transgenic mouse is born from the foster parent mouse. By crossing the chimeric transgenic mouse with a normal mouse, a heterotransgenic mouse can be obtained. By crossing the heterotransgenic mice, homotransgenic mice can be obtained.
[0025]
The progeny of the above-described transgenic mammal of the present invention, as well as some of the transgenic mammals, are also within the scope of the present invention. Part of the transgenic mammal includes tissues, organs and cells of the mammal.
[0026]
(4) Transgenic mouse production example
Hereinafter, an example of a method for producing a transgenic mouse using a mouse as a mammal will be described more specifically.
[0027]
1. Cloning of defensin gene
To produce the pathological model mouse of the present invention, first, a defensin gene is cloned. As the cloning method, various known methods can be used. For example, cDNA is prepared from mRNA of defensin, the cDNA is incorporated into DNA of a vector such as a plasmid, and the DNA recombinant vector is incorporated into a host such as Escherichia coli to grow Escherichia coli. A DNA recombination vector can be obtained from Escherichia coli produced in large quantities, and the defensin gene can be cloned by fractionating and purifying the gene by column chromatography or electrophoresis. It is desirable to confirm that the gene having the target base sequence has been purified by DNA sequencing or the like.
[0028]
2. Screening for genes encoding defensins
A cDNA encoding the present defensin obtained from a mouse gene library, or the same cDNA obtained directly from mouse mRNA by a method such as RT-PCR is inserted into a plasmid vector or the like by ligation.
[0029]
3. vector
As a cloning vector, a bacterial plasmid capable of amplifying a specific gene in a host, a yeast plasmid derived, a bacteriophage derived, a transposon derived and a combination thereof derived vectors, for example, plasmids such as cosmids and phagemids and bacteriophage. Mention may be made of those derived from genetic elements.
[0030]
4. Host
The gene encoding the present defensin is propagated by a host cell, preferably via a vector. Introduction of the gene encoding the present defensin into host cells is described in Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. NY, 1989), by transformation or infection as described in many standard laboratory manuals.
Examples of the host cell include bacterial prokaryotic cells such as Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus, and Staphylococcus, and fungal cells such as yeast and Aspergillus.
[0031]
5. Production of transgenic mice
In order to produce a defensin overexpressing mouse, for example, the defensin gene obtained by the above method is microinjected into the cell nucleus of a mouse fertilized egg. The amount of the gene to be injected is preferably 200 to 1000 copies per fertilized egg. Separately, a pseudo-pregnant foster mother is prepared by mating with a vasectomized male mouse, and the fertilized egg is transplanted into the oviduct of the foster parent to produce a mouse.
[0032]
As a method of introducing a defensin gene, a method of infecting a fertilized egg with a retroviral vector may be employed. In addition, a mouse overexpressing defensin can be produced by the same method using ES cells instead of fertilized eggs.
[0033]
The cDNA encoding the defensin cloned by the above-described method includes CAG (FASEBJ. 2001 Feb: 15 (2): 393-402), a mouse neurofilament, a promoter such as SV40, and a poly-A or intron such as rabbit β-globin and SV40. To construct a transgene to prepare a transgene vector. Of these, the CAG promoter is preferably used.
[0034]
The transgene vector thus prepared is linearized and introduced into a fertilized egg by microinjection, infection using a retrovirus, or the like.
[0035]
After microinjection, stable integration of the transgene into the chromosome generally occurs in 10-40% of born embryos. It is preferable to confirm whether integration of the gene of interest has occurred in the selected mouse chromosome by PCR, Southern blotting, or the like. When this mouse is cross-crossed with a wild-type mouse, a heterozygous mouse can be obtained, and the defensin overexpressing mouse of the present invention can be prepared.
[0036]
As a method for confirming whether or not the defensin overexpressing mouse of the present invention has occurred, for example, RNA is isolated from the mouse obtained by the above method and examined by Northern blotting or the like. There is a method to check by the method.
[0037]
6. Properties of transgenic mice
The transgenic mouse of the present invention is obtained by the above method, and has overexpressed defensin. As the defensin overexpressed mice grew, they showed poor growth, decreased muscle strength, and curvature of the spinal column, and the serum CPK activity increased. Such findings correspond to the symptoms and findings of human muscular dystrophy. Furthermore, on muscle pathological findings, most of the muscle fibers were replaced by regenerated fibers having a central nucleus, and necrotic muscle fibers were scattered among them, indicating the characteristic of muscular dystrophy. The relationship between defensins and muscular dystrophy was first clarified by the production of the mouse of the present invention.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
[0038]
【Example】
Example 1
Using the previously cloned mouse β-defensin-6 as a template, a mouse β-defensin-6-specific 5′-end primer (ACCATGAAGATCCCATTACCTG) (SEQ ID NO: 3) and a 3′-end primer designed to maintain a kozak sequence The entire length of the open reading frame was amplified by a PCR method using TGTGCATATTCACGAAGAAG (SEQ ID NO: 4). The PCR product was inserted into a plasmid vector (pCR4-TOPO, invitrogen) by the method of TA cloning, and incorporated into Escherichia coli (TOP10 One Shot cells, invitrogen) by transformation and cultured. After preparing plasmid DNA from the grown Escherichia coli, the plasmid DNA was cut with EcoRI, and after electrophoresis, a fragment containing mouse β-defensin-6 was purified from the gel. The nucleotide sequence of the cloned DNA (identical to SEQ ID NO: 1) was confirmed by DNA sequencing.
[0039]
On the other hand, a pCAGGS vector having a cloning site for EcoRI between the chicken β-actin promoter and the rabbit β-globin polyA signal (Niwa H, Yamamura K, Miyazaki J (1991) Efficient exchange technology for high-expression-reduction technology-examination exchange-high-expression-reaction. 108: 193-199.) Was digested with EcoRI, treated with alkaline phosphatase, and the above-mentioned DNA fragment containing mouse β-defensin-6 was subcloned.
[0040]
This new expression vector was again transformed into Escherichia coli (DH5α) by transformation, the Escherichia coli was cultured, and plasmid DNA was extracted. 100 μg of the extracted DNA was cleaved with an appropriate restriction enzyme so as to remove plasmid-derived DNA, purified by gel after electrophoresis, cytomegalovirus immediate-early enhancer, chicken β-actin promoter, mouse β-defensin-6, rabbit A transgene consisting of the β-globin poly A signal was prepared.
[0041]
Superovulation was induced by intraperitoneal administration of PMS and hCG to C57BL / 6 female mice, and they were cohabited and mated with C57BL / 6 male mice. The female mouse in which mating was established was killed by dislocation of the cervical vertebra, the abdominal cavity was opened, the salivary tube was cut out, transferred to M16 medium containing hyaluronidase, and the fertilized egg was transferred to the medium by tearing off the salivary tube under a stereoscopic microscope. Fertilized eggs were collected using a glass pipette.
[0042]
Next, M16 medium containing fertilized eggs and a drop of the injected DNA (concentration: 5 ng / μl) were respectively prepared in a glass dish, and covered with paraffin oil. Approximately 0.1 μl of the DNA to be injected was aspirated into an injection pipette under an inverted microscope equipped with a micromanipulator, and the DNA was injected into the pronucleus of a fertilized egg fixed with a holding pipette.
[0043]
Pseudopregnant female mice (ICR mice) for transplanting fertilized eggs into which DNA had been injected were prepared by mating with male mice (ICR mice) from which vas deferens were removed. On the day when the DNA was injected into the fertilized egg, the pseudopregnant female mouse was anesthetized, the ovaries and fallopian tubes were pulled out from the back, and the opening of the fallopian tubes was exposed. Approximately 10 fertilized eggs were sucked using a transplant pipette under a stereoscopic microscope, and the fertilized eggs were respectively transplanted from the left and right oviduct openings. After transplantation of fertilized eggs, pseudopregnant female mice gave birth to offspring in about 20 days.
[0044]
Genomic DNA was extracted from the tail of the born mouse, and a mouse into which a foreign gene had been incorporated was selected using PCR and Southern blotting. PCR was performed using a 5′-end primer (GGTTATTGTGCTGTCCATC) (SEQ ID NO: 5) and a 3′-end primer (ATTTGTGGAGCCAGGGCATTTG) (SEQ ID NO: 6) at 35 ° C. at 94 ° C. for 30 sec, 55 ° C. for 45 sec, 72 ° C. for 1 min. The detection was performed by detecting a PCR product having a size of 380 bp.
[0045]
In Southern blotting, genomic DNA was cut with BglII, electrophoresed on a 0.8% agarose gel, and transferred to a nylon membrane. Next, a DNA fragment consisting of exon 2 of the mouse β-defensin-6 gene was obtained using a random primed DNA labeling kit (Rosche Molecular Biochemicals).³²P labeling was performed, and hybridization and washing were performed according to a protocol using Express Hyb hybridization solution (CLONTECH). However, hybridization was performed for 16 hours. For the detection, a bioimaging analyzer BAS2000 (Fuji Film) was used. In the wild type, a strong band of 1.4 kb and a weak band of about 3.3 kb are observed, whereas in the mutant type, bands are observed in addition to these bands at 4 kb, 3.5 kb, and 2.1 kb. .
[0046]
This primary transgenic mouse was intercrossed with a wild mouse, and a Western blot of a muscle extract protein of the obtained heterozygous mouse was performed. Muscle proteins were extracted using isogen (Nippon Gene) according to the protocol, and then shaken overnight at room temperature in a 5% acetic acid solution to selectively extract positively charged proteins. After the extract was neutralized with 10% aqueous ammonia and quantified, 8 μg of wild-type and mutant-type muscle extract proteins were fractionated by SDS-PAGE using tricine and transferred to a PVDF membrane. The PVDF membrane was reacted with rabbit-derived anti-mouse β-defensin-6 serum overnight at 4 ° C., followed by reaction with a peroxidase conjugate anti-rabbit IgG antibody at room temperature for 1 hour. For detection, Enhanced Chemiluminescent kit (Amersham) was used.
FIG. 1 shows the results of the Western blot. From the results of FIG. 1, overexpression of mouse β-defensin-6 was confirmed.
[0047]
The situation at 25 weeks of age of the transgenic mouse obtained by the above method is shown in FIG. As is clear from FIG. 2, the transgenic mouse of the present invention exhibited a muscular dystrophy-like disease state.
[0048]
【The invention's effect】
According to the present invention, a pathological model of muscular dystrophy, which has been obtained only by spontaneous generation, can be easily manufactured. Therefore, the effect of elucidating the pathophysiology of muscular dystrophy, or facilitating the study of therapeutic agents is obtained. Can be In addition, since the distribution pattern and the degree of skeletal muscle necrosis are different from those of the spontaneous muscular dystrophy disease model mouse (mdx mouse), versatile analysis different from that of the mdx mouse is possible. Furthermore, by showing a new mechanism of skeletal muscle necrosis by the action of endogenous humoral factors on cell membranes, it will contribute greatly to elucidation of pathophysiology and drug discovery from a completely different viewpoint from previous findings. it can.
[0049]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the results of Western blotting of muscle extract proteins and staining with anti-mouse β-defensin-6 serum. The left lane contains 8 μg of muscle-extracted protein from a wild-type mouse, and the right lane contains 8 μg of muscle-extracted protein from the transgenic mouse of the present invention. In the present transgenic mouse, the expression of 4 kD mouse β-defensin-6 can be confirmed.
FIG. 2A shows the appearance of the transgenic mouse at 25 weeks of age. Prominent scoliosis was observed (arrow), indicating weakness of paraspinal muscles. FIG. 2B shows HE staining of the lower limb muscle of the transgenic mouse of the present invention at 25 weeks of age. Most of the muscle fibers are replaced by regenerated fibers with a central nucleus, in which necrotic muscle fibers are scattered (arrows), which exactly matches the pathological findings of muscular dystrophy.

Claims (10)

A muscular dystrophy disease model mammal overexpressing β-defensin, its offspring, or a part thereof. The mammal according to claim 1, which is a transgenic mammal into which the β-defensin gene has been introduced, a progeny thereof, or a part thereof. The mammal according to claim 2, which overexpresses the introduced β-defensin gene in muscle, a progeny thereof, or a part thereof. The mammal according to any one of claims 1 to 3, wherein the β-defensin is β-defensin-6, progeny thereof, or a part thereof. The mammal according to any one of claims 1 to 4, wherein the mammal is a rodent animal, a progeny thereof, or a part thereof. The mammal according to claim 5, wherein the rodent animal is a mouse, a progeny thereof or a part thereof. A method for producing a pathological model mammal for muscular dystrophy, comprising introducing a β-defensin gene into a mammalian egg cell or stem cell and using the cell to generate an animal individual. The production method according to claim 7, wherein the β-defensin is β-defensin-6. The method according to claim 7 or 8, wherein the mammal is a rodent. The production method according to claim 9, wherein the rodent animal is a mouse.

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