JP5250810B2 - Screening for substances that enhance utrophin gene expression - Google Patents

Description

  The present invention relates to a transgenic non-human animal that expresses a reporter gene using a utrophin gene expression control mechanism and a cell derived therefrom. The present invention also relates to a screening method for drugs that affect utrophin gene expression and a screening method for drug candidates for muscle diseases or disorders using the transgenic non-human animal and cells derived therefrom.

  Muscular dystrophy that is a typical example of myopathy, especially Duchenne muscular dystrophy (DMD) due to dystrophin deficiency, is a severe genetic muscular disease that lacks dystrophin, which is responsible for the cytoskeleton of muscle cells, due to mutations on the X chromosome. . This incidence is relatively high, one in 3,500 newborn boys, and one third is due to mutations in the maternal egg cell level, so prevention by genetic counseling is not always effective. Therefore, there is a demand for the development of a treatment for this severe and fatal genetic muscle disease. However, many problems remain to be solved regarding gene therapy and stem cell transplantation treatment, such as immune response and low effectiveness. Drug therapy is also being carried out, but it has been reported that side effects are strong. Therefore, it is sufficiently meaningful to aim at the development of pharmacotherapy based on the elucidation of molecular pathology that is relatively easy to approach.

  As one of drug development targets for DMD, utrophin, which is a homologue of dystrophin deficient in DMD, has attracted attention (see Non-Patent Document 1). Utrophin is a cytoskeletal protein that is similar in structure to dystrophin, and its expression is known to increase in the muscle cell membrane during the recovery process after muscle damage, but it is normally expressed in the muscle cell membrane of mature muscle. It is expressed only at the neuromuscular junction and the muscle tendon junction. Since it is known that the developing muscle and the regenerated muscle are transiently expressed in the sarcolemma, it is suggested that overexpression of utrophin may compensate for the dystrophin-deficient state. The effect is expected. However, Professor Kay Davies of Oxford University in the UK has reported that tens of thousands of drugs have been screened, but no effective drug has been found to increase utrophin expression.

  The inventor's research group has shown that in the process of gene transfer using an adenovirus vector, expression of utrophin is accompanied by an excessive immune response, and the phenotype of muscular dystrophy in dystrophin-deficient mdx mice is alleviated. (Non-Patent Document 2). Furthermore, the enhanced expression of utrophin could be reproduced by single introduction of interleukin 6 (IL-6) (Non-patent Document 3). However, when IL-6 alone is administered, it is difficult to put it to practical use as a therapeutic agent due to the short expression enhancement period of utrophin and the strong toxicity of IL-6. The construct used in the gene transfer using this adenovirus vector is obtained by adding the avian-derived β-actin gene promoter and cytomegalovirus enhancer upstream of the lacZ gene and the poly A signal downstream. It was shown that utrophin is also expressed in the muscle cell membrane as a result of in vivo immune response to adenoviral vector introduction, not a gene introduced.

  It has been shown that the utrophin enhancer (DUE) sequence downstream of the utrophin gene plays an important role in enhancing the expression of utrophin in the process of muscle regeneration (Non-patent Document 4). There is no report that a foreign animal is produced by linking a foreign gene under the control of this DUE sequence, and that the foreign gene is expressed in that animal.

  In addition, establishment of a screening system for drugs for the treatment of muscular dystrophy is also desired. For example, although a method for screening a drug that increases utrophin expression has been developed, it is difficult to control utrophin expression in skeletal muscle and myocardium, and it has not been effective for screening drugs for muscular dystrophy treatment. (Non-patent document 5).

Jonathan M et.al.Amelioration of the dystrophic phenotype of mdx mice using a truncated utrophin transgene.Nature 384, 349-353, 1996 Yamamoto K et al. Immune response to adenovirus-delivered antigens upregulates utrophin and results in mitigation of muscle pathology in mdx mice.Hum Gene Ther 11: 669-680, 2000 Fujimori K et al. Interleukin-6 induces overexpression of the sarcolemmal utrophin in neonatal mdx skeletal muscle.Hum Gene Ther 13: 509-518, 2002 Galvagni F et al. The utrophin gene Is transcriptionally up-regulated in regenerated muscle. J Biol Chem 2002; 277: 19106-13 Takahashi J et al. The utrophin promoter A drives high expression of the transgenic LacZ gene in liver, testis, colon, submandibular gland, and small intestine.J Gene Med 7: 237-248, 2005

  Since utrophin is a homologue of dystrophin and is a muscle cytoskeletal protein, it may be possible to treat or prevent muscle diseases or disorders by enhancing utrophin expression. Then, it aims at providing the method and tool for searching the substance which affects the expression of utrophin.

  As a result of intensive studies to solve the above problems, the present inventor has introduced an expression cassette in which a utrophin enhancer (DUE) sequence downstream of the utrophin gene and a reporter gene are linked downstream of the transcriptional regulatory region into a mouse. , Confirming that the reporter gene is expressed in both skeletal muscle and myocardium, and using this mouse, the knowledge that high-throughput screening of drugs, transcription factors, etc. that are linked to enhanced utrophin expression can be performed. As a result, the present invention has been completed.

  That is, this invention is the following (1)-(11).

(1) A transgenic non-human animal, wherein a reporter gene is introduced under the control of a utrophin enhancer (DUE) downstream of a utrophin gene and a transcriptional regulatory region, and the reporter gene is expressed.
In the transgenic non-human animal, the reporter gene is expressed in a tissue or cell of an organ selected from the group consisting of brain, large intestine, small intestine, kidney, liver, testis, lung, submandibular gland, heart and muscle. Preferably, the reporter gene is expressed in a tissue or cell selected from the group consisting of skeletal muscle, cardiac muscle and smooth muscle.
Examples of the reporter gene include, but are not limited to, a gene (lacZ) encoding β-galactosidase.
The transgenic non-human animals include, for example, invertebrates including Drosophila and nematodes, fish including zebrafish and medaka, birds including chicken and quail, mice, rats, dogs, minipigs, rabbits, goats, sheep, cows Mammals including marmoset and cynomolgus monkeys.
The transgenic non-human animal is one in which an expression cassette in which a reporter gene is linked downstream of a DUE sequence of a utrophin gene and a transcriptional regulatory region is introduced. In the expression cassette, a nuclear translocation signal sequence is preferably linked upstream of the reporter gene and / or a poly A signal sequence is preferably linked downstream of the reporter gene.

(2) A cell obtained from the transgenic non-human animal described in (1) above and expressing a reporter gene.
The cell is derived from an organ selected from the group consisting of brain, large intestine, small intestine, kidney, liver, testis, lung, submandibular gland, heart and muscle. Preferred are cells derived from skeletal muscle, cardiac muscle or smooth muscle, and more preferred are myoblasts or stationary muscle satellite cells. The cells may be macrophages, fibroblasts, glial cells, adipocytes, vascular endothelial cells, and the like.

(3) A method for screening a drug that increases or decreases utrophin gene expression, comprising the following steps:
(A) contacting the cell according to (2) with a test drug,
(B) measuring the expression of a reporter gene in the cell,
(C) selecting a test drug that increases or decreases the expression of the reporter gene.

(4) A method for screening a drug that increases or decreases utrophin gene expression, comprising the following steps:
(D) a step of administering a test drug to the transgenic non-human animal according to (1) above;
(E) measuring the expression of a reporter gene in the transgenic non-human animal,
(F) selecting a test drug that increases or decreases the expression of the reporter gene.
In the said method, after performing step (a)-(c) of the method as described in previous clause (3), step (d)-(f) can be performed.

(5) A method for screening drug candidates for treating or preventing a disease or disorder associated with increased or decreased utrophin gene expression, comprising the following steps:
(A) contacting the cell according to (2) with a test drug,
(B) measuring the expression of a reporter gene in the cell,
(C) selecting a test drug that increases or decreases reporter gene expression as a drug candidate.

(6) A method for screening drug candidates for treating or preventing a disease or disorder associated with increased or decreased utrophin gene expression, comprising the following steps:
(D) a step of administering a test drug to the transgenic non-human animal according to (1) above;
(E) measuring the expression of a reporter gene in the transgenic non-human animal,
(F) selecting a test drug that increases or decreases reporter gene expression as a drug candidate.
In the said method, after performing step (a)-(c) of the method as described in previous clause (5), step (d)-(f) can be performed.
In the methods (5) and (6), examples of the disease or disorder associated with increased or decreased utrophin gene expression include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and polymyositis. Raised.

(7) A method for screening a drug candidate for treating or preventing a muscular disease or disorder including the following steps:
(A) the step of contacting cells derived from skeletal muscle, cardiac muscle or smooth muscle (preferably myoblasts) according to (2) above with a test drug;
(B) measuring the expression of a reporter gene in the cell,
(C) selecting a test drug that increases or decreases reporter gene expression as a drug candidate.

(8) A method for screening a drug candidate for treating or preventing a muscular disease or disorder, comprising the following steps:
(D) a step of administering a test drug to the transgenic non-human animal according to (1) above;
(E) measuring the expression of a reporter gene in muscle tissue or muscle cells of the transgenic non-human animal,
(F) selecting a test drug that increases or decreases reporter gene expression as a drug candidate.
In the said method, after performing step (a)-(c) of the method as described in previous clause (7), step (d)-(f) can be performed.
The method according to item (7) or (8) further includes the following steps:
( G ) administering the selected drug candidate to a non-human model animal of muscle disease or disorder;
( H ) It may include a step of determining whether or not the drug candidate affects a muscle disease or disorder in the non-human model animal.
Moreover, in the method as described in said (7) and (8), a muscular dystrophy is mentioned as a muscular disease or a muscular disorder.

(9) A plasmid comprising a sequence consisting of a nuclear translocation signal, a reporter gene and a poly A signal sequence under the control of a utrophin enhancer (DUE) downstream of the utrophin gene and a transcriptional regulatory region.
In the above plasmid, as the transcriptional regulatory region of the utrophin gene, for example, one containing the base sequence shown in SEQ ID NO: 18 can be used.

(10) A cell which is transfected with the plasmid described in (9) above and expresses a reporter gene.
The cell is a cell derived from, for example, skeletal muscle, cardiac muscle or smooth muscle. The cells may be germ cells or embryonic stem cells.

(11) A method for producing a non-human animal that expresses a reporter gene under the control of a DUE sequence of a utrophin gene and a transcriptional regulatory region, comprising the following steps:
(A) producing a transgenic non-human animal using the plasmid described in (9) above;
(B) A step of selecting a transgenic non-human animal expressing a reporter gene from the produced transgenic non-human animals.

  INDUSTRIAL APPLICABILITY According to the present invention, a transgenic non-human animal that expresses a reporter gene using the utrophin expression control mechanism and cells thereof are provided. Since the transgenic non-human animal and its cells use a utrophin expression control mechanism, it is possible to conveniently and efficiently screen for drugs that affect utrophin gene expression based on the reporter gene expression. It becomes possible. Therefore, it is possible to screen for drug candidates that can be used for the treatment or prevention of diseases or disorders related to utrophin gene expression, as well as diseases or disorders related to dystrophin deficiency that can be supplemented with utrophin (for example, muscular dystrophy). Become.

  Hereinafter, the present invention will be described in detail.

  The present invention uses a utrophin enhancer (DUE) sequence downstream of a utrophin gene to express a foreign gene in various tissues and cells (particularly muscle cells and muscle tissues) of transgenic animals by the expression control mechanism of the utrophin gene. Based on the knowledge that it can. Therefore, by measuring the expression of the reporter gene in transgenic animals and cells into which the reporter gene has been introduced under the control of the DUE sequence and the utrophin gene transcriptional regulatory region, conditions or drugs that affect the expression of the utrophin gene are determined. It becomes possible to identify.

1. Regulation of utrophin gene expression (1) Function of transcriptional regulatory region of utrophin gene Utrophin is expressed in systemic organs such as brain, large intestine, small intestine, kidney, liver, testis, lung, submandibular gland, heart and muscle. -There are two types of utrophin and B-utrophin expressed in vascular endothelium, and two of A- and B-utrophin have different N-terminal sequences (Fig. 1 (B)). In order to elucidate the utrophin gene expression regulation mechanism in skeletal muscle and myocardium, the present inventor has obtained about 12 from upstream (5.4 kb) corresponding to the transcriptional regulatory region of A-utrophin gene to just before the translation start point of exon 2. A construct containing an expression cassette (Gnls mouse expression cassette) in which .9 kb and a lacZ gene to which a nuclear translocation signal (nls) was added as a reporter gene was linked was obtained. Next, transgenic mice (Gnls mice) into which the same construct was introduced were prepared to obtain 4 strains. Analysis of reporter gene expression in the mouse revealed lacZ gene expression in tissues such as liver, testis, submandibular gland, large intestine, and small intestine, and these tissues reproduced endogenous A-utrophin expression. (FIG. 3A). In the Gnls mouse, in the cerebrum and cerebellum, the expression of lacZ gene was endogenous in some strains, but the expression of lacZ gene in the heart and muscle was the endogenous A-utrophin gene. Could not be reproduced (FIG. 3 (A); Non-Patent Document 5). In addition, when skeletal muscles were damaged by cardiotoxin (CTX) injection and muscle regeneration was induced, the expression of lacZ gene was not induced despite the increase in endogenous utrophin expression (FIG. 8). (A); Non-patent document 5). Therefore, an upstream base sequence of about 12.9 kb including the A-utrophin gene transcriptional regulatory region is sufficient for controlling the expression of utrophin in tissues such as liver and testis. It has become clear that the expression control is not sufficient in (Comparative Example 1; Non-Patent Document 5).

(2) Function of downstream utrophin enhancer (DUE) sequence In order to clarify the regulation mechanism of utrophin expression in skeletal muscle and myocardium, the present inventor added a downstream utrophin enhancer in addition to an upstream base sequence of about 12.9 kb. It was found that by using the (DUE) sequence, expression control in skeletal muscle and myocardium of transgenic mice (DUE mice) becomes possible (Example 1).

  The downstream utrophin enhancer (DUE) sequence is an enhancer present in intron 2 of the utrophin gene (FIG. 2), and has been shown to play an important role in enhancing utrophin expression during muscle regeneration. (Galvagni F et al. The utrophin gene Is transcriptionally up-regulated in regenerated muscle. J Biol Chem 2002; 277: 19106-13).

  In DUE mice, stronger expression of the lacZ gene was observed in tissues such as liver, testis, submandibular gland, large intestine, and small intestine, and the expression of endogenous A-utrophin was reproduced (FIG. 3 (B)). On the other hand, in the cerebrum, cerebellum, skeletal muscle and myocardium, the expression of the lacZ gene was recognized in a manner that reproduced the expression of endogenous A-utrophin (FIG. 3 (B)). In addition, when skeletal muscles were damaged by CTX injection and muscle regeneration was induced, the expression of lacZ gene was enhanced at both the mRNA level and the protein level. Therefore, the DUE sequence was used to control the expression of utrophin in skeletal muscles. It was found that they were deeply involved (FIG. 8B).

  Therefore, the fact that the DUE sequence is important for the control of utrophin expression in skeletal muscle and myocardium as described above is not only an important finding in elucidating the expression control mechanism of utrophin, but also utrophin. Using a transgenic animal and cells that express a reporter gene under the control of DUE and a transcriptional regulatory region, a drug that enhances utrophin expression is screened conveniently and efficiently using the expression of the reporter gene It becomes possible.

2. Transgenic non-human animal and cells thereof The transgenic non-human animal according to the present invention can be obtained by introducing a reporter gene under the control of the DUE sequence of the utrophin gene and the transcriptional regulatory region using techniques known in the art. Can be produced.

  In producing a transgenic animal, it is preferable to use a vector (nucleic acid construct) for expressing a reporter gene under the control of the DUE sequence and the transcriptional regulatory region of the utrophin gene in the animal. Such a vector (nucleic acid construct) includes an expression cassette containing a DUE sequence of the utrophin gene, a transcriptional regulatory region of the utrophin gene, and a reporter gene.

  The vector may be either linear or circular as long as the expression cassette present on the vector is introduced into the animal or animal cell and the reporter gene can be expressed in the animal or cell. For example, a known vector such as a plasmid, phagemid, virus-based vector, artificial chromosome or the like can be used. Examples of plasmid DNA include bacterial-derived plasmids (eg, pBluescript II SK +), yeast-derived plasmids, and the like, and phagemid DNA includes λ phage. Furthermore, animal virus vectors such as retroviruses, adenoviruses and vaccinia viruses, insect virus vectors such as baculoviruses, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), human artificial chromosomes (HAC) and the like are used as vectors. be able to. A vector can be appropriately selected by those skilled in the art depending on the type of animal or cell to be introduced, the method of introduction, and the like.

  The DUE sequence of the utrophin gene is known and can be isolated by methods known in the art. For example, the DUE sequence of the mouse utrophin gene consists of 129 bp present in intron 2 of the utrophin gene and has, for example, the sequence shown in SEQ ID NO: 1 (Galvagni F et al. J Biol Chem 275: 3168-3172, 2000 Galvagni F et al., J Biol Chem 277: 19106-13, 2002). The mouse DUE sequence includes a DUE sequence by PCR using primers DUE-F / DUE-R containing the sequences shown in SEQ ID NOs: 2 and 3, using a genomic DNA or genomic DNA library extracted from mouse skeletal muscle as a template. A 689 bp fragment can be amplified. In addition, human DUE sequences (Galvagni F et al., J Biol Chem 277: 19106-13, 2002; SEQ ID NO: 14) have also been reported, rat DUE sequences (SEQ ID NO: 15), canine DUE sequences (SEQ ID NO: 16) Can be identified by homology search or the like based on DUE sequences of other species (eg, human DUE sequences). The homology search is known in the art, and can be performed using, for example, UCSC BLAT SEARCH (http://genome.ucsc.edu/cgi-bin/hgBlat).

  Also, DUE sequences are highly conserved among animals other than mice, such as humans, mice, rats, and dogs. Therefore, even when a DUE sequence other than a mouse is used, it is suggested that the same result as that obtained when a mouse DUE sequence is used can be obtained.

The transcriptional regulatory region of the utrophin gene is a region that exists in the upstream sequence of the transcription start point of the utrophin gene and includes a promoter sequence and the like. The transcriptional regulatory region of the utrophin gene is the mouse utrophin gene (chr 10: 12282993-12743312, NM 011682), human utrophin gene (chr 6: 144654658-145209657, NM 007124), rat utrophin gene (chr 1: 7099839-7614980, NM 013070), upstream sequence of the canine utrophin gene (chr 1: 38951584-38969015, AY583531). The transcription regulatory region can be identified by those skilled in the art. For example, the transcriptional regulatory region of the utrophin gene in mice is thought to be chr 10: 12281693-12282992 (Carina L. Dennis et al. Nucleic Acids Research 24: 1646-52, 1996).

  The transcriptional regulatory region may further include an exon and / or an intron of the utrophin gene in the upstream sequence of the utrophin gene. For example, the transcriptional regulatory region of the mouse utrophin gene includes the 5 ′ terminal side (5,385 bp) including the promoter, exon 1A (408 bp), intron 1 (7,016 bp), and the first untranslated region (59 bp) of exon 2. Including the approximately 12.9 kb sequence (SEQ ID NO: 18) can be used. The sequence of the transcriptional regulatory region of the mouse utrophin gene shown in SEQ ID NO: 18 should be found in the sequence registered as Ensemble Transcript ID: ENSMUST00000076817 in EnsEmbl (http://www.ensembl.org/index.html). Can do. In SEQ ID NO: 18, the 4690th to 4699th bases (gaaaa) are NFAT binding sites, the 4946th to 4951th bases (ttccgg) are N-box sequences, the 5038-5043rd bases (caggtg) are E-box sequences, 5188 The -5193th base corresponds to Sp1, the 5386-5793th base corresponds to exon 1A, and the 12810-12868th base corresponds to the 5'UTR region of exon 2. As long as the transcriptional regulatory region maintains transcriptional regulatory activity (promoter activity), one or more bases may be deleted, substituted, or added in the base sequence shown in SEQ ID NO: 18.

  A reporter gene refers to a gene generally used for determining the function of a transcriptional regulatory region (for example, the transcription activity of a promoter or enhancer), and the presence or absence of the expression or the expression level can be easily detected. The reporter gene that can be used in the present invention is not particularly limited. For example, genes encoding fluorescent proteins, enzymes, cell surface antigens and the like are included. Specifically, the reporter gene includes, but is not limited to, a lacZ gene encoding β-galactosidase, a gfp gene encoding green fluorescent protein (GFP), a luc gene encoding luciferase, and chloramphenicol acetyl. Examples thereof include a cat gene encoding transferase.

  In order to facilitate identification of the nucleus in which the reporter gene is expressed, it is preferable to add a nuclear translocation signal to the reporter gene that utilizes the utrophin expression control mechanism. A nuclear translocation signal is a sequence required for a protein synthesized in the cytoplasm to be transported into the nucleus, and is known in the art. In the present invention, for example, a nuclear translocation signal (nls) derived from the large T antigen of SV40 virus can be used (SEQ ID NO: 17; Kalderon D et al. Cell 39: 499-509, 1984).

  Furthermore, it is preferable to link a poly A signal sequence downstream of the reporter gene so that a poly A chain is added at the time of transcription of the reporter gene after introduction and mRNA is stabilized. Poly A signal sequences are also known in the art and any sequence can be used. For example, a poly A signal sequence derived from a rabbit β globin gene, SV40, or a bovine growth hormone gene can be used.

  In addition to the above sequences, a splicing signal, a selection marker, a ribosome binding sequence (SD sequence) and the like are preferably linked to the vector. Examples of the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.

  In order to link a target sequence to a vector, first, a method in which the purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of the vector, and linked to the vector is employed. Further, after all the target sequences are linked to the vector, a region containing the target sequence can be cut out from the vector and used.

  The target animal is not particularly limited as long as it is an animal. For example, invertebrates (such as Drosophila and nematodes), fish (such as zebrafish and medaka), birds (such as chicken and quail), mammals (such as mice, Rat, dog, minipig, rabbit, goat, sheep, cow, marmoset, cynomolgus monkey, etc.). In the present invention, mammals, particularly mice and rats that are easy to handle in experiments are preferred.

  In order to introduce a vector into an animal, it is preferable to inject the fertilized egg (germ cell) of the target animal. As a result, an expression cassette linked to a reporter gene under the control of the utrophin DUE and the transcriptional regulatory region is incorporated into a fertilized egg cell, and the expression cassette is copied to cells throughout the body with the subsequent division of the fertilized egg. Is done.

  As a method for introducing a vector into animal cells, in addition to a method of directly introducing into a fertilized egg, a method of introducing into an embryonic stem (ES) cell, a method of introducing a cell nucleus introduced into a cultured cell into a fertilized egg by nuclear transfer (Nuclear transplantation method). In the method of direct introduction into a fertilized egg, vector DNA can be introduced into the fertilized egg using microinjection, viral infection, or the like. Moreover, after introducing the DNA of a vector into ES cells or other cultured cells by electroporation, lipofection, etc., and selecting using a selectable marker, the desired introduced cells can be obtained. When vector DNA is introduced into ES cells, it is injected into embryonic placental vesicles or 8-cell stage embryos using a capillary tube or the like. The nuclear transfer method is performed by introducing a vector DNA into an arbitrary somatic cell, injecting the cell into which the vector DNA has been introduced into an enucleated fertilized egg, and cell fusion by electrical stimulation. When a fertilized egg is used, it may be developed into an embryo placental cyst or an 8-cell stage embryo. In addition, embryo placental blastocyst or 8-cell stage embryo may be further cultured for one day to develop to blastocyst.

  Thereafter, the fertilized egg or embryo placental cyst or 8-cell stage embryo is directly transferred into the foster tube of the foster parent, or the blastocyst is transferred to the foster parent's uterus. A foster parent is raised and born to obtain a transgenic animal or a chimeric animal that is a pup into which the target expression cassette has been introduced. In order to confirm the introduction of a desired reporter gene in the animal, a part of the animal body (for example, the tip of the tail) is cut, DNA in the somatic cell is extracted, and PCR or Southern blotting is used. Confirm the presence of the introduced sequence.

  In the case of a transgenic animal, if an individual in which the introduced sequence is confirmed to be the first generation, the expression cassette is transmitted to 50% of the offspring (F1). That is, a heterozygous animal (F1) can be obtained by mating the transgenic animal with a normal animal, and a homozygous animal (F2) can be obtained by mating the heterozygotes.

  In the case of a chimeric animal obtained using ES cells, by mating the chimeric animals, some of the offspring are heterozygotes having the target expression cassette on one chromosome. Therefore, by mating these heterozygous animals, homozygotes, that is, transgenic animals can be obtained.

  As long as the reporter gene is introduced, the transgenic animal may be a puppy obtained by mating transgenic animals, or a puppy obtained by mating a transgenic animal with another animal. For example, a pup (DUE / mdx) obtained by mating a transgenic mouse (DUE mouse) into which a reporter gene is introduced under the control of a DUE sequence and a transcriptional regulatory region, and a known muscular dystrophy model mouse (mdx mouse). Mouse). Such DUE / mdx mice are useful in examining the mechanism of utrophin expression regulation under dystrophin deficiency.

  In order to confirm the expression of the introduced reporter gene in the transgenic animal, RT-PCR method or Northern blot method using RNA derived from the cells of the transgenic animal, or detecting the expression of the reporter gene. Any possible method can be used. In the transgenic animal produced by the above method, the reporter gene will be expressed in tissues or cells of organs such as brain, large intestine, small intestine, kidney, liver, testis, lung, submandibular gland, heart and muscle. . In particular, in the transgenic animal according to the present invention, the reporter gene is expressed in a tissue or cell selected from skeletal muscle, cardiac muscle and smooth muscle.

  Thus, a transgenic animal that expresses a reporter gene by the utrophin expression control mechanism is used for screening for drugs that affect utrophin gene expression as described below, and for treating or preventing muscle diseases or disorders. It is useful as a tool for screening drug candidates.

  In addition, cells expressing a reporter gene can be established from the transgenic non-human animal produced as described above under the control of the utrophin DUE and the transcriptional regulatory region. Establishment of cells from the transgenic animal can be performed according to a method known in the art.

  Alternatively, an animal cell is transfected with an expression vector (plasmid) containing the expression cassette containing the DUE sequence of the utrophin gene, the transcriptional regulatory region of the utrophin gene, and the reporter gene, and the expression of the reporter gene in the cell is confirmed. Can establish cells that express the reporter gene under the control of the utrophin DUE and the transcriptional regulatory region. The expression vector (plasmid) to be used preferably contains a nuclear translocation signal upstream of the reporter gene and a poly A signal sequence downstream of the reporter gene. Transfection of an expression vector into cells can be performed using any method known in the art (for example, electroporation, lipofection, etc.).

  The cell to be established is preferably a cell in which the gene is expressed by the utrophin gene expression control mechanism, for example, brain, large intestine, small intestine, kidney, liver, testis, lung, submandibular gland, heart and muscle. Cells derived from an organ selected from the group consisting of: For example, there are macrophages, fibroblasts, glial cells, fat cells, vascular endothelial cells and the like. More preferably, it is a cell derived from skeletal muscle, cardiac muscle or smooth muscle, and specifically, a myoblast is established.

  For example, myoblasts can be obtained by amending the pre-plating method (TA Rando et al. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. JCB 1994; 125: 1275-87). Only purified and established as a primary culture.

The method for culturing the established cells is performed according to a usual method used for culturing animal cells. For example, a medium in which 20% fetal bovine serum, 0.005% bFGF, 1% penicillin / streptomycin is added to F10 medium is used. The culture is usually performed at about 37 ° C. in the presence of 5% CO ₂ .

  In addition, in order to confirm the introduction and expression of the reporter gene in the established cells, the RT-PCR method or Northern blot method using RNA or the expression of the reporter gene as described herein may be detected. Any possible method can be used.

  By establishing a cell system in this way, it becomes possible to proliferate cells in large quantities and to screen for test drugs and the like under the same conditions.

3. Screening Method The present invention provides a method for screening a drug associated with increased or decreased utrophin expression using transgenic animals and cells that express a reporter gene by a utrophin expression control mechanism.

  That is, by administering a test drug to the transgenic animal according to the present invention or bringing a cell into contact with the test drug and measuring the expression of the reporter gene in the animal or cell, the test drug becomes an utrophin gene expression control mechanism. It can be determined whether or not it has an effect.

  In the method for screening a drug related to the expression increase or decrease of utrophin according to the present invention, first, a test drug is administered to a transgenic animal or cells are brought into contact with the test drug.

  The kind of the test drug to be subjected to the screening method of the present invention is not particularly limited. For example, the test drug can be any material factor, specifically a naturally occurring molecule such as an amino acid, peptide, oligopeptide, polypeptide, protein, nucleic acid, lipid, carbohydrate (such as sugar), steroid, glycopeptide Synthetic analogs or derivatives of naturally occurring molecules, such as peptidomimetics, nucleic acid molecules (aptamers, antisense nucleic acids, double-stranded RNA (RNAi), etc.), etc .; non-naturally occurring molecules, such as And low molecular organic compounds (inorganic and organic compound libraries, combinatorial libraries, etc.) prepared using combinatorial chemistry techniques, and the like, and mixtures thereof. The test drug may be a single substance, a complex composed of a plurality of substances, a transcription factor, or the like. Furthermore, the test drug may be an environmental factor (radiation, ultraviolet light, etc.) in addition to the above-described physical factors.

  In addition, as a test drug, a single test drug may be tested independently, or a mixture of several candidate test drugs (including a library and the like) may be tested. Examples of the library containing a plurality of test drugs include a synthetic compound library (such as a combinatorial library) and a peptide library (such as a combinatorial library).

  When the cells are brought into contact with the test drug, the contact conditions vary depending on the type of the drug and the type of cell, but can be easily determined by those skilled in the art. For example, such contact is performed by culturing the cells in a medium containing the test drug, immersing the cells in a solution containing the test drug, or laminating the test drug on the cells. Can do. In addition, when a test drug is administered to an animal, administration conditions such as the dose, administration period, and administration route vary depending on the type of the test drug, but can be easily determined by those skilled in the art. For example, when administering a test drug to a transgenic animal, the route of administration is intramuscular injection, oral administration, intravenous injection, intraperitoneal injection, transdermal administration, depending on the type of test drug, the type of animal used, etc. Administration forms such as subcutaneous injection can be used as appropriate.

  In addition, the effect of the test drug can be examined under several conditions. Such conditions include the time or period of contact with or administration of the test drug, the amount (large or small), the number of times, and the like. For example, a plurality of doses can be set by preparing a dilution series of a test drug. The contact or administration period of the test drug can also be appropriately set. For example, it can be contacted or administered over a period of 1 day to several weeks.

  Furthermore, when examining the additive action and synergistic action of a plurality of drugs, test drugs may be used in combination.

  Subsequently, the expression of the reporter gene in the transgenic animal or cell is measured. The reporter gene can be measured by any method known in the art depending on the type of reporter gene used and the type of animal tissue or cell to be measured (for example, “Molecular Biology Experiment Protocol II”). "See Saigo and Sano, 1997, Maruzen Co., Ltd.).

  For example, when a gene encoding an enzyme protein (eg, a lacZ gene encoding β-galactosidase, a cat gene encoding chloramphenicol acetyltransferase, etc.) is used as a reporter gene, the expression of the reporter gene is expressed by the enzyme. Detection can be based on activity. For example, if a substance that can be detected by an enzyme is converted into an undetectable substance, or vice versa, the presence of such a substance is detected. By doing so, the expression of the reporter gene can be detected. For example, the expression of the lacZ gene is performed by reacting 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) as a substrate and detecting a blue insoluble substance generated by hydrolysis. Can be determined. The expression of the cat gene can be detected by separating and identifying acetylated chloramphenicol generated by the reaction of acetyl CoA and chloramphenicol, for example, by thin layer chromatography.

  Further, for example, when a gene encoding a fluorescent protein (eg, gfp gene encoding green fluorescent protein, luc gene encoding luciferase, etc.) is used as a reporter gene, the expression of the reporter gene is detected based on the fluorescence. be able to. For example, cells expressing a reporter gene can be quickly determined using a sorting method (such as FACS) using a flow cytometer.

  Alternatively, it is possible to detect the expression of the reporter gene using a reaction using an antibody against the protein encoded by the reporter gene, or using a reaction using a primer or probe for mRNA of the reporter gene. .

  After measuring the reporter gene expression, a test drug that increases or decreases the reporter gene expression is selected as compared to the control reporter gene expression. As a control, an animal or cell into which a reporter gene has not been introduced, an animal into which a reporter gene has been introduced but which has not been administered a test drug, or a cell that has not been in contact with the test drug can be used.

  In the present invention, first, a cell is brought into contact with a test drug, and the test drug showing enhanced or reduced expression is selected by measuring the expression of a reporter gene in the cell, and then selected as a transgenic animal. The test drug may be selected by administering the test drug and measuring the expression of the reporter gene in the transgenic animal.

  As a result, a test drug that increases the expression of the reporter gene is identified as a drug that increases the expression of the utrophin gene. In addition, the test drug that reduces the expression of the reporter gene is identified as a drug that reduces the expression of the utrophin gene.

  In another embodiment, the utrophin gene expression is increased by administering a test drug to the transgenic animal according to the present invention or contacting a cell with the test drug and measuring the expression of the reporter gene in the animal or the cell. Alternatively, drug candidates for treating or preventing a disease or disorder associated with reduced expression can be identified. Alternatively, the test drug is administered to the transgenic animal according to the present invention, or cells derived from the skeletal muscle, cardiac muscle or smooth muscle are contacted with the test drug, and the muscle tissue or muscle cell of the animal, or a reporter in the cell By measuring gene expression, drug candidates for treating or preventing muscle diseases or disorders can be identified.

  In the drug candidate screening method according to the present invention, first, a test drug is administered to a transgenic animal, or cells derived from skeletal muscle, cardiac muscle or smooth muscle are brought into contact with the test drug. Cells derived from skeletal muscle, cardiac muscle or smooth muscle can be prepared by methods known in the art. As such cells, for example, myoblasts are preferably used.

  The kind of drug (test drug) to be subjected to the screening method of the present invention is not particularly limited, and for example, the drugs described above can be used. In addition, administration of the test drug to the animal and contact between the cells and the test drug can be appropriately selected as described above.

  In one embodiment, the disease or disorder to be treated or prevented by a drug candidate is a disease or disorder associated with increased or decreased utrophin gene expression. For example, diseases or disorders associated with increased utrophin gene expression include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and polymyositis. On the other hand, there is no known disease or disorder related to utrophin gene expression reduction, but a disease or disorder to be screened may be found as research proceeds in the future.

  In another embodiment, the disease or disorder to be treated or prevented by the drug candidate is a muscle disease or disorder. Examples of the muscular disease or disorder include diseases or disorders caused by muscle degeneration, such as muscular dystrophy, specifically Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

  Measurement of reporter gene expression in cells or transgenic animals can be performed as described above. In the measurement of reporter gene expression in animals, the tissue whose expression is to be measured varies depending on the purpose of screening for a drug candidate for a disease or disorder associated with utrophin gene expression in the tissue. For example, when screening a drug candidate for a disease or disorder associated with utrophin expression in an organ such as the kidney or liver, the expression of a reporter gene in the organ is measured. In addition, when screening for drug candidates for treating or preventing muscular diseases or disorders by supplementing dystrophin deficiency with utrophin, reporter gene expression in muscle tissue or muscle cells, such as skeletal muscle, cardiac muscle or smooth muscle, is examined. taking measurement.

  After measuring the reporter gene expression, a test drug that increases or decreases the reporter gene expression is selected as compared to the control reporter gene expression. As a control, an animal or cell into which a reporter gene has not been introduced, an animal into which a reporter gene has been introduced but which has not been administered a test drug, or a cell that has not been in contact with the test drug can be used.

  As a result, a test drug that increases or decreases reporter gene expression can be selected as a drug candidate. For example, in the case of drug candidate screening aiming to compensate for dystrophin deficiency with utrophin, a test drug that increases reporter gene expression is selected as the drug candidate.

  In addition, screening for drug candidates is performed as a primary screening, in which cells are brought into contact with a test drug, and the test drug that shows an increase or decrease in expression is selected by measuring the expression of a reporter gene in the cell, and then as a secondary screening. The selected test drug may be selected as a drug candidate by administering the selected test drug to the transgenic animal and measuring the expression of the reporter gene in the transgenic animal to show expression enhancement or reduction.

Further, in the drug candidate screening, the selected drug candidate is further administered to a disease or disorder model animal to determine whether the drug candidate affects the disease or disorder in the model animal. Also good. The model animal is not particularly limited as long as it has a target disease or disorder factor (such as a genetic predisposition) or exhibits at least one symptom. For example, muscular dystrophy model mice (mdx mice: Sicinski, P. et al., Science 244: 1578-1580, 1989), muscular dystrophy model dogs (c-xmd dogs: Komegay, JN et al., Muscle Nerve 11: 1056-1064, 1988; CXMD _J dog: Shimatsu Y et al., Exp Anim. 52: 93-7. 2003, Shimatsu Y et al., Acta Myol. 24: 145-54 2005) can be used.

  The determination of whether a drug candidate affects a disease or disorder in a model animal depends on the type, factor, symptom, etc. of the model animal's disease or disorder, but those skilled in the art will have an effect on the disease or disorder. Can be appropriately determined. For example, to determine the effect on muscle disease or disorder, measure muscle strength, serum creatine kinase level, isolated skeletal muscle tension, histological maximum muscle diameter and central core fiber frequency, In addition, immunohistological staining using a utrophin antibody can be performed.

  As described above, a drug candidate when improvement of a disease or disorder is observed can be selected as a drug candidate for treatment or prevention of the disease or disorder.

  From the above, by using the transgenic animal or cell according to the present invention, a drug for treating or preventing a disease or disorder associated with increased or decreased utrophin expression, or treating or preventing a muscular disease or disorder. Can be found, and further, the effect of the medicine or treatment method can be confirmed.

  Hereinafter, the present embodiment will be described in more detail with reference to examples. The present invention is not limited to these.

[Comparative Example 1]
Transgenic mice (Gnls) were generated using an expression cassette that did not contain a downstream utrophin enhancer (DUE).

(1) Production of Gnls Mouse A genomic sequence (about 12.9 kb) containing the 5 ′ end of mouse A-utrophin was obtained from a 129Sv mouse gene library. This clone of about 12.9 kb contains the mouse A-utrophin gene (chr10: 122282993 (NM 01682)) containing the promoter of 5 'end (5,385 bp), exon 1A (408 bp), intron 1 (7,016 bp), including the first untranslated region (59 bp) of exon 2 (SEQ ID NO: 18; FIG. 2 (corresponding to “5 ′ terminal sequence of utrophin gene”). This fragment (about 12.9 kb) was linked to a nuclear translocation signal (nls) and a lacZ gene as a reporter gene, and a rabbit β globin polyA was attached to the 3 ′ end to express a transgene expression cassette (expression for Gnls mouse in FIG. 2). Cassette). The DNA fragment containing the expression cassette was separated and purified from an agarose gel using Gel Extraction Kit (QIAGEN). This DNA fragment was injected into fertilized eggs of C57BL / 6J mice by microinjection and transplanted into the oviduct of pseudopregnant mice to obtain offspring mice.

Genomic DNA was extracted from the tails of pups and subjected to Southern blotting. Specifically, genomic DNA was digested with proteinase K (0.15 mg / ml) and pronase E (1 mg / ml) and digested with lysis buffer (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 20 mM EDTA, 1% SDS). ). Subsequently, genomic DNA (10 μg) was digested with BamHI, separated on a 0.8% agarose gel and transferred to Hybond-N ⁺ membrane (Amersham Biosciences). A 3072 bp DNA fragment of the lacZ gene was labeled with ³² P-dCTP as a Southern probe and hybridized with the membrane at 65 ° C. overnight. Subsequently, the membrane was washed to 65 ° C. by washing with 2 × SSPE, 0.1% SDS, 1 × SSPE, 0.1% SDS, and 0.1 × SSPE, 0.1% SDS. The sample was thoroughly washed and analyzed by BAS 2500 (Fuji Film). As a result, the selected mice expressing LacZ Transgenic _{F 0} mice (Gnls mice).

(2) Expression of β-galactosidase In order to confirm the expression of β-galactosidase in Gnls mice, reverse transcriptase-polymerase amplification reaction (RT-PCR), histological analysis and immunohistological analysis were performed.

First, 3 weeks old transgenic mice were euthanized, and each tissue (cerebrum, cerebellum, skeletal muscle, myocardium, small intestine, large intestine, testis, liver, submandibular gland, kidney, lung, diaphragm) was collected and liquid nitrogen was collected. Frozen at. From these frozen tissues, total RNA was isolated using TRIsol reagent (Invitrogen Life Technologies). This total RNA was treated with 1 U of RNase-free DNase I (Invitrogen Life Technologies) per 1 μg of RNA at room temperature for 15 minutes, and 25 mM EDTA (pH 8.0) was added to inactivate DNase I. And heated for 10 minutes. Using 3.5 μg of this total RNA, a reverse transcription reaction was performed by SuperScriptII ^™ (Invitrogen Life Technologies). 1 minute denaturation at 95 ° C. using utrophin exon 1A sense primer (5′-GGCGTTTCCAATCGGGTGTC-3 ′; SEQ ID NO: 10) and lacZ gene antisense primer (5′-CGTTCTGCCCATCATCAGGC-3 ′; SEQ ID NO: 11) PCR was performed with 35 cycles of annealing at 59 ° C. for 30 seconds and extension at 72 ° C. for 1 minute. As a control, the expression level of 18S rRNA, a housekeeping gene, was also examined.

  The results are shown in FIG. In this Gnls mouse, expression of the lacZ gene was observed in tissues such as the liver and testis, and the expression reproduced the expression of endogenous A-utrophin, but the expression in the heart and muscle was that of endogenous A-utrophin. The expression could not be reproduced (Takahashi J et al. The utrophin promoter A drives high expression of the transgenic LacZ gene in liver, testis, colon, submandibular gland, and small intestine. J Gene Med 2005; 7: 237- 248). In addition, histochemical analysis using hematoxylin and eosin (H & E) staining and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; Wako Chemicals), antibodies against utrophin Similar results were obtained with the immunohistochemical analysis used (data not shown).

In this example, a transgenic mouse (DUE mouse) was prepared using an expression cassette containing DUE.
(1) Preparation of DUE mouse Enhancer 129 bp (downstream utrophin enhancer: DUE) present in mouse utrophin gene intron 2 (Galvagni F et al. The utrophin gene is transcriptionally up-regulated in regenerated muscle. J Biol Chem 2002; 277: 19106-13) was inserted upstream of the Gnls mouse expression cassette described in Comparative Example 1 to construct a new expression cassette (DUE mouse expression cassette in FIG. 2). . Using this expression cassette, a transgenic mouse (DUE mouse) was prepared.

Briefly, genomic DNA is extracted from mouse skeletal muscle, and the DUE sequence is amplified by the PCR method using primers DUE-F / DUE-R (Table 1, SEQ ID NOs: 2 and 3), followed by electrophoresis. A 689 bp band containing the DUE sequence was confirmed. The amplified 689 bp PCR product was purified by Gel Extraction Kit and inserted into the vector pCR2.1 (3.9 kb, manufactured by Invitrogen) (FIG. 4 (A)). In order to amplify a 219 bp fragment containing DUE in the same clone, a primer (DUE-F-NotI) (Table 1, SEQ ID NO: 4) added with restriction enzyme NotI on the 5 ′ side and restriction enzyme EagI on the 5 ′ side. A primer (DUE-R-EagI) (Table 1, SEQ ID NO: 5) was prepared, subjected to PCR using LA Taq polymerase, and the sequence of the restriction enzyme site added to the 219 bp sequence by electrophoresis. A 232 bp band was confirmed. The 232 bp PCR product was purified by Gel Extraction Kit, inserted again into vector pCR2.1 (3.9 kb), and transformed into competent cells (TOYOBO). White colonies were picked up by blue-white selection, cultured using ampicillin-containing Luria-Bertani medium (LB medium), and the plasmid was purified by the miniprep method. The plasmid was cleaved with the restriction enzyme EcoRI, colonies in which a 232 bp fragment was incorporated were selected by electrophoresis, cultured in ampicillin-containing LB medium, and purified by the maxi-prep method. After cutting this plasmid with the restriction enzyme EagI, a linear DNA band containing the DUE sequence was confirmed by electrophoresis, then cleaved with the restriction enzyme NotI and electrophoresed to obtain a 219 bp DNA fragment containing the DUE sequence from the gel. It refine | purified by Gel Extraction Kit (FIG. 4 (B)).

  Next, in order to create a DUE mouse by inserting the DUE sequence upstream of the Gnls mouse expression cassette, a plasmid (Gnls mouse expression cassette) in which the Gnls transgene expression cassette is incorporated into pBluescriptIISK + is cut with NotI. After treatment with calf intestinal alkaline phosphatase (CIAP), a fragment containing the DUE sequence was inserted (FIG. 4C). A plasmid into which a fragment containing this DUE sequence was inserted was transduced into competent cells (TOYOBO). A colony of E. coli was picked up and cultured in ampicillin-containing LB medium, the plasmid was purified by the miniprep method, and 235 bp PCR using T3 (20mer) primer and DUE-inR primer (Table 1, SEQ ID NOs: 6 and 7). Based on the presence or absence of the product, a plasmid incorporating the DUE sequence was selected (FIG. 4D).

Subsequently, the plasmid in which the fragment containing the DUE sequence is inserted is cultured in ampicillin-containing LB medium, the plasmid is purified by the maxi-prep method, and the nucleotide sequence is confirmed, followed by restriction enzymes NotI and XhoI for Gnls mice. A fragment (DUE mouse expression cassette in FIG. 2) having a DUE sequence added to the 5 ′ side of the expression cassette was cleaved from pBluescriptIISK + (FIG. 4 (E)). This fragment was purified by Gel Extraction Kit, and injected into C57BL / 6J mice fertilized eggs by microinjection, to obtain a 2 mice _{F 0} mice (DUE mice). Were crossed with F ₀ mouse and C57BL / 6J mice was obtained pups. Genomic DNA was extracted from the tail of a pup, and a transgene positive mouse was selected by PCR using primers LacZ-F / LacZ-R (Table 1, SEQ ID NOs: 8 and 9). Lines were established by repeated mating with transgenic mice of the same generation or C57BL / 6J mice.

(2) Expression of β-galactosidase Using the obtained DUE mouse, expression of β-galactosidase was confirmed. First, in order to confirm the expression at the mRNA level, RT-PCR was performed as described in (2) of Comparative Example 1. The primer is lacZ sense 1 primer (5′-CGACATTGCCGTAAGTGAAG-3 ′; SEQ ID NO: 12) and lacZ antisense 1 primer (5′-ATCGCCATTTGACCACTACC-3 ′; SEQ ID NO: 13), and 5 minutes at 95 ° C. After denaturation, 30 cycles of denaturation at 95 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and extension at 72 ° C for 1 minute were performed 30 times, and then an extension reaction at 72 ° C for 7 minutes Stored at 4 ° C.

  The result is shown in FIG. In DUE mice, stronger expression of the lacZ gene was observed in tissues such as liver, testis, submandibular gland, large intestine, and small intestine, and endogenous A-utrophin was also expressed in cerebrum, cerebellum, skeletal muscle and myocardium. Expression of the lacZ gene was observed in a reproducible manner.

  In addition, tissues (cerebrum, cerebellum, submandibular gland, liver, small intestine, large intestine, testis, anterior tibial (TA) muscle, heart) were collected from DUE mice and frozen in liquid nitrogen cooled isopentane. Frozen sections (10 μm) were stained with hematoxylin and eosin (H & E), and stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; Wako Chemicals) It was.

  A frozen section (6 μm) was placed on a slide, dried, and fixed in acetone at −20 ° C. for 6 minutes. Immunohistochemical analysis was performed using a rabbit polyclonal antibody (Imamura M. et al., Proc Nal Acad Sci USA 95: 6139-6144, 1998) that recognizes amino acids 1768-2078 of human utrophin (UT-2). went. Primary antibodies were detected using goat anti-rabbit IgG (Molecular Probes) labeled with Alexa488. Nuclei were stained with iodinated TOTO-3 (Molecular Probes). The neuromuscular junction (NMJ) was stained with α-bungarotoxin (α-BTX) (Molecular Probes) labeled with Alexa594. The signal was recorded on a photograph using a laser scanning confocal imaging system (TCCSSP ™, Leica).

  These results are shown in FIGS. In FIG. 5, the symbols are V = terminal hepatic venule, Si = sinus capillaries, H = hepatocytes, ST = tubules, L = Leydig cells, S = Sertri cells, C = capillary, CM = Inner ring muscle layer, G = goblet cell, LM = outer longitudinal muscle layer, SD = striate duct, Sc = serous secretory cell, SA = serous acinar, Vi = fur, Cr = crypt, P = perineto cell, MM = muscle mucosa. In FIG. 6, symbols are CP = choroid plexus, PM = cerebral buffy coat, CC = cerebral cortex, N = neuron, ML = molecular layer, GL = granular layer, P = Purkinje cell, ID = intercalated disk , NMJ = representing the neuromuscular junction. Also from this result, in DUE mice, stronger expression of β-galactosidase was observed in tissues such as liver, testis, submandibular gland, large intestine and small intestine, and β-galactosidase was also observed in cerebrum, cerebellum, skeletal muscle and myocardium. Expression was observed.

(3) β-galactosidase expression during muscle regeneration In order to cause muscle degeneration in DUE mice, a 29 gauge needle was used for the right anterior tibial (TA) muscle of 5-week-old DUE mice. Of cardiotoxin (CTX; 10 μM in saline, Wako Chemicals). The concentration of CTX was measured according to Couetteaux R. et al. (Biol Cell 62: 171-182, 1988). DUE mice injected with CTX were euthanized 1-7 days after CTX injection. TA muscle injected with CTX and non-injected contralateral TA muscle were collected and frozen in liquid nitrogen cooled isopentane. The frozen section (10 μm) was stained with X-Gal. At the same time, frozen sections (6 μm) were stained with UT-2 and Alexa594-labeled α-BTX. In addition, total RNA was isolated from these frozen sections, and the lacZ gene was amplified using lacZ sense 1 primer (SEQ ID NO: 12) and lacZ antisense 1 primer (SEQ ID NO: 13).

  The results are shown in FIGS. From this result, it was revealed that the expression of β-galactosidase is enhanced at both the mRNA level and the protein level when skeletal muscle is damaged by injecting CTX and muscle regeneration is induced. Therefore, it was found that the DUE sequence is deeply involved in the regulation of utrophin expression in skeletal muscle.

(4) Expression of β-galactosidase by mating with mdx mice It is known that the expression of utrophin increases in the muscle cell membrane of mdx mice. In order to obtain mdx mice into which an expression cassette containing a DUE sequence was introduced, male DUE mice and female mdx homo mice were mated to obtain pup mice (male). Genomic DNA was extracted from pups that were completely deficient in dystrophin (DUE / mdx mice), and the presence of the introduced expression cassette was confirmed by PCR and Southern blotting. By using DUE / mdx mice, it is possible to examine the utrophin expression regulation mechanism under dystrophin deficiency. Two-week-old DUE / mdx mice were euthanized, and the anterior tibial muscle (TA muscle), gastrocnemius muscle (GA muscle), heart, and diaphragm were collected and frozen in liquid nitrogen-cooled isopentane. The frozen section (10 μm) was stained with X-Gal. At the same time, frozen sections (6 μm) were stained with UT-2 and Alexa594-labeled α-BTX. In addition, total RNA was isolated from these frozen sections, and the lacZ gene was amplified using lacZ sense 1 primer (SEQ ID NO: 12) and lacZ antisense 1 primer (SEQ ID NO: 13).

  As a result, β-galactosidase expression was enhanced at both mRNA and protein levels under dystrophin deficiency. In a state where dystrophin is deficient as in muscular dystrophy patients and mdx mice, endogenous utrophin is expressed not only in the neuromuscular junction and the muscle tendon junction but also in the muscle cell membrane. Therefore, it is considered that the expression of β-galactosidase mRNA and protein level introduced as a transgene also increased in DUE / mdx mice lacking dystrophin. Moreover, the expression of β-galactosidase was observed not only at the neuromuscular junction and the muscle tendon junction but also at the muscle cell membrane. That is, it can be said that the expression level and expression site of β-galactosidase were able to reproduce the trend of endogenous utrophin. Therefore, it was suggested that drug screening using reporter gene expression as an index using DUE mice is useful for developing a therapeutic method for muscular dystrophy that aims to enhance utrophin expression.

  In this example, the pre-plating method (TA Rando et al. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. JCB 1994; 125: 1275- 87), a primary culture of myoblasts was established as follows.

The skeletal muscle obtained from the DUE mouse was chopped and then stirred in a 0.2% type 2 collagenase solution at 37 ° C. for 30 minutes. Thereafter, the tissue piece was pulverized by passing several times through an 18G injection needle, and further treated with collagenase for 15 minutes. PBS containing 2% serum at 4 ° C. was added to stop the action of collagenase, passed through a 100 μm filter and then a 40 μm filter, and the filter filtrate was centrifuged at 1,500 rpm for 5 minutes. Add hypotonic solution (0.83% NH ₄ Cl and 0.17M Tris-HCl (pH 7.65) in a ratio of 9: 1) to the pelleted cells to remove erythrocytes, and lightly for 1 minute. Shake. PBS containing 2% serum was added to equilibrate, and the cells were collected again by centrifugation at 1,500 rpm for 5 minutes, and suspended in a growth medium. The suspended cells were transferred to an uncoated culture dish, cultured at 37 ° C. and 5% CO ₂ concentration for 2 hours, and then the supernatant containing unattached cells was collected and transferred to a culture dish coated with type 1 collagen. ° C., and cultured overnight at 5% CO ₂ concentration. On the next day, the supernatant containing unattached cells was collected, transferred to a culture dish coated with type 1 collagen, and cultured. The growth medium was changed every day initially and every 2-3 days thereafter (Uezumi A et. Al. Functional heterogeneity of side population cells in skeletal muscle. BBRC 2006; 341: 864-873). When cells reached confluence, they were subcultured and cells that had continued to divide more than 100 times were regarded as established. A colony that appears to be derived from a single cell generated by seeding the established cell line at a very low density is isolated. This operation was repeated 2-3 times and cloned (colony isolation method).

  INDUSTRIAL APPLICABILITY According to the present invention, a transgenic non-human animal that expresses a reporter gene using the utrophin expression control mechanism and cells thereof are provided. Since the transgenic non-human animal and its cells use a utrophin expression control mechanism, a drug or drug that affects utrophin gene expression is conveniently and efficiently screened based on the expression of the reporter gene. It becomes possible. Therefore, it is possible to screen for drug candidates that can be used for the treatment or prevention of diseases or disorders related to utrophin gene expression, as well as diseases or disorders related to dystrophin deficiency that can be supplemented with utrophin (for example, muscular dystrophy). Become.

The structure of the A-utrophin and B-utrophin genes on the genome (A), and the structures of the A-utrophin and B-utrophin proteins and their expression sites (B) are shown. The structure of the expression cassette for Gnls mice and the expression cassette for DUE mice is shown. The expression of the lacZ gene in each tissue of Gnls mouse (A) and DUE mouse (B) is shown. The procedure for constructing an expression cassette for DUE mice is shown. The expression of β-galactosidase and utrophin in each tissue of DUE mice is shown. The expression of β-galactosidase and utrophin in each tissue of DUE mice is shown. The expression of (beta) -galactosidase and utrophin in the skeletal muscle tissue of the DUE mouse | mouth after cardiotoxin administration is shown. The expression of the lacZ gene in the skeletal muscle tissue of Gnls mice (A) and DUE mice (A) after cardiotoxin administration is shown.

SEQ ID NO: 1: Mouse downstream utrophin enhancer SEQ ID NO: 2-13: Synthetic oligonucleotide SEQ ID NO: 14: Human downstream utrophin enhancer SEQ ID NO: 15: Rat downstream utrophin enhancer SEQ ID NO: 16: Canine downstream utrophin enhancer SEQ ID NO: 17: SV40 Virus nuclear translocation signal

Claims (31)

  A transgenic non-human animal, wherein a reporter gene is introduced under the control of a utrophin enhancer (DUE) downstream of a utrophin gene and a transcriptional regulatory region, and the reporter gene is expressed.   The transgenic non-transgenic gene of claim 1, wherein the reporter gene is expressed in a tissue or cell of an organ selected from the group consisting of brain, large intestine, small intestine, kidney, liver, testis, lung, submandibular gland, heart and muscle. Human animals.   The transgenic non-human animal according to claim 1 or 2, wherein the reporter gene is expressed in a tissue or cell selected from the group consisting of skeletal muscle, cardiac muscle and smooth muscle.   The transgenic non-human animal according to any one of claims 1 to 3, wherein the reporter gene is a gene (lacZ) encoding β-galactosidase.   Invertebrates including Drosophila and nematodes, fish including zebrafish and medaka, birds including chicken and quail, mammals including mice, rats, dogs, minipigs, rabbits, goats, sheep, cows, marmoset and cynomolgus monkeys The transgenic non-human animal according to any one of claims 1 to 4, which is selected from the group.   The transgenic non-human animal according to any one of claims 1 to 5, wherein an expression cassette having a reporter gene linked thereto is introduced downstream of the DUE sequence of the utrophin gene and the transcriptional regulatory region.   The transgenic non-human animal according to claim 6, wherein a nuclear translocation signal sequence is linked upstream of the reporter gene in the expression cassette.   The transgenic non-human animal according to claim 6 or 7, wherein a poly A signal sequence is linked downstream of the reporter gene in the expression cassette.   A cell obtained from the transgenic non-human animal according to any one of claims 1 to 8, and expressing a reporter gene.   The cell according to claim 9, which is a cell derived from an organ selected from the group consisting of brain, large intestine, small intestine, kidney, liver, testis, lung, submandibular gland, heart and muscle.   The cell according to claim 9 or 10, which is a cell derived from skeletal muscle, cardiac muscle or smooth muscle.   The cell according to any one of claims 9 to 11, which is a myoblast or a quiescent muscle satellite cell.   The cell according to any one of claims 9 to 11, which is a cell selected from the group consisting of macrophages, fibroblasts, glial cells, adipocytes and vascular endothelial cells. (A) contacting the cell according to any one of claims 9 to 13 with a test drug;
(B) measuring the expression of a reporter gene in the cell,
(C) A method for screening for a drug that increases or decreases the expression of the utrophin gene, comprising the step of selecting a test drug that increases or decreases the expression of the reporter gene. (D) administering a test drug to the transgenic non-human animal according to any one of claims 1 to 8,
(E) measuring the expression of a reporter gene in the transgenic non-human animal,
(F) A method of screening for a drug that increases or decreases the expression of the utrophin gene, comprising selecting a test drug that increases or decreases the expression of the reporter gene.   The method according to claim 15, wherein steps (d) to (f) are performed after performing steps (a) to (c) of the method according to claim 14. (A) contacting the cell according to any one of claims 9 to 13 with a test drug;
(B) measuring the expression of a reporter gene in the cell,
(C) screening a drug candidate for treating or preventing a disease or disorder associated with increased or decreased utrophin gene expression, comprising selecting a test drug that increases or decreases reporter gene expression as a drug candidate. Method. (D) administering a test drug to the transgenic non-human animal according to any one of claims 1 to 8,
(E) measuring the expression of a reporter gene in the transgenic non-human animal,
(F) screening a drug candidate for treating or preventing a disease or disorder associated with increased or decreased utrophin gene expression, comprising selecting a test drug that increases or decreases reporter gene expression as a drug candidate. Method.   The method according to claim 18, wherein steps (d) to (f) are performed after performing steps (a) to (c) of the method according to claim 17.   The method according to any one of claims 17 to 19, wherein the disease or disorder associated with increased or decreased utrophin gene expression is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMB), or polymyositis. (A) contacting the cell according to claim 11 or 12 with a test drug;
(B) measuring the expression of a reporter gene in the cell,
(C) A method of screening a drug candidate for treating or preventing a muscular disease or disorder, comprising selecting a test drug that increases or decreases reporter gene expression as a drug candidate. (D) administering a test drug to the transgenic non-human animal according to any one of claims 1 to 8,
(E) measuring the expression of a reporter gene in muscle tissue or muscle cells of the transgenic non-human animal,
(F) A method for screening a drug candidate for treating or preventing a muscular disease or disorder, comprising selecting, as a drug candidate, a test drug that increases or decreases reporter gene expression.   The method of claim 22, wherein steps (d) to (f) are performed after performing steps (a) to (c) of the method of claim 21. (G) administering the selected drug candidate to a non-human model animal having muscle disease or disorder;
24. The method according to any one of claims 21 to 23, further comprising the step of (h) determining whether the drug candidate affects a muscle disease or disorder in the non-human model animal.   The method according to any one of claims 21 to 24, wherein the muscular disease or disorder is muscular dystrophy.   A plasmid comprising a sequence consisting of a nuclear translocation signal, a reporter gene and a poly A signal sequence under the control of a utrophin enhancer (DUE) downstream of the utrophin gene and a transcriptional regulatory region.   The plasmid according to claim 26, wherein the transcriptional regulatory region of the utrophin gene comprises the base sequence shown in SEQ ID NO: 18.   28. A cell transfected with the plasmid of claim 26 or 27 and expressing a reporter gene.   The cell according to claim 28, which is a cell derived from skeletal muscle, cardiac muscle or smooth muscle.   30. The cell of claim 28, which is a non-human germ cell or embryonic stem cell. (A) producing a transgenic non-human animal using the plasmid of claim 26 or 27;
(B) a non-human that expresses a reporter gene under the control of the DUE sequence of the utrophin gene and a transcriptional regulatory region, comprising selecting a transgenic non-human animal that expresses the reporter gene from the produced transgenic non-human animals How to make animals.

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