WO2010010887A1 - Tissue expression promoter - Google Patents

Tissue expression promoter Download PDF

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WO2010010887A1
WO2010010887A1 PCT/JP2009/063088 JP2009063088W WO2010010887A1 WO 2010010887 A1 WO2010010887 A1 WO 2010010887A1 JP 2009063088 W JP2009063088 W JP 2009063088W WO 2010010887 A1 WO2010010887 A1 WO 2010010887A1
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dna
seq
base sequence
promoter
adipose tissue
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PCT/JP2009/063088
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French (fr)
Japanese (ja)
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真一郎 堀
和彦 前川
鈴木　稔
五紀 大島
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塩野義製薬株式会社
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Priority to JP2010521710A priority Critical patent/JPWO2010010887A1/en
Publication of WO2010010887A1 publication Critical patent/WO2010010887A1/en

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
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    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention relates to a novel promoter useful for specific expression in adipose tissue and / or pancreatic tissue and use thereof.
  • adipocyte P2 promoter (hereinafter also referred to as aP2 promoter) region is known to specifically express a gene connected downstream thereof in adipose tissue (cell), and a fusion gene with this promoter region.
  • MCP-1 monocytochemotractant protein-1
  • CDO cyste dioxidase
  • taurine synthesis can be promoted in the adipose tissue of transgenic mice prepared by introducing a gene in which this enzyme is fused with the aP2 promoter. It has been.
  • CDO is overexpressed 2 to 3 times that of normal mice, and is released as a research tool for the association of taurine with obesity and metabolic syndrome.
  • a sequence of about 520 bp (SEQ ID NO: 21) located at the most upstream of the aP2 promoter (SEQ ID NO: 24) derived from a mouse of about 5.4 kb has been shown to be adipose tissue-specific expression in vivo by experiments with transgenic mice. It has been reported to be important. However, as a result of comparing the in vivo expression intensity with the case where the full length of the promoter is used, the activity is greatly reduced (Non-patent Document 1), and only this region is used as a promoter for adipose tissue-specific expression. Even if it was used, its expression was weak, so that a practical transgenic mouse could not be produced.
  • transgenic mice can be produced with very high efficiency by introducing genes into fertilized eggs using a lentiviral vector, compared to the conventional microinjection method.
  • aP2 gene promoter has a sequence length of about 5.4 kb, and when incorporated into a lentiviral vector, its large size becomes a barrier, making it difficult to produce a transgenic mouse using this promoter. there were.
  • the reason is that, due to the large length of the promoter sequence, the packaging efficiency of the virus is reduced, and it is not possible to prepare a virus having the titer necessary for the production of a transgenic mouse.
  • the same reason can be considered for other viral vectors used for gene therapy such as adenovirus and AAV.
  • An object of the present invention is to provide an element capable of specific expression in adipose tissue and / or pancreatic tissue and having high introduction efficiency.
  • a partial region in the sequence derived from the mouse aP2 gene promoter is important for promoting expression in adipose tissue, Even when a transgenic mouse is produced using a truncated promoter containing this region, it has a specific activity and sufficient expression level in adipose tissue and pancreatic tissue, and human-derived sequences are similar to those in mice. It has been found that it has activity, and the present invention has been completed.
  • the present invention (1) an isolated DNA having at least the base sequence shown in SEQ ID NO: 22 and having specific promoter activity in adipose tissue and / or pancreas, (2) (a) DNA having at least one base substitution, deletion, insertion or addition in the base sequence shown in SEQ ID NO: 22; (b) complementary DNA consisting of the base sequence shown in SEQ ID NO: 22 A DNA selected from the group consisting of DNA that can hybridize with the strand under stringent conditions, and (c) a DNA having a sequence identity of at least 90% to the DNA consisting of the base sequence represented by SEQ ID NO: 22 Isolated DNA having specific promoter activity in adipose and / or pancreatic tissue, (3) The isolated DNA according to (1) or (2), which is further DNA having the base sequence represented by SEQ ID NO: 21; (4) The isolated DNA according to any one of (1) to (3), which is further DNA having the base sequence represented by SEQ ID NO: 23, (5) an isolated DNA having at least the base sequence shown in SEQ ID NO: 28 and
  • transgenic mouse capable of specifically expressing a target gene in adipose tissue and / or pancreatic tissue, and to perform gene therapy targeting these tissues. Become.
  • FIG. 1 is a schematic diagram of a reporter assay construct for the aP2 promoter-derived sequence prepared this time.
  • FIG. 2 is a graph showing the luciferase activity when co-expressing a reporter assay construct of an aP2 promoter-derived sequence and a pRL-Tk vector, which is a vector expressing Renilla luciferase as an internal control reporter. The horizontal axis shows the relative value of the emission intensity when other constructs are used with the emission intensity when aP2-11-luc is introduced at 293 being 1.
  • FIG. 3 is a graph showing the luciferase activity when reporter vectors of human and mouse aP2 promoter-derived sequences are coexpressed with the pRL-Tk vector, respectively.
  • the abscissa represents the relative value of the luminescence intensity when other constructs are used with the luminescence intensity when aP2-11 is introduced into undifferentiated 3T3-L1 being 1.
  • the “promoter” includes an expression regulatory region capable of associating with RNA polymerase in a cell, and initiates transcription of a coding sequence existing downstream (toward the 3 ′ end) of the region. It includes a region having a function to be performed. Furthermore, the “promoter” refers to a region including a so-called enhancer region responsible for tissue-specific expression regulation.
  • adipose tissue and / or pancreatic tissue means that expression in adipose tissue and / or pancreatic tissue is clearly higher compared to other tissues.
  • the adipose tissue is not particularly limited as long as it is a mammalian adipose tissue, such as subcutaneous adipose tissue, visceral adipose tissue, white adipose tissue, brown adipose tissue, perigenital adipose tissue, mesenteric adipose tissue, scapula Contains brown adipose tissue.
  • the DNA of the present invention is an DNA having at least the base sequence shown in SEQ ID NO: 22 and having a specific promoter activity in adipose tissue and / or pancreatic tissue. DNA. Since the DNA of the present invention has the base sequence shown in SEQ ID NO: 22, it has an excellent property that it can be expressed specifically in adipose tissue and / or pancreatic tissue even in vivo.
  • the base sequence shown in SEQ ID NO: 22 is a sequence of the region consisting of positions 1662 to 2357 in the mouse-derived adipocyte P2 gene promoter.
  • the inventors have now found that these sequences play an important role in the specific expression of adipose and / or pancreatic tissue in vivo. In general, gene expression trends in vitro often differ greatly from actual gene expression trends in vivo.
  • the promoter activity of the DNA of the present invention is, for example, the following steps I to III: I a sequence in which a reporter gene (for example, luciferase gene, alkaline phosphatase gene, etc.) is operably linked under the control of the DNA to be tested.
  • a step of preparing a plasmid for activity measurement retaining II, a step of transforming appropriate adipose tissue cells (or cells of pancreatic tissue) and control cells with the activity measurement plasmid of I above, and III This includes a step of detecting the presence or absence of expression of a reporter gene in the obtained cells and measuring the expression intensity, and can be determined by a reporter gene assay or the like.
  • the DNA is an indicator that it is a promoter specific to adipose tissue (pancreatic tissue) It becomes.
  • the amount of the expression product of the reporter gene is an indicator of the strength of the promoter activity.
  • the DNA of the present invention is a DNA having a substitution, deletion, insertion or addition of at least one base, specifically, one or more bases in the base sequence shown in SEQ ID NO: 22, and It may be an isolated DNA having specific promoter activity in adipose tissue and / or pancreatic tissue.
  • the number of substitutions, deletions, insertions or additions (hereinafter may be simply referred to as “mutation”) and the position of introduction are determined by performing a reporter gene assay comprising the steps I to III above and / or Any promoter that is determined to be a pancreatic tissue-specific promoter may be used.
  • mutation substitutions, deletions, insertions or additions are included.
  • Artificial mutation can be introduced by a conventional site-specific mutation introduction method.
  • methods for introducing site-specific mutation include a method using amber mutation (gapped duplex method, Nucleic Acids Research, 12, 9441-9456 (1984)) and a PCR method using amber mutation (international publication). 98/02535) or the like can be used.
  • the DNA of the present invention has a specific promoter activity in adipose tissue and / or pancreatic tissue, it hybridizes under stringent conditions with a complementary strand of DNA consisting of the base sequence shown in SEQ ID NO: 22.
  • Isolated DNA having specific promoter activity in adipose tissue and / or pancreatic tissue is also encompassed by the present invention.
  • Such DNA is not particularly limited as long as it is determined to be a promoter specific to adipose tissue and / or pancreatic tissue by performing a reporter gene assay including the steps I to III.
  • hybridization operation can be performed, for example, according to the method described in Molecular Cloning: A Laboratory Manual Second Edition (issued by Cold Spring Harbor Laboratory, 1989).
  • stringent conditions include hybridization conditions described in the above-mentioned books and the like. Specifically, 10% dextran sulfate, 1M NaCl, 1% SDS, 100 mg / ml salmon sperm DNA, 1 After performing a hybridization reaction under the conditions of ⁇ 10 6 cpm / ml 32 P-labeled probe (SEQ ID NO: 22), 2 ⁇ SSC, 0.1% SDS, twice at room temperature, 0.1 ⁇ SSC, 0. The conditions are such that washing is performed twice at 1% SDS and 60 ° C.
  • the DNA of the present invention includes DNA having at least 90% sequence identity to the DNA consisting of the base sequence shown in SEQ ID NO: 22, preferably 95% or more, more preferably 98% or more. Also included is isolated DNA having specific enhancer activity in adipose tissue and / or pancreatic tissue. If DNA having such sequence identity is determined to be a promoter specific to adipose tissue and / or pancreatic tissue by performing a reporter gene assay including the steps I to III described above, for example. Good.
  • sequence identity can be determined using a homology search program (for example, BLAST etc.) commonly used in this field by default.
  • sequence identity of nucleotide sequences is calculated by aligning the two types of nucleotide sequences to be compared and dividing the number of nucleotide sequences matched by the alignment by the total number of reference nucleotide sequences. It is the number which showed the ratio which was done in%. Note that the gap generated by the alignment is calculated as a mismatch.
  • the base sequence shown in SEQ ID NO: 21 is further included.
  • This DNA is operably linked, but it is generally desirable that the DNA is linked so that SEQ ID NO: 21 is upstream.
  • the base sequence shown in SEQ ID NO: 21 is a sequence of a region consisting of about 520 bases at the most upstream of the mouse-derived adipocyte P2 gene promoter. This region is generally called an “enhancer” and has a function of increasing the transfer speed. It has also been reported to have an important role in adipose tissue-specific expression (Journal of Cellular Biochemistry, Vol. 49, 1992, pages 219-224).
  • the base sequence shown in SEQ ID NO: 23 is a sequence of about 200 bp in the region from 5359 to 5548 in the mouse-derived adipocyte P2 gene promoter (aP2 promoter).
  • This region is called a proximal-promoter and includes a transcription factor binding region such as AP-1 or C / EBP (CCAAT / enhancer binding protein).
  • AP-1 or C / EBP CCAAT / enhancer binding protein
  • This region plays an important role in adipose differentiation, it has been reported that it is not an essential region for adipose tissue-specific expression in vivo such as in transgenic animals. (Proc. Natl. Acad. Sci. USA, Vol. 87, pp 9590-9594, 1990).
  • This downstream part contains sequences called TATA box and CAAT box, and it is known to lead RNA polymerase to its normal start site and to promote transcription.
  • the DNA of the present invention provides an expression vector capable of expressing a foreign gene specifically in adipose tissue and / or pancreatic tissue.
  • expression vector refers to a vector comprising a sequence of a foreign gene that encodes at least a portion of a gene product that can be transcribed.
  • the sequence of the foreign gene is optionally translated into a protein, polypeptide or peptide. In other cases, these sequences are not translated.
  • siRNA that specifically cleaves mRNA using RNA interference action such as miRNA (microRNA) and shRNA (short hairpin structure RNA).
  • miRNA miRNA
  • shRNA short hairpin structure RNA
  • the expression vector of the present invention can be obtained by inserting the DNA of the present invention into an appropriate vector.
  • the vector used in the present invention include plasmids, phages, cosmids, viruses (bacteriophages, animal viruses and plant viruses) and artificial chromosomes (for example, YAC).
  • the expression vector of the present invention is characterized in that the DNA of the present invention is operably linked upstream of the foreign gene sequence in the transcription direction, and is specific to adipose tissue and / or pancreatic tissue. In which foreign genes can be expressed.
  • a preferred embodiment is an expression vector in which a foreign gene is operably linked downstream of DNA having the base sequence shown in SEQ ID NO: 22 and the base sequence shown in SEQ ID NO: 21.
  • an expression in which a foreign gene is operably linked downstream of a DNA having the base sequence shown in SEQ ID NO: 22, the base sequence shown in SEQ ID NO: 21, and the base sequence shown in SEQ ID NO: 23 It is a vector.
  • the expression vector of the present invention can itself be a very useful therapeutic tool for diseases in adipose tissue and / or pancreatic tissue.
  • the transgenic non-human animal having the expression vector of the present invention and cells derived therefrom are adipose tissues (or fat cells) such as anti-obesity drugs, diabetes drugs, hyperlipidemia drugs, hypertension drugs, It can be a tool for searching for drugs that improve diseases associated with pancreatic tissue (or pancreatic cells) (such as metabolic syndrome-related diseases).
  • the base sequences shown in SEQ ID NOs: 21 to 23 are all derived from mice, but the present inventors have found a base sequence having the same action from a human-derived gene. Specifically, the base sequence represented by SEQ ID NO: 21 is the base sequence represented by SEQ ID NO: 27, the base sequence represented by SEQ ID NO: 22 is the base sequence represented by SEQ ID NO: 28, and the base sequence represented by SEQ ID NO: 23. Since the sequence corresponds to the base sequence represented by SEQ ID NO: 29, it is within the common sense of those skilled in the art to obtain the same effect by replacing these sequences.
  • Examples of the foreign gene include (A) a gene encoding a protein for treating the target disease, (B) a nucleic acid for treating the target disease, and (C) a marker gene.
  • target disease examples include diseases that can be treated by expressing a foreign gene specifically in adipose tissue and / or pancreatic tissue. Specific examples include adipose tissue atrophy, type II diabetes, hyperlipidemia, obesity and the like.
  • proteins for treating a target disease include insulin, leptin, and glucose transporter.
  • Nucleic acids for treating the target disease include synthetic or non-naturally occurring forms of nucleic acids, such as phosphorothioate antisense oligonucleotides, aptamers, siRNAs, or double stranded RNAi, and nucleic acids well known in the art Other forms of nucleic acids such as the chemical derivatives of can also be used.
  • nucleic acids such as the chemical derivatives of can also be used.
  • Various nucleotide sequences can be used for designing siRNA and other interference sequences. For example, siDirect (http://design.RNAi.jp/) can be used for sequence design.
  • marker gene examples include ⁇ -galactosidase gene, alkaline phosphatase gene, chloramphenicol acetyltransferase gene, growth hormone gene, luciferase gene, green fluorescent protein gene and derivatives thereof.
  • the “derivative” includes an artificially produced mutant.
  • the foreign gene when the foreign gene is “the gene encoding a protein for treating the target disease” or “the nucleic acid for treating the target disease”, it is specific to adipose tissue and / or pancreatic tissue. It can be used as an active ingredient of a therapeutic agent. Such therapeutic agents are also included in the present invention.
  • the therapeutic agent of the present invention is characterized by containing the expression vector as an active ingredient, and encodes a foreign gene specifically for a fat tissue and / or pancreatic tissue, specifically a protein for treating a target disease. Or a nucleic acid for treating a target disease. Since such a therapeutic agent contains the expression vector as an active ingredient, it has an excellent property of exhibiting high specificity for adipose tissue and / or pancreatic tissue.
  • the therapeutic agent of the present invention may appropriately contain components for maintaining the expression vector, which is an active ingredient, in a stable state, for example, buffer components, degradation protective agents (for example, inhibitors of nucleolytic enzymes, etc.) and the like. Good.
  • the gene expression agent of the present invention may further contain a drug suitable for introduction into cells and / or tissues.
  • the amount of the expression vector in the therapeutic agent of the present invention can be appropriately set depending on the purpose of use. For example, when used in the therapeutic agent described later, it can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient.
  • the expression vector amount is 0.0001 to 100 mg, preferably 0.001 to 10 mg. It is desirable. It is desirable to administer such a dose once every few days to several months.
  • the therapeutic agent of the present invention can be introduced into cells by, for example, a lipofection method, a phosphate-calcium coprecipitation method; a DEAE-dextran method; a direct injection method using a micro glass tube.
  • Examples of the method for introducing the therapeutic agent of the present invention into a tissue include introduction method using encapsulated liposome, introduction method using electrostatic liposome, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), particle gun Examples thereof include a method of transferring an active ingredient together with a carrier (metal particles) into a cell, a method of introduction with a positively charged polymer, and the like.
  • the therapeutic agent of the present invention since it has high specificity for adipose tissue and pancreatic tissue, diseases that can be treated by expressing foreign genes specifically for adipose tissue and / or pancreatic tissue (for example, , Obesity, type II diabetes, adipose tissue atrophy, pancreatic cancer, etc.), and can exhibit a symptom reduction or curative effect. Therefore, at the same time, it has an excellent property that it becomes possible to reduce the influence of the therapeutic agent on tissues other than adipose tissue and pancreatic tissue.
  • diseases that can be treated by expressing foreign genes specifically for adipose tissue and / or pancreatic tissue for example, Obesity, type II diabetes, adipose tissue atrophy, pancreatic cancer, etc.
  • Examples of the “disease that can be treated by expressing a foreign gene specifically in adipose tissue and / or pancreatic tissue” include the same as the “target disease”.
  • the therapeutic agent of the present invention is administered, for example, intravenously, artery, subcutaneously, intradermally, intramuscularly, or directly locally to the subject adipose tissue and / or pancreatic tissue itself. be able to.
  • various preparation forms for example, a liquid agent etc.
  • the injection can be prepared by a conventional method, for example, after dissolving in an appropriate solvent (buffer solution such as PBS, physiological saline, sterilized water, etc.)
  • buffer solution such as PBS, physiological saline, sterilized water, etc.
  • it can be prepared by sterilizing by filtration with a filter or the like and then filling into an aseptic container.
  • a conventional carrier or the like may be added to the injection as necessary.
  • Liposomes such as HVJ-liposomes can be in the form of liposome preparations such as suspensions, freezing agents, and centrifugal concentrated freezing agents.
  • a sustained-release preparation (mini-pellet preparation or the like) can be prepared and implanted near the affected area, or can be gradually and gradually administered to the affected area using an osmotic pump or the like.
  • the dosage form of the therapeutic agent of the present invention is described in detail, for example, in the experiment manual, etc., its preparation method, administration method, etc. (separate volume experimental medicine, basic technology of acupuncture gene therapy, Yodosha, 1996, separate volume experiment) Medicine, Acupuncture Gene Introduction & Expression Analysis Experimental Method, Yodosha, 1997).
  • experiment manual etc.
  • administration method etc.
  • the therapeutic agent of the present invention can be introduced into cells or tissues by the following technique.
  • Examples of the method for introducing a gene into a cell include a lipofection method, a phosphate-calcium coprecipitation method, and a direct injection method using a micro glass tube.
  • Gene transfer methods to tissues include gene transfer method using encapsulated liposomes, gene transfer method using electrostatic liposomes, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), receptor-mediated gene transfer And a method of transferring an active ingredient together with a carrier (metal particles) with a particle gun, a direct introduction method of naked-DNA, an introduction method using a positively charged polymer, and the like.
  • a viral vector When a viral vector is used in the construction of the expression vector of the present invention, examples of such a viral vector include recombinant adenovirus, retrovirus and the like. More specifically, for example, lentivirus, detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, shinbis virus, Sendai virus, SV40, immunodeficiency virus ( HIV) and other DNA viruses or RNA viruses can be introduced into a cell by introducing the DNA of the present invention and a foreign gene operably linked to the DNA, and infecting the resulting expression vector. Is possible. Of the viral vectors, it is desirable to use a lentivirus to infect fertilized eggs.
  • Lentiviruses are a subgroup of retroviruses, various primate viruses such as human immunodeficiency viruses HIV-1 and HIV-2, simian immunodeficiency virus (SIV), and non-primate viruses (eg, maedi bisna virus ( MW), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), goat arthritis encephalitis virus (CAEV) and bovine immunodeficiency virus (BIV)).
  • various primate viruses such as human immunodeficiency viruses HIV-1 and HIV-2, simian immunodeficiency virus (SIV), and non-primate viruses (eg, maedi bisna virus ( MW), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), goat arthritis encephalitis virus (CAEV) and bovine immunodeficiency virus (BIV)).
  • the method for introducing the therapeutic agent of the present invention is the same as described above.
  • the dose can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient.
  • the amount of the expression vector contained in the therapeutic agent is 0.0001 to 100 mg, preferably 0.001 to 10 mg. It is desirable to be. It is desirable to administer such a dose once every few days to several months.
  • a transgenic non-human mammal carrying such an expression vector is provided.
  • the transgenic non-human mammal of the present invention is a transgenic non-human mammal having the expression vector, wherein a foreign gene in the expression vector is expressed specifically in adipose tissue and / or pancreatic tissue.
  • Transgenic non-human mammals that retain an expression vector containing a marker gene as a foreign gene retain a specific promoter in adipose tissue or pancreatic tissue. Using the presence / absence and strength as an index, it has an excellent property that it can easily screen for therapeutic agents for metabolic syndrome-related diseases including diabetes drugs and anti-obesity drugs.
  • a transgenic non-human mammal having an expression vector containing a gene encoding a protein for treating the target disease or a nucleic acid such as miRNA or shRNA as a foreign gene is used in adipose tissue and / or pancreatic tissue. Since a specific promoter is retained, the therapeutic effect of the protein or the nucleic acid when expressed in these tissues can be evaluated.
  • the transgenic non-human mammal of the present invention is used to examine the influence of a specific gene on these tissues in the living body. For example, if there are genes with different expression levels in adipose tissue between obese patients and healthy individuals, the effects can be examined in vivo by expressing proteins encoding these in adipose tissue.
  • the therapeutic effect of a nucleic acid such as miRNA expressed specifically in adipose tissue can be examined.
  • the expression vector may be incorporated and retained in the chromosome of the mammal.
  • non-human mammal examples include mammals other than humans, for example, mammals such as mice, rats, rabbit pigs, dogs, sheep and goats. Above all, there is a long history of use in medical research and accumulation of data, and there is a track record that many pathological models have been created so far, and the production technology of genetically modified animals has been established. Therefore, rodents represented by mice, rats and the like are preferable, and mice are particularly preferable.
  • the transgenic non-human mammal of the present invention can be produced by a known method.
  • it can be produced by a method including the following steps (a) to (c), for example.
  • (A) a step of introducing a vector or the like into a fertilized egg and transplanting the transgenic fertilized egg into a foster animal;
  • (B) selecting a transgenic animal from offspring born from the animal; and
  • (c) establishing a line from the selected animal (founder).
  • step (a) When introducing the vector of step (a) into a fertilized egg, a microinjection method (manual operation manual for mouse embryo (Modern Publishing, 1989), molecular biology protocol (Nanedo, 1994)) or lentivirus
  • a microinjection method manual operation manual for mouse embryo (Modern Publishing, 1989), molecular biology protocol (Nanedo, 1994)
  • lentivirus The lentivirus method for infecting fertilized eggs (Lois C et al., Science, 295, 2002, pages 868-872) is used.
  • transgenic non-human mammals In general, however, it is desirable to produce transgenic non-human mammals by the lentiviral method. This is because in the microinjection method, several tens to several hundreds of transgenes are inserted into one unspecified chromosome location and inserted, and depending on the insertion location, an individual with no transgene expression is born. Therefore, it takes a period of 1 to 2 years after selection of useful strains after production of mice and production of mice for use in experiments. On the other hand, the lentivirus method has a short production period and high production efficiency of transgenic mice, and can obtain the number of mice used for experiments in the first-generation offspring.
  • the transgene is inserted one copy at a different location on the genome, a plurality of mice showing various expression levels can be obtained at a time.
  • the promoter of the present invention is characterized in that its sequence length is shorter than that of the natural type, the packaging efficiency of the lentiviral vector is improved, and it becomes possible to prepare a high titer virus. Therefore, it is possible to produce a transgenic non-human mammal by the lentivirus method with high efficiency.
  • a transgenic non-human mammal carrying an expression vector containing a marker gene or a cell derived from the transgenic non-human mammal is subjected to treatment.
  • a method for evaluating the expression level of a marker gene in animal tissues or cells after administration of a test substance can be mentioned.
  • a test substance to the transgenic non-human mammal of the present invention to suppress the expression of a marker gene is evaluated as an index for reducing adipocytes in each tissue in vivo. be able to.
  • the test substance may be any known substance or novel substance, for example, a nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound library prepared using combinatorial chemistry technology And random peptide libraries prepared by solid phase synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.
  • a nucleic acid carbohydrate, lipid, protein, peptide, low molecular organic compound
  • compound library prepared using combinatorial chemistry technology
  • random peptide libraries prepared by solid phase synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.
  • the mixture of 2 or more types of these compounds can also be provided as a sample.
  • an amplified fragment into which BglII and HindIII restriction enzyme sites were introduced was obtained.
  • a promoter region at about 200 bp from the transcription start point was used at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds using a primer consisting of the base sequence of SEQ ID NO: 3 and a primer consisting of the base sequence of SEQ ID NO: 4.
  • a fragment of the enhancer region was treated with BglII and HindIII, and inserted into the BglII and HindIII sites of pEGFP-N1 (Clontech) to obtain a ligated plasmid.
  • This plasmid was treated with BglII and NotI and inserted into the BamHI and NotI sites of pENTR4 (Invitrogen) to obtain a ligated plasmid.
  • This plasmid was treated with KpnI and BamHI, and a promoter fragment previously introduced with a KpnI and BamHI restriction enzyme site was ligated to prepare aP2-0-EGFP / pENTR.
  • This DNA includes a sequence of about 200 bp of a region consisting of about 520 bases at the most upstream and a region consisting of positions 5359-5548 of the full length of the aP2 promoter shown in SEQ ID NO: 24.
  • aP2-0-EGFP / pENTR was treated with SalI to remove the fragment containing the enhancer region, and then ligated with itself to prepare aP2-11-EGFP / pENTR.
  • This DNA includes a sequence of about 200 bp in the region consisting of positions 5359-5548 of the full length aP2 promoter shown in SEQ ID NO: 24.
  • the following primer pair (primer consisting of the base sequence of SEQ ID NO: 5 and primer consisting of the base sequence of SEQ ID NO: 6), (primer consisting of the base sequence of SEQ ID NO: 7) , And a primer consisting of the base sequence of SEQ ID NO: 8), (a primer consisting of the base sequence of SEQ ID NO: 9 and a primer consisting of the base sequence of SEQ ID NO: 10), (a primer consisting of the base sequence of SEQ ID NO: 11, and SEQ ID NO: A primer comprising 12 nucleotide sequences), (SEQ ID NO: 13) A primer consisting of a base sequence and a primer consisting of a base sequence of SEQ ID NO: 14) (a primer consisting of a base sequence of SEQ ID NO: 15 and a primer consisting of a base sequence of SEQ ID NO: 16), (consisting of a base sequence of SEQ ID
  • DNAs include the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 23 in common, and are located at positions 481 to 1137 in the full length of the aP2 promoter shown in SEQ ID NO: 24, respectively. It includes an array of regions consisting of 1069 to 1780, 1662 to 2357, 2296 to 2988, 2908 to 3558, 3496 to 4199, 4122 to 4823, and 4728 to 5380.
  • the vector aP2 promoter / pBluescript II containing the entire length of the aP2 promoter region of about 5.5 kb was used as a template, and the above primer pairs were used under the conditions of repeating 94 ° C. for 30 seconds, 55 ° C.
  • This DNA includes the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 23, and includes the sequence of the region consisting of positions 481 to 5380 in the full length of the aP2 promoter shown in SEQ ID NO: 24. It is out.
  • This fragment was treated with AgeI and NotI, and aP2-0-EGFP / pENTR, aP2-1-EGFP / pENTR, aP2-2-EGFP / pENTR, aP2-3-EGFP / pENTR, aP2-4-EGFP / pENTR, AgeI of aP2-5-EGFP / pENTR, aP2-6-EGFP / pENTR, aP2-7-EGFP / pENTR, aP2-8-EGFP / pENTR, aP2-11-EGFP / pENTR, aP2-12-EGFP / pENTR Plasmids inserted into NotI sites to convert EGFP into luciferase gene, aP2-0-luc / pENTR, aP2-1-luc / pENTR, aP2-2-luc / pENTR, aP2-3-luc / pENTR, aP
  • the pCDH- ⁇ CMV-GW vector was obtained by introduction into the NheI and SalI sites of pCDH-MCS1- ⁇ CMV.
  • aP2-0-EGFP / pENTR aP2-3-EGFP / pENTR, aP2-5-EGFP / pENTR, and aP2-12-EGFP / pENTR with LR clonase II enzyme mix (Invitrogen)
  • AP2-0-EGFP / pCDH, aP2-3-EGFP / pCDH, aP2-5-EGFP / pCDH, aP2-12-EGFP / pCDH were obtained.
  • aP2-8-luc was introduced, the transcriptional activity of 2 times or more was obtained in 3T3-L1 adipose differentiated cells, but the specific activity differed by about 6 to 14 times.
  • an approximately 520 bp sequence located at the most upstream of the aP2 promoter shown in SEQ ID NO: 21 is necessary for specific expression in adipocytes, but other regions are also expected to affect the promoter activity. It was.
  • aP2-12-luc contained a sequence of about 520 bp located at the most upstream of the aP2 promoter, it was less active in adipocytes than other constructs.
  • transgenic mice using lentivirus and expression in each tissue Using DMEM containing 10% FCS (fetal calf serum), 293TN cells (System Biosciences) under conditions of 5% CO 2 and 37 ° C. Cultured. For transfection, FuGene6 (Roche) was used and the attached protocol was followed. Among the vectors that were found to be specific in adipocytes from the results of Example 2, the vectors used were aP2-0-EGFP / pCDH, 3T3-L1 undifferentiated cells with the smallest promoter size, and relatively active in 293 cells.
  • aP2-3-EGFP / pCDH, aP2-5-EGFP / pCDH, and aP2-12-EGFP / pCDH containing the full length of the aP2 promoter were selected and co-introduced with pPACKH1 (SBI). 72 hours after the transfection, the medium was collected, concentrated by ultracentrifugation, and purified by sucrose to prepare a lentivirus solution for producing a transgenic mouse. Virus titer was measured using p24 Antigen ELIZA kit (ZeptoMetrix).
  • Superovulation was induced by administering 5 IU of PSG (pregnant horse serum gonadotropin) to BDF1 female mice and 48 I HCG (placental gonadotropin) 48 hours later, and mated with BDF1 male mice.
  • PSG pregnant horse serum gonadotropin
  • I HCG placental gonadotropin
  • the oviducts of female mice whose vaginal plug was confirmed the day after mating were collected approximately 40 hours after mating and perfused with 0.4% BSA solution.
  • the recovered 2-cell embryo was cultured in kSOM medium (Arc Resource) for a short period, and then treated with acidic Tyrode (Arc Resource) for about 1 to 2 minutes to remove the embryo transparency.
  • Embryos from which the zona pellucida was successfully removed were washed in kSOM medium and cultured in the same medium until viral infection. Viral infection of embryos is performed by culturing embryos from which the zona pellucida has been removed for 2 days with a drop (6 ⁇ l / spot) of kSOM medium diluted to contain about 150 pg / ⁇ l of viral particles or 1500 pg / ⁇ l. It was. All embryos were cultured under conditions of 5% CO 2 and 37 ° C.
  • Genomic DNA was purified from the tail of a litter collected at 4 weeks of age using DNeasy Blood & Tissue Kit (QIAGEN). Using purified DNA as a template, PCR was performed under the conditions of 95 ° C 15 seconds, 57 ° C 15 seconds, 72 ° C 30 seconds 40 times using a primer consisting of the base sequence of SEQ ID NO: 25 and a primer consisting of the base sequence of SEQ ID NO: 26.
  • the transgene was detected by performing agarose gel electrophoresis, and the genotype of the transgenic mouse was determined.
  • a portion of perigenital fat was biopsyed from the transgenic mice and observed for EGFP fluorescence under a fluorescent stereomicroscope. Individuals in which EGFP fluorescence was observed were necropsied, and EGFP fluorescence was observed in major organs and 4 types of adipose tissue under a fluorescent stereomicroscope. The results are shown in Table 1 below.
  • the efficiency of producing the transgenic mouse is reduced to 1/10 or less compared to the case of using other truncated promoters.
  • no fluorescence was observed in any tissue. This is thought to be due to a decrease in virus packaging efficiency.
  • fluorescence was confirmed in the adipose tissue, but its intensity was weak, and it was similar in the pancreas.
  • primer pair primer consisting of the base sequence of SEQ ID NO: 30 and primer consisting of the base sequence of SEQ ID NO: 31
  • primer consisting of the base sequence of SEQ ID NO: 33 primer consisting of the base sequence of SEQ ID NO: 34 and a primer consisting of the base sequence of SEQ ID NO: 35
  • NheI, NheI and XhoI, and BglII and HindIII restriction enzyme sites were obtained.
  • the amplified fragment containing SEQ ID NO: 29 was treated with BglII and HindIII, and ligated to the BglII and HindIII sites of pGL3-basic (Promega) to obtain human aP2-11 / pGL3.
  • this plasmid was treated with SacI and NheI, and the amplified fragment containing SEQ ID NO: 27 was ligated with the fragment treated with SacI and NheI to prepare human aP2-0 / pGL3.
  • this plasmid was treated with NheI and XhoI, and the amplified fragment containing SEQ ID NO: 28 was ligated with the fragment treated with NheI and XhoI to prepare human aP2-3 / pGL3.
  • 3T3-L1 adipose differentiated cells Reaction of each promoter in cultured cells Using a transient assay in 3T3-L1 adipose differentiated cells, the transcriptional activity of each construct derived from the human aP2 promoter in adipocytes was examined. For comparison, 3T3-L1 cells, HeLa cells, and 293 cells that were not induced to differentiate were used. Using DMEM containing 10% FCS (fetal calf serum), 3T3-L1 cells are cultured under conditions of 5% CO 2 and 37 ° C. After 2 days of confluence, differentiation induction is performed in a medium containing insulin, dexamethasone and IBMX. Started. Transfection was performed by electroporation.
  • FCS fetal calf serum
  • This figure shows the relative value of the luminescence intensity when other constructs were used with the luminescence intensity when aP2-11 was introduced into undifferentiated 3T3-L1 cells (indicated as Luciferase activity in the figure) as 1. It was.

Abstract

Conventional promoters have long sequences and therefore cannot be introduced into living bodies with high efficiency.  Thus, disclosed is a novel promoter.  The promoter has a promoter activity specific to an adipose tissue and/or a pancreatic tissue.

Description

組織発現プロモーターTissue expression promoter
本発明は、脂肪組織および／又は膵臓組織での特異的発現に有用である新規プロモーターおよびその利用に関する。 The present invention relates to a novel promoter useful for specific expression in adipose tissue and / or pancreatic tissue and use thereof.
アディポサイト（ａｄｉｐｏｃｙｔｅ）Ｐ２プロモーター（以下、ａＰ２プロモーターともいう）領域は、その下流につないだ遺伝子を脂肪組織（細胞）に特異的に発現させることが知られており、このプロモーター領域との融合遺伝子を導入したトランスジェニックマウスの報告が過去に多くなされている。例えば、ＭＣＰ－１（モノサイトケモアトラクタントプロテインー１）は脂肪細胞で発現するケモカインの一種である。このケモカインをａＰ２プロモーターと融合させた遺伝子を導入して作製したトランスジェニックマウスは、全身性のインスリン抵抗性を示すことが知られている。このマウスの脂肪組織ではマクロファージが浸潤し、炎症反応が誘発されていることから、脂肪組織における炎症反応とインスリン抵抗性との関連を研究する上で有用といえる。また、ＣＤＯ（システインジオキシダーゼ）はタウリン合成の律速酵素であるが、この酵素をａＰ２プロモーターと融合させた遺伝子を導入して作製したトランスジェニックマウスの脂肪組織においては、タウリン合成を促進できることが知られている。このマウスの脂肪組織では、ＣＤＯが正常マウスの２～３倍過剰発現しており、タウリンと肥満やメタボリックシンドロームの関連のための研究ツールとして発売されている。 An adipocyte P2 promoter (hereinafter also referred to as aP2 promoter) region is known to specifically express a gene connected downstream thereof in adipose tissue (cell), and a fusion gene with this promoter region There have been many reports of transgenic mice that have introduced the above. For example, MCP-1 (monocytochemotractant protein-1) is a kind of chemokine expressed in adipocytes. It is known that a transgenic mouse produced by introducing a gene in which this chemokine is fused with the aP2 promoter exhibits systemic insulin resistance. In this mouse adipose tissue, macrophages infiltrate and induce an inflammatory response, which is useful for studying the relationship between the inflammatory response in adipose tissue and insulin resistance. CDO (cysteine dioxidase) is a rate-limiting enzyme for taurine synthesis, but it is known that taurine synthesis can be promoted in the adipose tissue of transgenic mice prepared by introducing a gene in which this enzyme is fused with the aP2 promoter. It has been. In this mouse adipose tissue, CDO is overexpressed 2 to 3 times that of normal mice, and is released as a research tool for the association of taurine with obesity and metabolic syndrome.
一方、約５．４ｋｂのマウス由来のａＰ２プロモーター（配列番号２４）の最上流に位置する約５２０ｂｐの配列（配列番号２１）は、トランスジェニックマウスによる実験により、インビボでの脂肪組織特異的発現に重要であることが報告されている。ところが、プロモーターの全長を用いた場合と、インビボでの発現強度を比較した結果、その活性は大きく低下しており（非特許文献１）、この領域のみを脂肪組織特異的発現のためのプロモーターとして使用しても、その発現は弱いため実用的なトランスジェニックマウスの作製はできなかった。 On the other hand, a sequence of about 520 bp (SEQ ID NO: 21) located at the most upstream of the aP2 promoter (SEQ ID NO: 24) derived from a mouse of about 5.4 kb has been shown to be adipose tissue-specific expression in vivo by experiments with transgenic mice. It has been reported to be important. However, as a result of comparing the in vivo expression intensity with the case where the full length of the promoter is used, the activity is greatly reduced (Non-patent Document 1), and only this region is used as a promoter for adipose tissue-specific expression. Even if it was used, its expression was weak, so that a practical transgenic mouse could not be produced.
近年、レンチウイルスベクターを用いてマウス受精卵への遺伝子導入を行うことにより、従来から汎用されてきた顕微注入法と比較して、非常に高い効率でトランスジェニックマウスが作製できることが報告されている（実験医学、２１巻、４号、２００３年、５０９－５１４ページ）。しかしながら、ａＰ２遺伝子プロモーターは約５．４ｋｂの配列長を有しており、レンチウイルスベクターに組み込む場合は、その大きなサイズが障壁となることから、本プロモーターを用いたトランスジェニックマウスの作製は困難であった。プロモーター配列長が大きい故に、ウイルスのパッケージング効率が低下し、トランスジェニックマウスの作製に必要な力価のウイルスを調製できないことが、その理由である。アデノウイルスやＡＡＶなど遺伝子治療に用いられる他のウイルスベクターでも同様の理由が考えられる。 In recent years, it has been reported that transgenic mice can be produced with very high efficiency by introducing genes into fertilized eggs using a lentiviral vector, compared to the conventional microinjection method. (Experimental Medicine, Vol. 21, No. 4, 2003, pp. 509-514). However, the aP2 gene promoter has a sequence length of about 5.4 kb, and when incorporated into a lentiviral vector, its large size becomes a barrier, making it difficult to produce a transgenic mouse using this promoter. there were. The reason is that, due to the large length of the promoter sequence, the packaging efficiency of the virus is reduced, and it is not possible to prepare a virus having the titer necessary for the production of a transgenic mouse. The same reason can be considered for other viral vectors used for gene therapy such as adenovirus and AAV.
本発明の課題は、脂肪組織及び／又は膵臓組織での特異的発現が可能かつ導入効率のよいエレメントを提供することである。 An object of the present invention is to provide an element capable of specific expression in adipose tissue and / or pancreatic tissue and having high introduction efficiency.
本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、マウスａＰ２遺伝子プロモーター由来の配列の中の一部の領域が脂肪組織での発現を促進するために重要であること、この領域を含む短縮型のプロモーターを用いてトランスジェニックマウスを作製した場合にも、脂肪組織及び膵臓組織に特異的な活性ならびに十分な発現量を有すること、ヒト由来の配列においてもマウスと同様の活性を有することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have found that a partial region in the sequence derived from the mouse aP2 gene promoter is important for promoting expression in adipose tissue, Even when a transgenic mouse is produced using a truncated promoter containing this region, it has a specific activity and sufficient expression level in adipose tissue and pancreatic tissue, and human-derived sequences are similar to those in mice. It has been found that it has activity, and the present invention has been completed.
すなわち本発明は、
（１）　配列番号：２２に示される塩基配列を少なくとも有するＤＮＡであって、かつ脂肪組織および／もしくは膵臓において特異的なプロモーター活性を有する、単離されたＤＮＡ、
（２）（ａ）配列番号：２２に示される塩基配列において、少なくとも１塩基の置換、欠失、挿入又は付加を有するＤＮＡ、（ｂ）配列番号：２２に示される塩基配列からなるＤＮＡの相補鎖とストリンジェントな条件下でハイブリダイズしうるＤＮＡ、及び（ｃ）配列番号：２２に示される塩基配列からなるＤＮＡに少なくとも９０％の配列同一性を有するＤＮＡからなる群より選択されたＤＮＡであって、かつ脂肪組織および／または膵臓組織において特異的なプロモーター活性を有する、単離されたＤＮＡ、
（３）　さらに配列番号：２１に示される塩基配列を有するＤＮＡである、前記（１）又は（２）記載の単離されたＤＮＡ、
（４）　さらに配列番号：２３に示される塩基配列を有するＤＮＡである、前記（１）～（３）のいずれかに記載の単離されたＤＮＡ、
（５）　配列番号：２８に示される塩基配列を少なくとも有するＤＮＡであって、かつ脂肪組織および／もしくは膵臓において特異的なプロモーター活性を有する、単離されたＤＮＡ、　
（６） （ａ）配列番号：２８に示される塩基配列において、少なくとも１塩基の置換、欠失、挿入又は付加を有するＤＮＡ、（ｂ）配列番号：２８に示される塩基配列からなるＤＮＡの相補鎖とストリンジェントな条件下でハイブリダイズしうるＤＮＡ、及び（ｃ）配列番号：２８に示される塩基配列からなるＤＮＡに少なくとも９０％の配列同一性を有するＤＮＡからなる群より選択されたＤＮＡであって、かつ脂肪組織および／または膵臓組織において特異的なプロモーター活性を有する、単離されたＤＮＡ、
（７）　さらに配列番号：２７に示される塩基配列を有するＤＮＡである、請求項５又は６記載の単離されたＤＮＡ、　
（８）　さらに配列番号：２９に示される塩基配列を有するＤＮＡである、請求項５～７のいずれかに記載の単離されたＤＮＡ、
（９）　前記（１）～（８）のいずれかに記載の単離されたＤＮＡに外来遺伝子が連結されたＤＮＡ、
（１０） 　前記（１）～（８）のいずれかに記載の単離されたＤＮＡと外来遺伝子が作動可能に連結されてなる発現ベクター、
（１１） 　外来遺伝子が、対象疾患を治療するためのタンパク質をコードする遺伝子である、前記（１０）に記載の発現ベクター、
（１２） 外来遺伝子が、対象疾患を治療するための核酸である、前記（１０）に記載の発現ベクター、
（１３）前記（１１）または（１２）のいずれかに記載の発現ベクターを有効成分として含有してなる治療剤、
（１４）前記（１０）～（１３）のいずれかに記載の発現ベクターを保持してなるトランスジェニック非ヒト哺乳動物、
（１５） 発現ベクターが哺乳動物の染色体中に組み込まれてなる、前記（１４）記載のトランスジェニック非ヒト哺乳動物、
（１６）　前記（１４）または請求項（１５）のいずれかに記載されたトランスジェニック非ヒト哺乳動物に由来する細胞、または
（１７）前記（１４）または（１５）のいずれかに記載のトランスジェニック非ヒト哺乳動物を用いることを特徴とする、スクリーニング方法、
に関する。 That is, the present invention
(1) an isolated DNA having at least the base sequence shown in SEQ ID NO: 22 and having specific promoter activity in adipose tissue and / or pancreas,
(2) (a) DNA having at least one base substitution, deletion, insertion or addition in the base sequence shown in SEQ ID NO: 22; (b) complementary DNA consisting of the base sequence shown in SEQ ID NO: 22 A DNA selected from the group consisting of DNA that can hybridize with the strand under stringent conditions, and (c) a DNA having a sequence identity of at least 90% to the DNA consisting of the base sequence represented by SEQ ID NO: 22 Isolated DNA having specific promoter activity in adipose and / or pancreatic tissue,
(3) The isolated DNA according to (1) or (2), which is further DNA having the base sequence represented by SEQ ID NO: 21;
(4) The isolated DNA according to any one of (1) to (3), which is further DNA having the base sequence represented by SEQ ID NO: 23,
(5) an isolated DNA having at least the base sequence shown in SEQ ID NO: 28 and having specific promoter activity in adipose tissue and / or pancreas,
(6) (a) DNA having at least one base substitution, deletion, insertion or addition in the base sequence shown in SEQ ID NO: 28, (b) complementation of DNA consisting of the base sequence shown in SEQ ID NO: 28 A DNA selected from the group consisting of DNA that can hybridize with a strand under stringent conditions, and (c) a DNA having at least 90% sequence identity to the DNA consisting of the base sequence shown in SEQ ID NO: 28 Isolated DNA having specific promoter activity in adipose and / or pancreatic tissue,
(7) The isolated DNA according to claim 5 or 6, which further has a base sequence represented by SEQ ID NO: 27,
(8) The isolated DNA according to any one of claims 5 to 7, which is a DNA having the base sequence represented by SEQ ID NO: 29,
(9) DNA in which a foreign gene is linked to the isolated DNA of any one of (1) to (8),
(10) an expression vector in which the isolated DNA according to any one of (1) to (8) and a foreign gene are operably linked;
(11) The expression vector according to (10), wherein the foreign gene is a gene encoding a protein for treating the target disease,
(12) The expression vector according to (10), wherein the foreign gene is a nucleic acid for treating the target disease,
(13) A therapeutic agent comprising the expression vector according to any of (11) or (12) as an active ingredient,
(14) a transgenic non-human mammal comprising the expression vector according to any one of (10) to (13),
(15) The transgenic non-human mammal according to (14), wherein the expression vector is integrated into the chromosome of the mammal,
(16) A cell derived from the transgenic non-human mammal described in any one of (14) or (15), or (17) the trans described in any one of (14) or (15). A screening method, characterized by using a transgenic non-human mammal,
About.
本発明の新規のプロモーターを用いることにより、目的とする遺伝子を脂肪組織および／又は膵臓組織において特異的発現できるトランスジェニックマウスの作製や、これらの組織をターゲットとした遺伝子治療を行うことが可能となる。 By using the novel promoter of the present invention, it is possible to produce a transgenic mouse capable of specifically expressing a target gene in adipose tissue and / or pancreatic tissue, and to perform gene therapy targeting these tissues. Become.
図１は、今回作製したａＰ２プロモーター由来配列のレポーターアッセイ用のコンストラクトの模式図である。FIG. 1 is a schematic diagram of a reporter assay construct for the aP2 promoter-derived sequence prepared this time. 図２は、ａＰ２プロモーター由来配列のレポーターアッセイ用コンストラクトと、内部コントロールレポーターとしてウミシイタケルシフェラーゼを発現するベクターであるｐＲＬ－Ｔｋベクターとを共発現した時のルシフェラーゼ活性を示すグラフである。横軸は、２９３でａＰ２－１１－ｌｕｃを導入したときの発光強度を１として他のコンストラクトを用いたときの発光強度の相対値を示している。FIG. 2 is a graph showing the luciferase activity when co-expressing a reporter assay construct of an aP2 promoter-derived sequence and a pRL-Tk vector, which is a vector expressing Renilla luciferase as an internal control reporter. The horizontal axis shows the relative value of the emission intensity when other constructs are used with the emission intensity when aP2-11-luc is introduced at 293 being 1. 図３は、ヒトとマウスのａＰ２プロモーター由来配列のレポーターベクターをそれぞれｐＲＬ－Ｔｋベクターと共発現した時のルシフェラーゼ活性を示すグラフである。横軸は、未分化３Ｔ３－Ｌ１でａＰ２－１１を導入したときの発光強度を１として他のコンストラクトを用いたときの発光強度の相対値を示している。FIG. 3 is a graph showing the luciferase activity when reporter vectors of human and mouse aP2 promoter-derived sequences are coexpressed with the pRL-Tk vector, respectively. The abscissa represents the relative value of the luminescence intensity when other constructs are used with the luminescence intensity when aP2-11 is introduced into undifferentiated 3T3-L1 being 1.
本明細書において、「プロモーター」とは、細胞内でＲＮＡポリメラーゼとの会合が可能な発現調節領域を含むものであり、かかる領域の下流（３'末端方向）に存在するコーディング配列の転写を開始させる機能を有する領域を含むものをいう。さらに、前記「プロモーター」は、組織特異的な発現調節を担う、いわゆるエンハンサー領域をも含む領域をいう。 In the present specification, the “promoter” includes an expression regulatory region capable of associating with RNA polymerase in a cell, and initiates transcription of a coding sequence existing downstream (toward the 3 ′ end) of the region. It includes a region having a function to be performed. Furthermore, the “promoter” refers to a region including a so-called enhancer region responsible for tissue-specific expression regulation.
脂肪組織および／又は膵臓組織において特異的な発現とは、脂肪組織および／又は膵臓組織における発現が、他の組織と比較して明らかに高いことを意味する。 Specific expression in adipose tissue and / or pancreatic tissue means that expression in adipose tissue and / or pancreatic tissue is clearly higher compared to other tissues.
脂肪組織とは、哺乳動物の脂肪組織 であれば特に限定はなく、例えば、皮下脂肪組織 、内臓脂肪組織 、白色脂肪組織 、褐色脂肪組織 、生殖器周囲脂肪組織、腸間膜脂肪組織、肩甲骨間褐色脂肪組織等を含む。 The adipose tissue is not particularly limited as long as it is a mammalian adipose tissue, such as subcutaneous adipose tissue, visceral adipose tissue, white adipose tissue, brown adipose tissue, perigenital adipose tissue, mesenteric adipose tissue, scapula Contains brown adipose tissue.
本発明のＤＮＡは、配列番号：２２に示される塩基配列を少なくとも有してなるＤＮＡであってかつ脂肪組織および／又は膵臓組織において特異的なプロモーター活性を有することを１つの特徴とする単離されたＤＮＡである。本発明のＤＮＡは、配列番号：２２に示される塩基配列を有しているため、インビボにおいても脂肪組織および／又は膵臓組織特異的に発現できるという優れた性質を有する。 The DNA of the present invention is an DNA having at least the base sequence shown in SEQ ID NO: 22 and having a specific promoter activity in adipose tissue and / or pancreatic tissue. DNA. Since the DNA of the present invention has the base sequence shown in SEQ ID NO: 22, it has an excellent property that it can be expressed specifically in adipose tissue and / or pancreatic tissue even in vivo.
前記配列番号：２２に示される塩基配列は、マウス由来のアディポサイトＰ２遺伝子プロモーターにおいて１６６２～２３５７位からなる領域の配列である。今回発明者らはこれらの配列が、インビボにおける脂肪組織および／又は膵臓組織の特異的発現に重要な働きをすることを見出した。一般に、インビトロにおける遺伝子発現の動向はインビボにおける実際の遺伝子発現の動向と大きく異なる場合が多い。 The base sequence shown in SEQ ID NO: 22 is a sequence of the region consisting of positions 1662 to 2357 in the mouse-derived adipocyte P2 gene promoter. The inventors have now found that these sequences play an important role in the specific expression of adipose and / or pancreatic tissue in vivo. In general, gene expression trends in vitro often differ greatly from actual gene expression trends in vivo.
本発明のＤＮＡのプロモーター活性は、例えば、下記のＩ～ＩＩＩのステップ：Ｉ 被検対象のＤＮＡの制御下に、レポーター遺伝子（例えば、ルシフェラーゼ遺伝子、アルカリホスファターゼ遺伝子など）を作動可能に連結した配列を保持した活性測定用プラスミドを作製するステップ、ＩＩ 前記Ｉの活性測定用プラスミドを用いて適切な脂肪組織の細胞（もしくは膵臓組織の細胞）及び対照細胞を形質転換するステップ、並びにＩＩＩ 前記ＩＩで得られた細胞におけるレポーター遺伝子の発現の有無を検出し、発現強度を測定するステップを含み、レポータージーンアッセイなどにより決定できる。かかる測定方法により、脂肪組織の細胞（もしくは膵臓組織の細胞）において対照細胞と比較して強い発現が検出された場合、かかるＤＮＡは、脂肪組織(膵臓組織)特異的なプロモーターであることの指標となる。ここでレポーター遺伝子の発現産物量がプロモーター活性の強度の指標となる。 The promoter activity of the DNA of the present invention is, for example, the following steps I to III: I a sequence in which a reporter gene (for example, luciferase gene, alkaline phosphatase gene, etc.) is operably linked under the control of the DNA to be tested. A step of preparing a plasmid for activity measurement retaining II, a step of transforming appropriate adipose tissue cells (or cells of pancreatic tissue) and control cells with the activity measurement plasmid of I above, and III This includes a step of detecting the presence or absence of expression of a reporter gene in the obtained cells and measuring the expression intensity, and can be determined by a reporter gene assay or the like. When a strong expression is detected in adipose tissue cells (or pancreatic tissue cells) as compared to control cells by such a measurement method, the DNA is an indicator that it is a promoter specific to adipose tissue (pancreatic tissue) It becomes. Here, the amount of the expression product of the reporter gene is an indicator of the strength of the promoter activity.
本発明のＤＮＡは、前記配列番号：２２に示される塩基配列において、少なくとも１塩基、具体的には、１又は複数個の塩基の置換、欠失、挿入又は付加を有するＤＮＡであって、かつ脂肪組織及び／又は膵臓組織において特異的なプロモーター活性を有する、単離されたＤＮＡであってもよい。置換、欠失、挿入又は付加（以下、単に「変異」と称する場合がある）の数及び導入位置は、前記Ｉ～ＩＩＩのステップを含む、レポータージーンアッセイを実施することによって、脂肪組織及び／又は膵臓組織特異的なプロモーターであることが決定されるものであればよい。好ましくは、１又は数個の置換、欠失、挿入又は付加が挙げられる。 The DNA of the present invention is a DNA having a substitution, deletion, insertion or addition of at least one base, specifically, one or more bases in the base sequence shown in SEQ ID NO: 22, and It may be an isolated DNA having specific promoter activity in adipose tissue and / or pancreatic tissue. The number of substitutions, deletions, insertions or additions (hereinafter may be simply referred to as “mutation”) and the position of introduction are determined by performing a reporter gene assay comprising the steps I to III above and / or Any promoter that is determined to be a pancreatic tissue-specific promoter may be used. Preferably, one or several substitutions, deletions, insertions or additions are included.
人為的な変異の導入方法としては、慣用の部位特異的変異導入法などにより行なうことができる。部位特異的変異を導入する方法としては、例えば、アンバー変異を利用する方法 （ギャップド・デュプレックス法、Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ、 １２、９４４１～９４５６ (１９８４)）やアンバー変異を利用したＰＣＲによる方法（国際公開第９８／０２５３５）等を用いることができる。 Artificial mutation can be introduced by a conventional site-specific mutation introduction method. Examples of methods for introducing site-specific mutation include a method using amber mutation (gapped duplex method, Nucleic Acids Research, 12, 9441-9456 (1984)) and a PCR method using amber mutation (international publication). 98/02535) or the like can be used.
本発明のＤＮＡは、脂肪組織及び／又は膵臓組織において特異的なプロモーター活性を有するＤＮＡであれば、配列番号：２２に示される塩基配列からなるＤＮＡの相補鎖とストリンジェントな条件下でハイブリダイズしうるＤＮＡであって、かつ脂肪組織および／または膵臓組織において特異的なプロモーター活性を有する単離されたＤＮＡも本発明に包含される。かかるＤＮＡは、例えば、前記Ｉ～ＩＩＩのステップを含む、レポータージーンアッセイを実施することによって、脂肪組織および／または膵臓組織特異的なプロモーターであることが決定されるものであればよい。 If the DNA of the present invention has a specific promoter activity in adipose tissue and / or pancreatic tissue, it hybridizes under stringent conditions with a complementary strand of DNA consisting of the base sequence shown in SEQ ID NO: 22. Isolated DNA having specific promoter activity in adipose tissue and / or pancreatic tissue is also encompassed by the present invention. Such DNA is not particularly limited as long as it is determined to be a promoter specific to adipose tissue and / or pancreatic tissue by performing a reporter gene assay including the steps I to III.
ここで、ハイブリダイゼーション操作は、例えば、モレキュラー・クローニング：ア・ラボラトリー・マニュアル 第２版（コールド・スプリング・ハーバー・ラボラトリー発行,１９８９年） などに記載の方法に従って、実施することができる。「ストリンジェントな条件」としては、前記の書籍などに記載のハイブリダイゼーションの条件が挙げられ、具体的には、１０% デキストラン硫酸、１Ｍ ＮａCｌ 、１％ ＳＤＳ、１００ mg/ml サケ精子ＤＮＡ 、１×106 ｃｐｍ／ｍｌ 32Ｐ 標識プローブ（配列番号２２）の条件下でハイブリダイズ反応を行った後、２×ＳＳＣ 、０．１％ＳＤＳ 、室温で２ 回、０．１×ＳＳＣ 、０．１％ＳＤＳ 、６０℃で２回の洗浄を行うような条件が挙げられる。 Here, the hybridization operation can be performed, for example, according to the method described in Molecular Cloning: A Laboratory Manual Second Edition (issued by Cold Spring Harbor Laboratory, 1989). Examples of “stringent conditions” include hybridization conditions described in the above-mentioned books and the like. Specifically, 10% dextran sulfate, 1M NaCl, 1% SDS, 100 mg / ml salmon sperm DNA, 1 After performing a hybridization reaction under the conditions of × 10 6 cpm / ml 32 P-labeled probe (SEQ ID NO: 22), 2 × SSC, 0.1% SDS, twice at room temperature, 0.1 × SSC, 0. The conditions are such that washing is performed twice at 1% SDS and 60 ° C.
本発明のＤＮＡには、配列番号：２２に示される塩基配列からなるＤＮＡに少なくとも９０％の配列同一性を有するＤＮＡ、好ましくは９５％以上、より好ましくは９８％以上の配列同一性を有するＤＮＡであって、脂肪組織および／または膵臓組織において特異的なエンハンサー活性を有する単離されたＤＮＡも含まれる。かかる配列同一性を有するＤＮＡは、例えば、前記Ｉ～ＩＩＩのステップを含む、レポータージーンアッセイを実施することによって、脂肪組織および／または膵臓組織特異的なプロモーターであることが決定されるものであればよい。 The DNA of the present invention includes DNA having at least 90% sequence identity to the DNA consisting of the base sequence shown in SEQ ID NO: 22, preferably 95% or more, more preferably 98% or more. Also included is isolated DNA having specific enhancer activity in adipose tissue and / or pancreatic tissue. If DNA having such sequence identity is determined to be a promoter specific to adipose tissue and / or pancreatic tissue by performing a reporter gene assay including the steps I to III described above, for example. Good.
本明細書において、「配列同一性」とは、当該分野で慣用のホモロジー検索プログラム（例えば、ＢＬＡＳＴ等）を初期設定で用いて決定することができる。本発明において、塩基配列の「配列同一性」とは、比較する２種の塩基配列を整列（アラインメント）させ、整列により一致した塩基配列の数を基準となる塩基配列の総数で除して算出した割合を％で示した数字である。なお、整列により生じたギャップは、不一致と見なして算出する。 In the present specification, “sequence identity” can be determined using a homology search program (for example, BLAST etc.) commonly used in this field by default. In the present invention, “sequence identity” of nucleotide sequences is calculated by aligning the two types of nucleotide sequences to be compared and dividing the number of nucleotide sequences matched by the alignment by the total number of reference nucleotide sequences. It is the number which showed the ratio which was done in%. Note that the gap generated by the alignment is calculated as a mismatch.
本発明の特定の態様としては、さらに配列番号：２１に示される塩基配列を含むものとする。このＤＮＡは作動可能なように連結されるものとするが、一般には配列番号：２１が上流側となるように連結されていることが望ましい。配列番号：２１に示される塩基配列は、マウス由来のアディポサイトＰ２遺伝子プロモーターの最上流の約５２０塩基からなる領域の配列である。この領域は一般に「エンハンサー」と呼ばれる領域であり、転写速度を増大させる働きがある。また、脂肪組織特異的発現に重要な役割を有することが報告されている（Ｊｏｕｒｎａｌ　ｏｆ　Ｃｅｌｌｕｌａｒ　Ｂｉｏｃｈｅｍｉｓｔｒｙ，第４９巻、１９９２年、２１９－２２４ページ）。 As a specific aspect of the present invention, the base sequence shown in SEQ ID NO: 21 is further included. This DNA is operably linked, but it is generally desirable that the DNA is linked so that SEQ ID NO: 21 is upstream. The base sequence shown in SEQ ID NO: 21 is a sequence of a region consisting of about 520 bases at the most upstream of the mouse-derived adipocyte P2 gene promoter. This region is generally called an “enhancer” and has a function of increasing the transfer speed. It has also been reported to have an important role in adipose tissue-specific expression (Journal of Cellular Biochemistry, Vol. 49, 1992, pages 219-224).
本発明の特定の態様としては、さらに配列番号：２３に示される塩基配列を有するＤＮＡを含むものとする。前記配列番号：２３に示される塩基配列は、マウス由来のアディポサイトＰ２遺伝子プロモーター（ａＰ２プロモーター） において５３５９～５５４８位の領域の約２００ｂｐの配列である。この領域は、近位プロモーター（ｐｒｏｘｉｍａｌ－ｐｒｏｍｏｔｅｒ）と呼ばれ、ＡＰ－１やＣ／ＥＢＰ（ＣＣＡＡＴ／ｅｎｈａｎｃｅｒ　ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎ）の転写因子の結合領域が含まれている。この領域は、脂肪分化には重要な働きをするが、トランスジェニック動物などインビボにおける脂肪組織特異的発現においての必須領域でないことが報告がされている。（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，Ｖｏｌ．８７，ｐｐ９５９０－９５９４，１９９０年）。この下流部には、ＴＡＴＡボックスやＣＡＡＴボックスと呼ばれる配列を含んでおり、ＲＮＡポリメレースをその正常な開始部位に導いたり、転写を促進したりする働きをすることが知られている。 As a specific embodiment of the present invention, it further includes DNA having the base sequence shown in SEQ ID NO: 23. The base sequence shown in SEQ ID NO: 23 is a sequence of about 200 bp in the region from 5359 to 5548 in the mouse-derived adipocyte P2 gene promoter (aP2 promoter). This region is called a proximal-promoter and includes a transcription factor binding region such as AP-1 or C / EBP (CCAAT / enhancer binding protein). Although this region plays an important role in adipose differentiation, it has been reported that it is not an essential region for adipose tissue-specific expression in vivo such as in transgenic animals. (Proc. Natl. Acad. Sci. USA, Vol. 87, pp 9590-9594, 1990). This downstream part contains sequences called TATA box and CAAT box, and it is known to lead RNA polymerase to its normal start site and to promote transcription.
本発明のＤＮＡにより、脂肪組織及び／もしくは膵臓組織特異的に外来遺伝子を発現させ得る発現ベクターが提供される。「発現ベクター」という用語は、転写されうる遺伝子産物の少なくとも一部をコードする外来遺伝子の配列を含むベクターのことを指す。外来遺伝子の配列は、場合によって最終的にはタンパク質、ポリペプチドまたはペプチドへと翻訳される。また別の場合には、これらの配列は翻訳されない。例えば、アンチセンス分子、リボザイムの他、ｍｉＲＮＡ(マイクロＲＮＡ)やｓｈＲＮＡ（ショートヘアピン構造ＲＮＡ）のようなＲＮＡ干渉作用を利用してｍＲＮＡを特異的に切断するｓｉＲＮＡの場合である。かかる発現ベクターも本発明に含まれる。 The DNA of the present invention provides an expression vector capable of expressing a foreign gene specifically in adipose tissue and / or pancreatic tissue. The term “expression vector” refers to a vector comprising a sequence of a foreign gene that encodes at least a portion of a gene product that can be transcribed. The sequence of the foreign gene is optionally translated into a protein, polypeptide or peptide. In other cases, these sequences are not translated. For example, in addition to antisense molecules and ribozymes, siRNA that specifically cleaves mRNA using RNA interference action such as miRNA (microRNA) and shRNA (short hairpin structure RNA). Such expression vectors are also included in the present invention.
本発明の発現ベクターは、本発明のＤＮＡを適切なベクターに挿入することにより得ることができる。本発明に用いられるベクターとしては、例えば、プラスミド、ファージ、コスミド、ウイルス（バクテリオファージ、動物ウイルスおよび植物ウイルス）および人工染色体（例えば、ＹＡＣ）が含まれる。 The expression vector of the present invention can be obtained by inserting the DNA of the present invention into an appropriate vector. Examples of the vector used in the present invention include plasmids, phages, cosmids, viruses (bacteriophages, animal viruses and plant viruses) and artificial chromosomes (for example, YAC).
本発明の発現ベクターは、転写の方向に関して、外来遺伝子配列の上流側に、上記の本発明のＤＮＡが作動可能に連結されていることを１つの特徴とし、脂肪組織及び／もしくは膵臓組織特異的に外来遺伝子を発現させ得るものである。好ましい態様は、配列番号２２に示される塩基配列、および配列番号２１に示される塩基配列を有するＤＮＡの下流に外来遺伝子が作動可能に連結されてなる発現ベクターである。さらに好ましい態様としては、配列番号２２に示される塩基配列、配列番号２１に示される塩基配列、および配列番号２３に示される塩基配列を有するＤＮＡの下流に外来遺伝子が作動可能に連結されてなる発現ベクターである。かかる発現ベクターは、外来遺伝子の上流に本発明のＤＮＡが局在するため、本発明のＤＮＡ中における組織特異的なプロモーターの制御下に外来遺伝子を発現させることができるので、前記外来遺伝子を脂肪組織特異的及び／もしくは膵臓組織特異的に発現させ得る。そのため、本発明の発現ベクターは、それ自体が脂肪組織及び／もしくは膵臓組織における疾患における極めて有用な治療剤のツールとなりうる。また、本発明の発現ベクターを保持してなるトランスジェニック非ヒト動物、及びこれに由来する細胞は、抗肥満薬や糖尿病薬、高脂血症薬、高血圧薬など脂肪組織（もしくは脂肪細胞）や膵臓組織（もしくは膵臓細胞）と関連を有する疾患（メタボリックシンドローム関連疾患など）を改善する薬剤の探索のためのツールになりうる。 The expression vector of the present invention is characterized in that the DNA of the present invention is operably linked upstream of the foreign gene sequence in the transcription direction, and is specific to adipose tissue and / or pancreatic tissue. In which foreign genes can be expressed. A preferred embodiment is an expression vector in which a foreign gene is operably linked downstream of DNA having the base sequence shown in SEQ ID NO: 22 and the base sequence shown in SEQ ID NO: 21. As a more preferred embodiment, an expression in which a foreign gene is operably linked downstream of a DNA having the base sequence shown in SEQ ID NO: 22, the base sequence shown in SEQ ID NO: 21, and the base sequence shown in SEQ ID NO: 23 It is a vector. In such an expression vector, since the DNA of the present invention is localized upstream of the foreign gene, the foreign gene can be expressed under the control of a tissue-specific promoter in the DNA of the present invention. It can be expressed tissue-specific and / or pancreatic tissue-specific. Therefore, the expression vector of the present invention can itself be a very useful therapeutic tool for diseases in adipose tissue and / or pancreatic tissue. Moreover, the transgenic non-human animal having the expression vector of the present invention and cells derived therefrom are adipose tissues (or fat cells) such as anti-obesity drugs, diabetes drugs, hyperlipidemia drugs, hypertension drugs, It can be a tool for searching for drugs that improve diseases associated with pancreatic tissue (or pancreatic cells) (such as metabolic syndrome-related diseases).
なお、上記の配列番号２１～２３で示される塩基配列はいずれもマウス由来であるが、本発明者らは、同様の作用を有する塩基配列をヒト由来の遺伝子より見出した。具体的には配列番号２１で示される塩基配列は配列番号２７で示される塩基配列に、配列番号２２で示される塩基配列は配列番号２８で示される塩基配列に、そして配列番号２３で示される塩基配列は配列番号２９で示される塩基配列に対応しているので、これらに置き換えることにより、同様の効果を得ることは当業者の常識の範囲内である。 The base sequences shown in SEQ ID NOs: 21 to 23 are all derived from mice, but the present inventors have found a base sequence having the same action from a human-derived gene. Specifically, the base sequence represented by SEQ ID NO: 21 is the base sequence represented by SEQ ID NO: 27, the base sequence represented by SEQ ID NO: 22 is the base sequence represented by SEQ ID NO: 28, and the base sequence represented by SEQ ID NO: 23. Since the sequence corresponds to the base sequence represented by SEQ ID NO: 29, it is within the common sense of those skilled in the art to obtain the same effect by replacing these sequences.
外来遺伝子としては、（Ａ）対象疾患を治療するためのタンパク質をコードする遺伝子、（Ｂ）対象疾患を治療するための核酸、および（Ｃ）マーカー遺伝子などが挙げられる。 Examples of the foreign gene include (A) a gene encoding a protein for treating the target disease, (B) a nucleic acid for treating the target disease, and (C) a marker gene.
前記「対象疾患」とは、例えば、脂肪組織及び／もしくは膵臓組織特異的に外来遺伝子を発現させることにより治療され得る疾患が挙げられる。具体的には、脂肪組織萎縮症、　II型糖尿病、高脂血症、肥満症及び類似のもの等が挙げられる。 Examples of the “target disease” include diseases that can be treated by expressing a foreign gene specifically in adipose tissue and / or pancreatic tissue. Specific examples include adipose tissue atrophy, type II diabetes, hyperlipidemia, obesity and the like.
（Ａ）対象疾患を治療するためのタンパク質としては、例えばインスリン、レプチン、及びグルコーストランスポーターなどが挙げられる。 (A) Examples of proteins for treating a target disease include insulin, leptin, and glucose transporter.
（Ｂ）対象疾患を治療するための核酸としては、ホスホロチオエートアンチセンスオリゴヌクレオチド、アプタマー、ｓｉＲＮＡ、または二本鎖ＲＮＡｉのような合成または天然に存在しない形態の核酸、および当該技術分野で周知の核酸の化学誘導体のような他の形態の核酸を用いることもできる。ｓｉＲＮＡなど干渉作用を利用した塩基配列の設計は、様々なものが利用できるが、一例としてｓｉＤｉｒｅｃｔ（ｈｔｔｐ：／／ｄｅｓｉｇｎ．ＲＮＡｉ．ｊｐ／）により配列設計が可能である。 (B) Nucleic acids for treating the target disease include synthetic or non-naturally occurring forms of nucleic acids, such as phosphorothioate antisense oligonucleotides, aptamers, siRNAs, or double stranded RNAi, and nucleic acids well known in the art Other forms of nucleic acids such as the chemical derivatives of can also be used. Various nucleotide sequences can be used for designing siRNA and other interference sequences. For example, siDirect (http://design.RNAi.jp/) can be used for sequence design.
（Ｃ）マーカー遺伝子としては、β－ガラクトシダーゼ遺伝子、アルカリホスファターゼ遺伝子、クロラムフェニコールアセチルトランスフェラーゼ遺伝子、成長ホルモン遺伝子、ルシフェラーゼ遺伝子、緑色蛍光タンパク質遺伝子並びにそれらの誘導体等が挙げられる。前記「誘導体」には、人工的に作製された変異体が含まれる。 (C) Examples of the marker gene include β-galactosidase gene, alkaline phosphatase gene, chloramphenicol acetyltransferase gene, growth hormone gene, luciferase gene, green fluorescent protein gene and derivatives thereof. The “derivative” includes an artificially produced mutant.
本発明の発現ベクターにおいて、外来遺伝子が前記「対象疾患を治療するためのタンパク質をコードする遺伝子」や「対象疾患を治療するための核酸」である場合、脂肪組織及び／もしくは膵臓組織特異的な治療剤の有効成分として使用されうる。かかる治療剤も本発明に含まれる。 In the expression vector of the present invention, when the foreign gene is “the gene encoding a protein for treating the target disease” or “the nucleic acid for treating the target disease”, it is specific to adipose tissue and / or pancreatic tissue. It can be used as an active ingredient of a therapeutic agent. Such therapeutic agents are also included in the present invention.
本発明の治療剤は、前記発現ベクターを有効成分として含有することを１つの特徴とし、脂肪組織及び／もしくは膵臓組織特異的に外来遺伝子、具体的には対象疾患を治療するためのタンパク質をコードする遺伝子や対象疾患を治療するための核酸を発現させ得るものである。かかる治療剤は、前記発現ベクターを有効成分として含有しているため、脂肪組織及び／もしくは膵臓組織に対して高い特異性を発揮するという優れた性質を有する。 The therapeutic agent of the present invention is characterized by containing the expression vector as an active ingredient, and encodes a foreign gene specifically for a fat tissue and / or pancreatic tissue, specifically a protein for treating a target disease. Or a nucleic acid for treating a target disease. Since such a therapeutic agent contains the expression vector as an active ingredient, it has an excellent property of exhibiting high specificity for adipose tissue and / or pancreatic tissue.
本発明の治療剤は、有効成分である発現ベクターを安定な状態に保持するための成分、例えば、緩衝成分、分解保護剤（例えば、核酸分解酵素の阻害因子など）などを適宜含有してもよい。本発明の遺伝子発現剤は、細胞及び／又は組織への導入に適した薬剤をさらに含有してもよい。 The therapeutic agent of the present invention may appropriately contain components for maintaining the expression vector, which is an active ingredient, in a stable state, for example, buffer components, degradation protective agents (for example, inhibitors of nucleolytic enzymes, etc.) and the like. Good. The gene expression agent of the present invention may further contain a drug suitable for introduction into cells and / or tissues.
本発明の治療剤における発現ベクターの量は、使用目的により適宜設定することができる。例えば、後述の治療剤に用いる場合、治療目的の疾患、患者の年齢、体重等により適宜調節することができ、例えば、発現ベクター量として０．０００１～１００ｍｇ、好ましくは０．００１～１０ｍｇであることが望ましい。かかる投与用量を数日ないし数ヶ月に１回投与することが望ましい。 The amount of the expression vector in the therapeutic agent of the present invention can be appropriately set depending on the purpose of use. For example, when used in the therapeutic agent described later, it can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient. For example, the expression vector amount is 0.0001 to 100 mg, preferably 0.001 to 10 mg. It is desirable. It is desirable to administer such a dose once every few days to several months.
本発明の治療剤は、例えば、リポフェクション法、リン酸－カルシウム共沈法；ＤＥＡＥ－デキストラン法；微小ガラス管を用いた直接注入法などの方法により、細胞に導入することができる。 The therapeutic agent of the present invention can be introduced into cells by, for example, a lipofection method, a phosphate-calcium coprecipitation method; a DEAE-dextran method; a direct injection method using a micro glass tube.
本発明の治療剤の組織への導入法としては、内包型リポソームによる導入法、静電気型リポソームによる導入法、ＨＶＪ－リポソーム法、改良型ＨＶＪ－リポソーム法（ＨＶＪ－ＡＶＥ リポソーム法）、パーティクル銃で担体（金属粒子）とともに有効成分を細胞に移入する方法、正電荷ポリマーによる導入法等が挙げられる。 Examples of the method for introducing the therapeutic agent of the present invention into a tissue include introduction method using encapsulated liposome, introduction method using electrostatic liposome, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), particle gun Examples thereof include a method of transferring an active ingredient together with a carrier (metal particles) into a cell, a method of introduction with a positively charged polymer, and the like.
本発明の治療剤によれば、脂肪組織や膵臓組織に高い特異性を有しているため、脂肪組織特異的及び／もしくは膵臓組織特異的に外来遺伝子を発現させることにより治療され得る疾患（例えば、肥満、ＩＩ型糖尿病、脂肪組織萎縮症、膵臓癌など）に対して特異的に、症状の軽減又は治癒効果を発揮しうる。したがって、同時に、脂肪組織や膵臓組織以外の組織に対する、治療剤の影響を低減することが可能になるという優れた性質を有する。 According to the therapeutic agent of the present invention, since it has high specificity for adipose tissue and pancreatic tissue, diseases that can be treated by expressing foreign genes specifically for adipose tissue and / or pancreatic tissue (for example, , Obesity, type II diabetes, adipose tissue atrophy, pancreatic cancer, etc.), and can exhibit a symptom reduction or curative effect. Therefore, at the same time, it has an excellent property that it becomes possible to reduce the influence of the therapeutic agent on tissues other than adipose tissue and pancreatic tissue.
「脂肪組織特異的及び／もしくは膵臓組織特異的に外来遺伝子を発現させることにより治療され得る疾患」としては、前記「対象疾患」と同様のものが挙げられる。 Examples of the “disease that can be treated by expressing a foreign gene specifically in adipose tissue and / or pancreatic tissue” include the same as the “target disease”.
本発明の治療剤の患者への導入法としては、該治療剤を直接体内に導入するインビボ法、及び、ヒトからある種の細胞を取り出して体外で治療剤を該細胞に導入し、その細胞を体内に戻すエクスビボ法がある（日本遺伝子治療学会編遺伝子治療開発研究ハンドブック, エヌ・ティー・エス,１９９９年 ）。 As a method for introducing the therapeutic agent of the present invention into a patient, an in vivo method in which the therapeutic agent is directly introduced into the body, and a certain cell is taken out from a human and introduced into the cell outside the body, and the cell There is an ex vivo method to return the substance to the body (Genetic Therapy Development Handbook edited by the Japanese Society for Gene Therapy, NTS, 1999).
インビボ法により投与する場合は、本発明の治療剤は、例えば、静脈、動脈、皮下、皮内、筋肉内などに投与するか、又は対象の脂肪組織及び／もしくは膵臓組織そのものに直接局所投与することができる。 When administered by an in vivo method, the therapeutic agent of the present invention is administered, for example, intravenously, artery, subcutaneously, intradermally, intramuscularly, or directly locally to the subject adipose tissue and / or pancreatic tissue itself. be able to.
製剤形態としては、上記の各投与形態に合った種々の製剤形態（例えば液剤など）をとり得る。例えば、治療剤を含有した注射剤の場合、当該注射剤は常法により調製することができ、例えば、適切な溶剤（ＰＢＳ等の緩衝液、生理食塩水、滅菌水等）に溶解した後、場合によっては、フィルター等で濾過滅菌し、次いで無菌的な容器に充填することにより調製することができる。当該注射剤には必要に応じて慣用の担体等を加えても良い。ＨＶＪ－リポソーム等のリポソームにおいては、懸濁剤、凍結剤、遠心分離濃縮凍結剤などのリポソーム製剤の形態とすることができる。 As a preparation form, various preparation forms (for example, a liquid agent etc.) suitable for each of the above administration forms can be taken. For example, in the case of an injection containing a therapeutic agent, the injection can be prepared by a conventional method, for example, after dissolving in an appropriate solvent (buffer solution such as PBS, physiological saline, sterilized water, etc.) In some cases, it can be prepared by sterilizing by filtration with a filter or the like and then filling into an aseptic container. A conventional carrier or the like may be added to the injection as necessary. Liposomes such as HVJ-liposomes can be in the form of liposome preparations such as suspensions, freezing agents, and centrifugal concentrated freezing agents.
その他に徐放性の製剤（ミニペレット製剤等）を調製し患部近くに埋め込むこと、あるいはオスモチックポンプなどを用いて患部に連続的に徐々に投与することも可能である。 In addition, a sustained-release preparation (mini-pellet preparation or the like) can be prepared and implanted near the affected area, or can be gradually and gradually administered to the affected area using an osmotic pump or the like.
本発明の治療剤の投与形態は、例えば、実験手引書などにその調製法、投与法などが詳しく解説されている（別冊実験医学, 遺伝子治療の基礎技術, 羊土社,１９９６年 、別冊実験医学, 遺伝子導入＆発現解析実験法, 羊土社,１９９７年）。以下、具体例を挙げて説明する。 The dosage form of the therapeutic agent of the present invention is described in detail, for example, in the experiment manual, etc., its preparation method, administration method, etc. (separate volume experimental medicine, basic technology of acupuncture gene therapy, Yodosha, 1996, separate volume experiment) Medicine, Acupuncture Gene Introduction & Expression Analysis Experimental Method, Yodosha, 1997). Hereinafter, a specific example will be described.
本発明の発現ベクターの構築において、非ウイルスベクターが用いられた場合、以下のような手法により、本発明の治療剤を細胞や組織に導入することができる。 When a non-viral vector is used in the construction of the expression vector of the present invention, the therapeutic agent of the present invention can be introduced into cells or tissues by the following technique.
細胞への遺伝子導入法としては、リポフェクション法、リン酸－カルシウム共沈法、微小ガラス管を用いた直接注入法などが挙げられる。 Examples of the method for introducing a gene into a cell include a lipofection method, a phosphate-calcium coprecipitation method, and a direct injection method using a micro glass tube.
組織への遺伝子導入法としては、内包型リポソームによる遺伝子導入法、静電気型リポソームによる遺伝子導入法、ＨＶＪ－リポソーム法、改良型ＨＶＪ－リポソーム法（ＨＶＪ－ＡＶＥリポソーム法）、受容体介在性遺伝子導入法、パーティクル銃で担体（金属粒子）とともに有効成分を細胞に移入する方法、ｎａｋｅｄ－ＤＮＡの直接導入法、正電荷ポリマーによる導入法等が挙げられる。 Gene transfer methods to tissues include gene transfer method using encapsulated liposomes, gene transfer method using electrostatic liposomes, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), receptor-mediated gene transfer And a method of transferring an active ingredient together with a carrier (metal particles) with a particle gun, a direct introduction method of naked-DNA, an introduction method using a positively charged polymer, and the like.
本発明の発現ベクターの構築において、ウイルスベクターを用いる場合、かかるウイルスベクターとしては、組換えアデノウイルス、レトロウイルス等が挙げられる。より具体的には、例えば、レンチウイルス、無毒化したレトロウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルス、ワクシニアウイルス、ポックスウイルス、ポリオウイルス、シンビスウイルス、センダイウイルス、ＳＶ４０、免疫不全症ウイルス（ＨＩＶ）等のＤＮＡウイルス又はＲＮＡウイルスに本発明のＤＮＡと該ＤＮＡに作動可能に連結した外来遺伝子とを導入し、得られた発現ベクターを感染させることによって、細胞内に遺伝子を導入することが可能である。前記ウイルスベクターのうち、受精卵への感染はレンチウイルスを用いることが望ましい。これは、レンチウイルスが宿主細胞ＤＮＡに不可逆的に組み込まれるというレトロウイルスの一般的な特徴を有するだけでなく、非増殖細胞を感染させる能力も有するからである。レンチウイルスはレトロウイルスの亜群であり、ヒト免疫不全ウイルスＨＩＶ－１およびＨＩＶ－２、サル免疫不全ウイルス（ＳＩＶ）などの種々の霊長類ウイルスならびに非霊長類ウイルス（たとえば、マエディビスナウイルス（ＭＷ）、ネコ免疫不全ウイルス（ＦＩＶ）、ウマ伝染性貧血ウイルス（ＥＩＡＶ）、ヤギ関節炎脳炎ウイルス（ＣＡＥＶ）およびウシ免疫不全ウイルス（ＢＩＶ））が含まれる。 When a viral vector is used in the construction of the expression vector of the present invention, examples of such a viral vector include recombinant adenovirus, retrovirus and the like. More specifically, for example, lentivirus, detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, shinbis virus, Sendai virus, SV40, immunodeficiency virus ( HIV) and other DNA viruses or RNA viruses can be introduced into a cell by introducing the DNA of the present invention and a foreign gene operably linked to the DNA, and infecting the resulting expression vector. Is possible. Of the viral vectors, it is desirable to use a lentivirus to infect fertilized eggs. This is because not only has the general characteristics of retroviruses that lentiviruses are irreversibly incorporated into host cell DNA, but also the ability to infect non-proliferating cells. Lentiviruses are a subgroup of retroviruses, various primate viruses such as human immunodeficiency viruses HIV-1 and HIV-2, simian immunodeficiency virus (SIV), and non-primate viruses (eg, maedi bisna virus ( MW), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), goat arthritis encephalitis virus (CAEV) and bovine immunodeficiency virus (BIV)).
本発明の治療剤の導入方法は、前記と同様である。投与量については、治療目的の疾患、患者の年齢、体重等により適宜調節することができ、例えば、治療剤中に含まれる発現ベクター量として０．０００１～１００ｍｇ、好ましくは０．００１～１０ｍｇであることが望ましい。かかる投与用量を数日ないし数ヶ月に１回投与することが望ましい。 The method for introducing the therapeutic agent of the present invention is the same as described above. The dose can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient. For example, the amount of the expression vector contained in the therapeutic agent is 0.0001 to 100 mg, preferably 0.001 to 10 mg. It is desirable to be. It is desirable to administer such a dose once every few days to several months.
本発明の発現ベクターによれば、かかる発現ベクターを保持したトランスジェニック非ヒト哺乳動物が提供される。 According to the expression vector of the present invention, a transgenic non-human mammal carrying such an expression vector is provided.
本発明のトランスジェニック非ヒト哺乳動物は、前記発現ベクターを保持してなるトランスジェニック非ヒト哺乳動物であって、該発現ベクター中の外来遺伝子が脂肪組織および／もしくは膵臓組織特異的に発現することを１つの特徴とする。外来遺伝子として、マーカー遺伝子を含有した発現ベクターを保持したトランスジェニック非ヒト哺乳動物は、脂肪組織や膵臓組織において特異的なプロモーターを保持しているため、これらの組織特異的なマーカー遺伝子の発現の有無及び強度を指標として、簡便に糖尿病薬や抗肥満薬などを含むメタボリックシンドローム関連疾患の治療薬をスクリーニングすることができるという優れた性質を有する。また、外来遺伝子として、対象疾患を治療するためのタンパク質をコードする遺伝子や、ｍｉＲＮＡ、ｓｈＲＮＡなどの核酸を含有した発現ベクターを保持したトランスジェニック非ヒト哺乳動物は、脂肪組織および／もしくは膵臓組織において特異的なプロモーターを保持しているため、これらの組織において発現させた場合の該タンパク質や該核酸の治療効果を評価することができる。そのほか、本発明のトランスジェニック非ヒト哺乳動物は、特定の遺伝子の生体中のこれらの組織への影響を調べるものとして使用される。例えば、肥満患者と健常人の間において脂肪組織における発現量が異なる遺伝子があれば、これらをコードするタンパク質を脂肪組織において発現させることにより生体内でその影響を調べることができる。また、脂肪組織などに特異的に発現させたｍｉＲＮＡなどの核酸の治療効果を調べることができる。 The transgenic non-human mammal of the present invention is a transgenic non-human mammal having the expression vector, wherein a foreign gene in the expression vector is expressed specifically in adipose tissue and / or pancreatic tissue. Is one feature. Transgenic non-human mammals that retain an expression vector containing a marker gene as a foreign gene retain a specific promoter in adipose tissue or pancreatic tissue. Using the presence / absence and strength as an index, it has an excellent property that it can easily screen for therapeutic agents for metabolic syndrome-related diseases including diabetes drugs and anti-obesity drugs. In addition, a transgenic non-human mammal having an expression vector containing a gene encoding a protein for treating the target disease or a nucleic acid such as miRNA or shRNA as a foreign gene is used in adipose tissue and / or pancreatic tissue. Since a specific promoter is retained, the therapeutic effect of the protein or the nucleic acid when expressed in these tissues can be evaluated. In addition, the transgenic non-human mammal of the present invention is used to examine the influence of a specific gene on these tissues in the living body. For example, if there are genes with different expression levels in adipose tissue between obese patients and healthy individuals, the effects can be examined in vivo by expressing proteins encoding these in adipose tissue. In addition, the therapeutic effect of a nucleic acid such as miRNA expressed specifically in adipose tissue can be examined.
本発明のトランスジェニック非ヒト哺乳動物においては、発現ベクターが哺乳動物の染色体中に組み込まれて保持されていてもよい。 In the transgenic non-human mammal of the present invention, the expression vector may be incorporated and retained in the chromosome of the mammal.
「非ヒト哺乳動物」としては、ヒト以外の哺乳動物、例えば、マウス、ラット、ウサギブタ、イヌ、ヒツジ、ヤギなどの哺乳動物などが挙げられる。なかでも、医薬研究用途での長い利用実績とデータの蓄積があり、これまでに数多くの病態モデルが作製されてきた実績があることや、遺伝子改変動物の作製技術が確立されている等の観点から、マウス、ラットなどに代表されるげっ歯類動物が好ましく、特にマウスが好ましい。 Examples of the “non-human mammal” include mammals other than humans, for example, mammals such as mice, rats, rabbit pigs, dogs, sheep and goats. Above all, there is a long history of use in medical research and accumulation of data, and there is a track record that many pathological models have been created so far, and the production technology of genetically modified animals has been established. Therefore, rodents represented by mice, rats and the like are preferable, and mice are particularly preferable.
本発明のトランスジェニック非ヒト哺乳動物は、公知の方法により作製することができる。例えば、例えば下記の工程（ａ）～（ｃ）を含む方法により製造することができる。
（ａ）ベクター等を受精卵に導入し、遺伝子導入受精卵を仮親動物に移植する工程；
（ｂ）前記動物から出生した子孫からトランスジェニック動物を選別する工程；および
（ｃ）前記選別した動物（ファウンダー）から系統を樹立する工程。 The transgenic non-human mammal of the present invention can be produced by a known method. For example, it can be produced by a method including the following steps (a) to (c), for example.
(A) a step of introducing a vector or the like into a fertilized egg and transplanting the transgenic fertilized egg into a foster animal;
(B) selecting a transgenic animal from offspring born from the animal; and (c) establishing a line from the selected animal (founder).
（ａ）の工程のベクターなどを受精卵に導入する場合にはマイクロインジェクション法（マウス胚の操作マニュアル（近代出版、１９８９年）、分子生物学プロトコール（南江堂、１９９４年））や、レンチウイルスを受精卵に感染させるレンチウイルス法（Ｌｏｉｓ Ｃら，サイエンス、第２９５巻、２００２年、８６８－８７２ページ）が用いられる。 When introducing the vector of step (a) into a fertilized egg, a microinjection method (manual operation manual for mouse embryo (Modern Publishing, 1989), molecular biology protocol (Nanedo, 1994)) or lentivirus The lentivirus method for infecting fertilized eggs (Lois C et al., Science, 295, 2002, pages 868-872) is used.
しかし一般的には、レンチウイルス法でトランスジェニック非ヒト哺乳動物を作製することが望ましい。なぜなら、マイクロインジェクション法は導入遺伝子が染色体の不特定の１ヵ所に数十～数百個連結して挿入されるため、挿入箇所によっては、導入遺伝子の発現が全くない個体が生まれてしまう。したがって、マウスを作製後に有用な系統の選抜を行い、実験に用いるマウスを生産するまでに１～２年の期間を要する。一方、レンチウイルス法は作製期間が短いうえにトランスジェニックマウスの作製効率が高く、１世代目の産仔で実験に用いるマウス個体数を得ることができる。また、導入遺伝子がゲノム上の異なる箇所に１コピーずつ挿入されるため、様々な発現量を示すマウスを一度に複数匹得ることができるという利点を有するからである。本発明のプロモーターは、天然型に比してその配列長が短いことを特徴とすることから、レンチウイルスベクターのパッケージング効率が改善され、高タイターのウイルスを調製することが可能となり、受精卵への遺伝子導入効率が上がるので、レンチウイルス法によるトランスジェニック非ヒト哺乳動物の作製を高い効率で行うことが可能となる。 In general, however, it is desirable to produce transgenic non-human mammals by the lentiviral method. This is because in the microinjection method, several tens to several hundreds of transgenes are inserted into one unspecified chromosome location and inserted, and depending on the insertion location, an individual with no transgene expression is born. Therefore, it takes a period of 1 to 2 years after selection of useful strains after production of mice and production of mice for use in experiments. On the other hand, the lentivirus method has a short production period and high production efficiency of transgenic mice, and can obtain the number of mice used for experiments in the first-generation offspring. Another reason is that since the transgene is inserted one copy at a different location on the genome, a plurality of mice showing various expression levels can be obtained at a time. Since the promoter of the present invention is characterized in that its sequence length is shorter than that of the natural type, the packaging efficiency of the lentiviral vector is improved, and it becomes possible to prepare a high titer virus. Therefore, it is possible to produce a transgenic non-human mammal by the lentivirus method with high efficiency.
本発明のメタボリックシンドローム関連疾患の治療薬のスクリーニング方法の態様の一つとして、マーカー遺伝子を含有した発現ベクターを保持したトランスジェニック非ヒト哺乳動物もしくは該トランスジェニック非ヒト哺乳動物由来の細胞に、被検物質を投与した後、動物の組織や細胞におけるマーカー遺伝子の発現レベルを評価する方法を挙げることができる。 As one aspect of the method for screening a therapeutic agent for a metabolic syndrome-related disease of the present invention, a transgenic non-human mammal carrying an expression vector containing a marker gene or a cell derived from the transgenic non-human mammal is subjected to treatment. A method for evaluating the expression level of a marker gene in animal tissues or cells after administration of a test substance can be mentioned.
具体的には、本発明のトランスジェニック非ヒト哺乳動物に被検物質を投与し、マーカー遺伝子の発現を抑制させることが、生体内での各組織における脂肪細胞が低減することの指標として評価することができる。 Specifically, administration of a test substance to the transgenic non-human mammal of the present invention to suppress the expression of a marker gene is evaluated as an index for reducing adipocytes in each tissue in vivo. be able to.
このとき、被験物質には、いかなる公知物質および新規物質であってもよく、例えば、核酸、糖質、脂質、蛋白質、ペプチド、有機低分子化合物、コンビナトリアルケミストリー技術を用いて作製された化合物ライブラリー、固相合成やファージディスプレイ法により作製されたランダムペプチドライブラリー、あるいは微生物、動植物、海洋生物等由来の天然成分などがあげられる。また、これらの化合物の２種以上の混合物を試料として供することもできる。 At this time, the test substance may be any known substance or novel substance, for example, a nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound library prepared using combinatorial chemistry technology And random peptide libraries prepared by solid phase synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like. Moreover, the mixture of 2 or more types of these compounds can also be provided as a sample.
以下に実施例を挙げて本発明を説明するが、実施例は本発明をより良く理解するために例示するものであって、本発明の範囲がこれらの実施例に限定されることを意図するものではない。 EXAMPLES The present invention will be described below with reference to examples. However, the examples are provided for better understanding of the present invention, and the scope of the present invention is intended to be limited to these examples. It is not a thing.
各種プラスミドの作製
　ａＰ２－０、ａＰ２－１１－ＥＧＦＰ／ｐＥＮＴＲの作製
ａＰ２プロモーター／エンハンサー領域全長約５．５ｋｂを含むベクターａＰ２　ｐｒｏｍｏｔｅｒ／ｐＢｌｕｅｓｃｒｉｐｔＩＩを鋳型にして、転写開始点から約５．５ｋｂにあるエンハンサー領域約５２０ｂｐを、配列番号１の塩基配列からなるプライマーおよび配列番号２の塩基配列からなるプライマーを用いて９４℃　３０秒、５５℃　３０秒、７２℃　３０秒を３０回繰り返す条件でＰＣＲを行うことにより、ＢｇｌＩＩとＨｉｎｄＩＩＩ制限酵素部位を導入した増幅断片を得た。また、転写開始点から約２００ｂｐにあるプロモーター領域を配列番号３の塩基配列からなるプライマー、および配列番号４の塩基配列からなるプライマーを用いて９４℃　３０秒、５５℃　３０秒、７２℃　３０秒を３０回繰り返す条件でＰＣＲを行うことにより、ＫｐｎＩとＢａｍＨＩ制限酵素部位を導入した増幅断片を得た。エンハンサー領域の断片をＢｇｌＩＩとＨｉｎｄＩＩＩで処理し、ｐＥＧＦＰ－Ｎ１（Ｃｌｏｎｔｅｃｈ社）のＢｇｌＩＩ、ＨｉｎｄＩＩＩの部位に挿入して連結したプラスミドを得た。このプラスミドをＢｇｌＩＩ、ＮｏｔＩで処理し、ｐＥＮＴＲ４（インビトロジェン社）のＢａｍＨＩ、ＮｏｔＩの部位に挿入して連結したプラスミドを得た。このプラスミドをＫｐｎＩ、ＢａｍＨＩで処理し、先にＫｐｎＩとＢａｍＨＩ制限酵素部位を導入したプロモーター断片を連結してａＰ２－０－ＥＧＦＰ／ｐＥＮＴＲを作製した。このＤＮＡには、配列番号：２４に示されるａＰ２プロモーター全長のうち最上流の約５２０塩基からなる領域および５３５９～５５４８位からなる領域の約２００ｂｐの配列が含まれている。
ａＰ２－０－ＥＧＦＰ／ｐＥＮＴＲをＳａｌＩ処理し、エンハンサー領域を含む断片を除いた後、自己で連結し、ａＰ２－１１－ＥＧＦＰ／ｐＥＮＴＲを作製した。このＤＮＡには、配列番号：２４に示されるａＰ２プロモーター全長のうち５３５９～５５４８位からなる領域の約２００ｂｐの配列が含まれている。 Preparation of various plasmids Preparation of aP2-0 and aP2-11-EGFP / pENTR Using a vector aP2 promoter / pBluescript II containing about 5.5 kb of aP2 promoter / enhancer region as a template, an enhancer located about 5.5 kb from the transcription start point. PCR is performed on the region of about 520 bp under the conditions of repeating 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds 30 times using a primer consisting of the base sequence of SEQ ID NO: 1 and a primer consisting of the base sequence of SEQ ID NO: 2. Thus, an amplified fragment into which BglII and HindIII restriction enzyme sites were introduced was obtained. In addition, a promoter region at about 200 bp from the transcription start point was used at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds using a primer consisting of the base sequence of SEQ ID NO: 3 and a primer consisting of the base sequence of SEQ ID NO: 4. By performing PCR under the conditions of repeating 30 times, an amplified fragment into which KpnI and BamHI restriction enzyme sites were introduced was obtained. A fragment of the enhancer region was treated with BglII and HindIII, and inserted into the BglII and HindIII sites of pEGFP-N1 (Clontech) to obtain a ligated plasmid. This plasmid was treated with BglII and NotI and inserted into the BamHI and NotI sites of pENTR4 (Invitrogen) to obtain a ligated plasmid. This plasmid was treated with KpnI and BamHI, and a promoter fragment previously introduced with a KpnI and BamHI restriction enzyme site was ligated to prepare aP2-0-EGFP / pENTR. This DNA includes a sequence of about 200 bp of a region consisting of about 520 bases at the most upstream and a region consisting of positions 5359-5548 of the full length of the aP2 promoter shown in SEQ ID NO: 24.
aP2-0-EGFP / pENTR was treated with SalI to remove the fragment containing the enhancer region, and then ligated with itself to prepare aP2-11-EGFP / pENTR. This DNA includes a sequence of about 200 bp in the region consisting of positions 5359-5548 of the full length aP2 promoter shown in SEQ ID NO: 24.
ａＰ２－１、ａＰ２－２、ａＰ２－３、ａＰ２－４、ａＰ２－５、ａＰ２－６、ａＰ２－７、ａＰ２－８、ａＰ２－１２－ＥＧＦＰ／ｐＥＮＴＲの作製
ａＰ２プロモーター／エンハンサー領域全長約５．５ｋｂを含むベクターａＰ２　ｐｒｏｍｏｔｅｒ／ｐＢｌｕｅｓｃｒｉｐｔＩＩを鋳型にして、下記のプライマー対（配列番号５の塩基配列からなるプライマー、および配列番号６の塩基配列からなるプライマー）、（配列番号７の塩基配列からなるプライマー、および配列番号８の塩基配列からなるプライマー）、（配列番号９の塩基配列からなるプライマー、および配列番号１０の塩基配列からなるプライマー）、（配列番号１１の塩基配列からなるプライマー、および配列番号１２の塩基配列からなるプライマー）、（配列番号１３の塩基配列からなるプライマー、および配列番号１４の塩基配列からなるプライマー）、（配列番号１５の塩基配列からなるプライマー、および配列番号１６の塩基配列からなるプライマー）、（配列番号１７の塩基配列からなるプライマー、および配列番号１８の塩基配列からなるプライマー）、および（配列番号１９の塩基配列からなるプライマー、および配列番号２０の塩基配列からなるプライマー）を用いて、９４℃　３０秒、５５℃　３０秒、７２℃　４５秒を３０回繰り返す条件でＰＣＲを行うことにより、ＨｉｎｄＩＩＩとＫｐｎＩ制限酵素部位を導入した増幅断片を得た。 Production of aP2-1, aP2-2, aP2-3, aP2-4, aP2-5, aP2-6, aP2-7, aP2-8, aP2-12-EGFP / pENTR aP2 promoter / enhancer region total length of about 5. Using the vector aP2 promoter / pBluescript II containing 5 kb as a template, the following primer pair (primer consisting of the base sequence of SEQ ID NO: 5 and primer consisting of the base sequence of SEQ ID NO: 6), (primer consisting of the base sequence of SEQ ID NO: 7) , And a primer consisting of the base sequence of SEQ ID NO: 8), (a primer consisting of the base sequence of SEQ ID NO: 9 and a primer consisting of the base sequence of SEQ ID NO: 10), (a primer consisting of the base sequence of SEQ ID NO: 11, and SEQ ID NO: A primer comprising 12 nucleotide sequences), (SEQ ID NO: 13) A primer consisting of a base sequence and a primer consisting of a base sequence of SEQ ID NO: 14) (a primer consisting of a base sequence of SEQ ID NO: 15 and a primer consisting of a base sequence of SEQ ID NO: 16), (consisting of a base sequence of SEQ ID NO: 17 94 ° C for 30 seconds and 55 ° C for 30 seconds using a primer and a primer consisting of the base sequence of SEQ ID NO: 18) and (a primer consisting of the base sequence of SEQ ID NO: 19 and a primer consisting of the base sequence of SEQ ID NO: 20). PCR was performed under the conditions of repeating 45 ° C. at 72 ° C. for 30 times to obtain an amplified fragment into which HindIII and KpnI restriction enzyme sites were introduced.
これらの断片をそれぞれＨｉｎｄＩＩＩとＫｐｎＩで処理し、ａＰ２－０－ＥＧＦＰ／ｐＥＮＴＲのＨｉｎｄＩＩＩとＫｐｎＩの部位に挿入し、それぞれａＰ２－１－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－２－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－３－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－４－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－５－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－６－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－７－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－８－ＥＧＦＰ／ｐＥＮＴＲを得た。これらのＤＮＡには、配列番号：２１と配列番号：２３に示される塩基配列が共通して含まれており、かつ、配列番号：２４で示されるａＰ２プロモーター全長において、それぞれ、４８１～１１３７位、１０６９～１７８０位、１６６２～２３５７位、２２９６～２９８８位、２９０８～３５５８位、３４９６～４１９９位、４１２２～４８２３位、４７２８～５３８０位からなる領域の配列を含んでいる。また、ａＰ２プロモーター領域全長約５．５ｋｂを含むベクターａＰ２　ｐｒｏｍｏｔｅｒ／ｐＢｌｕｅｓｃｒｉｐｔＩＩを鋳型にして、上記のプライマー対を用いて、９４℃　３０秒、５５℃　３０秒、７２℃　４分を３０回繰り返す条件でＰＣＲを行うことにより、ＨｉｎｄＩＩＩとＫｐｎＩ制限酵素部位を導入した増幅断片を得た。この断片をＨｉｎｄＩＩＩとＫｐｎＩで処理し、ａＰ２－０－ＥＧＦＰ／ｐＥＮＴＲのＨｉｎｄＩＩＩとＫｐｎＩの部位に挿入し、ａＰ２プロモーター領域全長を含むａＰ２－１２－ＥＧＦＰ／ｐＥＮＴＲを得た。このＤＮＡには、配列番号：２１と配列番号：２３に示される塩基配列が含まれており、かつ、配列番号：２４で示されるａＰ２プロモーター全長において、４８１～５３８０位からなる領域の配列を含んでいる。 These fragments were treated with HindIII and KpnI, respectively, and inserted into the HindIII and KpnI sites of aP2-0-EGFP / pENTR, respectively, aP2-1-EGFP / pENTR, aP2-2-EGFP / pENTR, aP2-3- EGFP / pENTR, aP2-4-EGFP / pENTR, aP2-5-EGFP / pENTR, aP2-6-EGFP / pENTR, aP2-7-EGFP / pENTR, aP2-8-EGFP / pENTR were obtained. These DNAs include the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 23 in common, and are located at positions 481 to 1137 in the full length of the aP2 promoter shown in SEQ ID NO: 24, respectively. It includes an array of regions consisting of 1069 to 1780, 1662 to 2357, 2296 to 2988, 2908 to 3558, 3496 to 4199, 4122 to 4823, and 4728 to 5380. In addition, the vector aP2 promoter / pBluescript II containing the entire length of the aP2 promoter region of about 5.5 kb was used as a template, and the above primer pairs were used under the conditions of repeating 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 4 minutes 30 times. By performing PCR, an amplified fragment into which HindIII and KpnI restriction enzyme sites were introduced was obtained. This fragment was treated with HindIII and KpnI and inserted into the HindIII and KpnI sites of aP2-0-EGFP / pENTR to obtain aP2-12-EGFP / pENTR containing the full length of the aP2 promoter region. This DNA includes the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 23, and includes the sequence of the region consisting of positions 481 to 5380 in the full length of the aP2 promoter shown in SEQ ID NO: 24. It is out.
ａＰ２－０、ａＰ２－１、ａＰ２－２、ａＰ２－３、ａＰ２－４、ａＰ２－５、ａＰ２－６、ａＰ２－７、ａＰ２－８、ａＰ２－１１、ａＰ２－１２－ｌｕｃ／ｐＥＮＴＲの作製ｐＧＬ３－ｂａｓｉｃベクター（Ｐｒｏｍｅｇａ社）を鋳型にして、配列番号１の塩基配列からなるプライマーおよび配列番号２の塩基配列からなるプライマーを用いて９４℃　３０秒、５５℃　３０秒、７２℃　３０秒を３０回繰り返す条件でＰＣＲを行うことにより、ＡｇｅＩとＮｏｔＩ制限酵素部位を導入したルシフェラーゼ遺伝子の断片を得た。この断片をＡｇｅＩとＮｏｔＩで処理し、ａＰ２－０－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－１－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－２－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－３－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－４－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－５－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－６－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－７－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－８－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－１１－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－１２－ＥＧＦＰ／ｐＥＮＴＲのＡｇｅＩとＮｏｔＩの部位に挿入してＥＧＦＰをルシフェラーゼ遺伝子に変換したプラスミド、ａＰ２－０－ｌｕｃ／ｐＥＮＴＲ、ａＰ２－１－ｌｕｃ／ｐＥＮＴＲ、ａＰ２－２－ｌｕｃ／ｐＥＮＴＲ、ａＰ２－３－ｌｕｃ／ｐＥＮＴＲ、ａＰ２－４－ｌｕｃ／ｐＥＮＴＲ、ａＰ２－５－ｌｕｃ／ｐＥＮＴＲ、ａＰ２－６－ｌｕｃ／ｐＥＮＴＲ、ａＰ２－７－ｌｕｃ／ｐＥＮＴＲ、ａＰ２－８－ｌｕｃ／ｐＥＮＴＲ、ａＰ２－１１－ｌｕｃ／ｐＥＮＴＲ、ａＰ２－１２－ｌｕｃ／ｐＥＮＴＲを得た。これらのプラスミドの構造の模式図を図１に示す。 Preparation of aP2-0, aP2-1, aP2-2, aP2-3, aP2-4, aP2-5, aP2-6, aP2-7, aP2-8, aP2-11, aP2-12-luc / pENTR pGL3 -Using a basic vector (Promega) as a template and a primer consisting of the base sequence of SEQ ID NO: 1 and a primer consisting of the base sequence of SEQ ID NO: 2 for 30 seconds at 94 ° C, 30 seconds at 55 ° C, 30 seconds at 72 ° C By performing PCR under repeated conditions, a fragment of the luciferase gene into which the AgeI and NotI restriction enzyme sites were introduced was obtained. This fragment was treated with AgeI and NotI, and aP2-0-EGFP / pENTR, aP2-1-EGFP / pENTR, aP2-2-EGFP / pENTR, aP2-3-EGFP / pENTR, aP2-4-EGFP / pENTR, AgeI of aP2-5-EGFP / pENTR, aP2-6-EGFP / pENTR, aP2-7-EGFP / pENTR, aP2-8-EGFP / pENTR, aP2-11-EGFP / pENTR, aP2-12-EGFP / pENTR Plasmids inserted into NotI sites to convert EGFP into luciferase gene, aP2-0-luc / pENTR, aP2-1-luc / pENTR, aP2-2-luc / pENTR, aP2-3-luc / pENTR, aP2- 4-luc / pENTR, aP -5-luc / pENTR, aP2-6-luc / pENTR, aP2-7-luc / pENTR, aP2-8-luc / pENTR, aP2-11-luc / pENTR, to obtain a aP2-12-luc / pENTR. A schematic diagram of the structure of these plasmids is shown in FIG.
ａＰ２－０、ａＰ２－３、ａＰ２－５、ａＰ２－１２－ＥＧＦＰ／ｐＣＤＨの作製
レンチウイルスベクターｐＣＤＨ１－ＭＣＳ１（Ｓｙｓｔｅｍ　Ｂｉｏｓｃｉｅｎｃｅｓ社）をＳｐｅＩおよびＸｂａＩで処理し、ＣＭＶプロモーター断片を除いた後、平滑末端処理し、自己で連結することでＣＭＶプロモーターの欠損したｐＣＤＨ－ＭＣＳ１－δＣＭＶを得た。次にＧａｔｅｗａｙＣａｓｓｅｔｔｅの両端にＮｈｅＩおよびＫｐｎＩ制限酵素部位を導入した断片をｐＣＤＮＡ３．１（＋）（インビトロジェン社）のＮｈｅＩ、ＫｐｎＩ部位に導入した後、このベクターをＮｈｅＩ、ＸｈｏＩ処理し、切り出したＧａｔｅｗａｙＣａｓｓｅｔｔｅをｐＣＤＨ－ＭＣＳ１－δＣＭＶのＮｈｅＩ、ＳａｌＩ部位に導入することで、ｐＣＤＨ－δＣＭＶ－ＧＷベクターを得た。このベクターとａＰ２－０－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－３－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－５－ＥＧＦＰ／ｐＥＮＴＲ、ａＰ２－１２－ＥＧＦＰ／ｐＥＮＴＲをそれぞれＬＲクロナーゼＩＩ酵素ミックス（インビトロジェン社）で組み換えることにより、ａＰ２－０－ＥＧＦＰ／ｐＣＤＨ、ａＰ２－３－ＥＧＦＰ／ｐＣＤＨ、ａＰ２－５－ＥＧＦＰ／ｐＣＤＨ、ａＰ２－１２－ＥＧＦＰ／ｐＣＤＨを得た。 Preparation of aP2-0, aP2-3, aP2-5, aP2-12-EGFP / pCDH The lentiviral vector pCDH1-MCS1 (System Biosciences) was treated with SpeI and XbaI to remove the CMV promoter fragment Thereafter, blunt end treatment and self-ligation were performed to obtain pCDH-MCS1-δCMV lacking the CMV promoter. Next, after introducing a fragment into which NheI and KpnI restriction enzyme sites were introduced at both ends of GatewayCassette into the NheI and KpnI sites of pCDNA3.1 (+) (Invitrogen), this vector was treated with NheI and XhoI, and the extracted GatewayCassette was used. The pCDH-δCMV-GW vector was obtained by introduction into the NheI and SalI sites of pCDH-MCS1-δCMV. By recombining this vector with aP2-0-EGFP / pENTR, aP2-3-EGFP / pENTR, aP2-5-EGFP / pENTR, and aP2-12-EGFP / pENTR with LR clonase II enzyme mix (Invitrogen) AP2-0-EGFP / pCDH, aP2-3-EGFP / pCDH, aP2-5-EGFP / pCDH, aP2-12-EGFP / pCDH were obtained.
培養細胞における各プロモーターの反応
３Ｔ３－Ｌ１脂肪分化細胞におけるトランジェント・アッセイを用い、ａＰ２プロモーター／エンハンサー由来の各コンストラクトの脂肪細胞における転写活性を調べた。比較のために、分化誘導していない３Ｔ３－Ｌ１細胞、ＨｅＬａ細胞、２９３細胞を用いた。１０％ＦＣＳ（ウシ胎仔血清）を含有するＤＭＥＭを用い、５％ＣＯ２、３７℃の条件下で３Ｔ３－Ｌ１細胞を培養し、コンフルエント２日後、インスリン、デキサメタゾン、ＩＢＭＸを含む培地で分化誘導を開始した。トランスフェクションはエレクトロポレーションにて行った。また、未分化の３Ｔ３－Ｌ１細胞、ＨｅＬａ細胞、２９３細胞のトランスフェクションはＦｕＧｅｎｅ６（ロシュ社）を用い、添付のプロトコールに従った。レポーターとしてａＰ２－０－ｌｕｃ、ａＰ２－１－ｌｕｃ、ａＰ２－２－ｌｕｃ、ａＰ２－３－ｌｕｃ、ａＰ２－４－ｌｕｃ、ａＰ２－５－ｌｕｃ、ａＰ２－６－ｌｕｃ、ａＰ２－７－ｌｕｃ、ａＰ２－８－ｌｕｃ、ａＰ２－１１－ｌｕｃ、ａＰ２－１２－ｌｕｃと内部コントロールレポーターとしてウミシイタケルシフェラーゼを発現するベクターであるｐＲＬ－Ｔｋベクターを導入した。トランスフェクションを行ってから２４時間後、それぞれの細胞を溶解し、Ｄｕａｌ－Ｌｕｃｉｆｅｒａｓｅ　Ｒｅｐｏｒｔｅｒ　Ａｓｓａｙ　Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社）を用いて、発光強度を測った。結果を図２に示す。この図においては、２９３でａＰ２－１１－ｌｕｃを導入したときの発光強度（図中ではＬｕｃｉｆｅｒａｓｅ活性と示す）を１として他のコンストラクトを用いたときの発光強度の相対値を示した。 Reaction of each promoter in cultured cells Using a transient assay in 3T3-L1 adipose differentiated cells, the transcriptional activity of each aP2 promoter / enhancer-derived construct in adipocytes was examined. For comparison, 3T3-L1 cells, HeLa cells, and 293 cells that were not induced to differentiate were used. Using DMEM containing 10% FCS (fetal calf serum), 3T3-L1 cells are cultured under conditions of 5% CO 2 and 37 ° C. After 2 days of confluence, differentiation induction is performed in a medium containing insulin, dexamethasone and IBMX. Started. Transfection was performed by electroporation. For transfection of undifferentiated 3T3-L1 cells, HeLa cells, and 293 cells, FuGene6 (Roche) was used and the attached protocol was followed. AP2-0-luc, aP2-1-luc, aP2-3-luc, aP2-4-luc, aP2-5-luc, aP2-6-luc, aP2-7-luc as reporters aP2-8-luc, aP2-11-luc, aP2-12-luc and the pRL-Tk vector, which is a vector for expressing Renilla luciferase, as an internal control reporter was introduced. Twenty-four hours after transfection, each cell was lysed and the luminescence intensity was measured using a Dual-Luciferase Reporter Assay System (Promega). The results are shown in FIG. In this figure, the luminescence intensity when aP2-11-luc is introduced in 293 (indicated as Luciferase activity in the figure) is 1, and the relative value of the luminescence intensity when other constructs are used is shown.
この結果、ａＰ２－０－ｌｕｃ、ａＰ２－１－ｌｕｃ、ａＰ２－２－ｌｕｃ、ａＰ２－３－ｌｕｃ、ａＰ２－４－ｌｕｃ、ａＰ２－５－ｌｕｃ、ａＰ２－６－ｌｕｃ、ａＰ２－７－ｌｕｃ、ａＰ２－８－ｌｕｃを導入した場合、いずれも３Ｔ３－Ｌ１脂肪分化細胞で２倍以上の転写活性が得られたが、比活性は約６倍～１４倍と差が認められた。この結果から、配列番号：２１に示されるａＰ２プロモーター最上流に位置する約５２０ｂｐの配列が脂肪細胞での特異的発現に必要であるが、その他の領域もプロモーター活性に影響を与えることが予想された。一方、ａＰ２－１２－ｌｕｃはａＰ２プロモーター最上流に位置する約５２０ｂｐの配列を含んでいるにも関わらず、他のコンストラクトに比べて脂肪細胞での活性が低かった。 As a result, aP2-0-luc, aP2-1-luc, aP2-2-2-luc, aP2-3-luc, aP2-4-luc, aP2-5-luc, aP2-6-luc, aP2-7-luc When aP2-8-luc was introduced, the transcriptional activity of 2 times or more was obtained in 3T3-L1 adipose differentiated cells, but the specific activity differed by about 6 to 14 times. From this result, an approximately 520 bp sequence located at the most upstream of the aP2 promoter shown in SEQ ID NO: 21 is necessary for specific expression in adipocytes, but other regions are also expected to affect the promoter activity. It was. On the other hand, although aP2-12-luc contained a sequence of about 520 bp located at the most upstream of the aP2 promoter, it was less active in adipocytes than other constructs.
レンチウイルスを使ったトランスジェニックマウスの作製及び各組織での発現
１０％ＦＣＳ（ウシ胎仔血清）を含有するＤＭＥＭを用い、５％ＣＯ２、３７℃の条件下で２９３ＴＮ細胞（Ｓｙｓｔｅｍ　Ｂｉｏｓｃｉｅｎｃｅｓ社）を培養した。トランスフェクションはＦｕＧｅｎｅ６（ロシュ社）を用い、添付のプロトコールに従った。使用するベクターは、実施例２の結果から脂肪細胞で特異性が見られたもののうち、最もプロモーターサイズが小さいａＰ２－０－ＥＧＦＰ／ｐＣＤＨ、３Ｔ３－Ｌ１未分化細胞、２９３細胞で比較的活性の低かったａＰ２－３－ＥＧＦＰ／ｐＣＤＨ、ａＰ２－５－ＥＧＦＰ／ｐＣＤＨ、ａＰ２プロモーター全長を含むａＰ２－１２－ＥＧＦＰ／ｐＣＤＨを選択し、それぞれｐＰＡＣＫＨ１（ＳＢＩ社）と共導入した。トランスフェクションを行ってから７２時間後、培地を回収し、超遠心で濃縮後、スクロース精製を行い、トランスジェニックマウス作製用のレンチウイルス液を調製した。ウイルスタイターはｐ２４　Ａｎｔｉｇｅｎ　ＥＬＩＺＡ　ｋｉｔ（ＺｅｐｔｏＭｅｔｒｉｘ社）を用いて測定した。ＢＤＦ１雌マウスにＰＭＳＧ（妊馬血清性性腺刺激ホルモン）を５ＩＵ、その４８時間後にＨＣＧ（胎盤性性腺刺激ホルモン）を５ＩＵ投与して過排卵を誘発し、ＢＤＦ１雄マウスと交配した。交配翌日に膣栓を確認できた雌マウスの卵管を、交配後約４０時間で採取し、０．４％ＢＳＡ溶液で灌流した。回収できた２細胞期胚はｋＳＯＭ培地（アークリソース社）中で短期間培養した後、１分から２分程度酸性タイロード（アークリソース社）処理することで胚の透明体を除去した。首尾よく透明帯が除去された胚は、ｋＳＯＭ培地中で洗浄し、ウイルス感染まで同培地中で培養した。胚へのウイルス感染は、ウイルス粒子数を約１５０ｐｇ／μlまたは１５００ｐｇ／μl含むように希釈したｋＳＯＭ培地のドロップ（６μl／スポット）にて、透明帯を除去した胚を２日間培養することにより行った。胚の培養は、全て５％ＣＯ２、３７℃の条件下で行った。ウイルス感染後に、胚盤胞期もしくは桑実胚期まで発生した胚を、膣栓確認後２．５日の偽妊娠マウス子宮内へ移植し、移植１７日後に自然分娩もしくは帝王切開により産仔を得た。生後４週齢で採取した産仔の尾端より、ＤＮｅａｓｙ　Ｂｌｏｏｄ　＆　Ｔｉｓｓｕｅ　Ｋｉｔ（ＱＩＡＧＥＮ）を用いてゲノムＤＮＡを精製した。精製したＤＮＡをテンプレートとして配列番号２５の塩基配列からなるプライマーおよび配列番号２６の塩基配列からなるプライマーを用いて９５℃　１５秒、５７℃　１５秒、７２℃　３０秒を４０回繰り返す条件でＰＣＲを行い、ＥＧＦＰ遺伝子の一部を増幅した後、アガロースゲル電気泳動を行うことで、導入遺伝子を検出し、トランスジェニックマウスの遺伝子型を判定した。生後８週齢前後でトランスジェニックマウスから生殖器周囲脂肪の一部を生検採取し、蛍光実体顕微鏡下でＥＧＦＰ蛍光の観察を行なった。ＥＧＦＰ蛍光が認められた個体の剖検を行い、蛍光実体顕微鏡下で主要臓器と４種の脂肪組織におけるＥＧＦＰ蛍光を観察し、結果を以下の表１に示した。 Generation of transgenic mice using lentivirus and expression in each tissue Using DMEM containing 10% FCS (fetal calf serum), 293TN cells (System Biosciences) under conditions of 5% CO 2 and 37 ° C. Cultured. For transfection, FuGene6 (Roche) was used and the attached protocol was followed. Among the vectors that were found to be specific in adipocytes from the results of Example 2, the vectors used were aP2-0-EGFP / pCDH, 3T3-L1 undifferentiated cells with the smallest promoter size, and relatively active in 293 cells. The aP2-3-EGFP / pCDH, aP2-5-EGFP / pCDH, and aP2-12-EGFP / pCDH containing the full length of the aP2 promoter were selected and co-introduced with pPACKH1 (SBI). 72 hours after the transfection, the medium was collected, concentrated by ultracentrifugation, and purified by sucrose to prepare a lentivirus solution for producing a transgenic mouse. Virus titer was measured using p24 Antigen ELIZA kit (ZeptoMetrix). Superovulation was induced by administering 5 IU of PSG (pregnant horse serum gonadotropin) to BDF1 female mice and 48 I HCG (placental gonadotropin) 48 hours later, and mated with BDF1 male mice. The oviducts of female mice whose vaginal plug was confirmed the day after mating were collected approximately 40 hours after mating and perfused with 0.4% BSA solution. The recovered 2-cell embryo was cultured in kSOM medium (Arc Resource) for a short period, and then treated with acidic Tyrode (Arc Resource) for about 1 to 2 minutes to remove the embryo transparency. Embryos from which the zona pellucida was successfully removed were washed in kSOM medium and cultured in the same medium until viral infection. Viral infection of embryos is performed by culturing embryos from which the zona pellucida has been removed for 2 days with a drop (6 μl / spot) of kSOM medium diluted to contain about 150 pg / μl of viral particles or 1500 pg / μl. It was. All embryos were cultured under conditions of 5% CO 2 and 37 ° C. Embryos that have developed to the blastocyst stage or morula stage after virus infection are transplanted into the uterus of pseudopregnant mice 2.5 days after confirmation of vaginal plug, and the offspring are born by natural delivery or caesarean section 17 days after transplantation. Obtained. Genomic DNA was purified from the tail of a litter collected at 4 weeks of age using DNeasy Blood & Tissue Kit (QIAGEN). Using purified DNA as a template, PCR was performed under the conditions of 95 ° C 15 seconds, 57 ° C 15 seconds, 72 ° C 30 seconds 40 times using a primer consisting of the base sequence of SEQ ID NO: 25 and a primer consisting of the base sequence of SEQ ID NO: 26. Then, after a part of the EGFP gene was amplified, the transgene was detected by performing agarose gel electrophoresis, and the genotype of the transgenic mouse was determined. Around 8 weeks of age, a portion of perigenital fat was biopsyed from the transgenic mice and observed for EGFP fluorescence under a fluorescent stereomicroscope. Individuals in which EGFP fluorescence was observed were necropsied, and EGFP fluorescence was observed in major organs and 4 types of adipose tissue under a fluorescent stereomicroscope. The results are shown in Table 1 below.
[規則26に基づく補充 18.09.2009]　
Figure WO-DOC-TABLE-1
 [Supplement under rule 26 18.09.2009]
Figure WO-DOC-TABLE-1
この結果、全長プロモーターを搭載したａＰ２－１２－ＥＧＦＰ／ｐＣＤＨで作製したトランスジェニックマウスにおいては、トランスジェニックマウスの作製効率が他の短縮型プロモーターを使用した場合と比較して１／１０以下に低下し、得られたトランスジェニックマウスにおいては、どの組織でも蛍光が見られなかった。これはウイルスのパッケージング効率の低下が原因だと考えられる。また、ａＰ２－０－ＥＧＦＰ／ｐＣＤＨで作製したトランスジェニックマウスは、脂肪組織で蛍光が確認されたが、その強度が弱く、また、膵臓でも同程度であった。一方、ａＰ２－３－ＥＧＦＰ／ｐＣＤＨで作製したトランスジェニックマウスは、膵臓の他、脂肪組織で強い蛍光が観察された。このことから、ａＰ２－３－ＥＧＦＰ／ｐＣＤＨに含まれる配列には生体内で脂肪組織及び膵臓組織に導入遺伝子を十分な強度で発現させるために必要なエレメントが含まれると考えられる As a result, in the transgenic mouse produced with aP2-12-EGFP / pCDH loaded with the full-length promoter, the efficiency of producing the transgenic mouse is reduced to 1/10 or less compared to the case of using other truncated promoters. In the obtained transgenic mice, no fluorescence was observed in any tissue. This is thought to be due to a decrease in virus packaging efficiency. In addition, in the transgenic mouse prepared with aP2-0-EGFP / pCDH, fluorescence was confirmed in the adipose tissue, but its intensity was weak, and it was similar in the pancreas. On the other hand, in the transgenic mouse prepared with aP2-3-EGFP / pCDH, strong fluorescence was observed not only in the pancreas but also in the adipose tissue. From this, it is considered that the sequence contained in aP2-3-EGFP / pCDH contains elements necessary for expressing the transgene with sufficient strength in adipose tissue and pancreatic tissue in vivo.
ヒトａＰ２－０、ヒトａＰ２－１１、ヒトａＰ２－３／ｐＧＬ３の作製
ヒトａＰ２遺伝子の転写開始点より上流約１０ｋｂに対して、配列番号：２１、２２、２３に示される配列の相同領域をそれぞれ検索し、配列番号：２７、２８、２９に示される配列を得た。これらの領域をクローニングするため、ヒトゲノムＤＮＡを鋳型にして、下記のプライマー対（配列番号３０の塩基配列からなるプライマー、および配列番号３１の塩基配列からなるプライマー）、（配列番号３２の塩基配列からなるプライマー、および配列番号３３の塩基配列からなるプライマー）、および（配列番号３４の塩基配列からなるプライマー、および配列番号３５の塩基配列からなるプライマー）を用いて、ＰＣＲを行うことにより、それぞれＳａｃＩとＮｈｅＩ、ＮｈｅＩとＸｈｏＩ、ＢｇｌＩＩとＨｉｎｄＩＩＩの制限酵素部位を導入した増幅断片を得た。配列番号２９を含む増幅断片をＢｇｌＩＩとＨｉｎｄＩＩＩで処理し、ｐＧＬ３－ｂａｓｉｃ（Ｐｒｏｍｅｇａ社）のＢｇｌＩＩ、ＨｉｎｄＩＩＩの部位に連結してヒトａＰ２－１１／ｐＧＬ３を得た。次にこのプラスミドをＳａｃＩとＮｈｅＩで処理し、配列番号２７を含む増幅断片をＳａｃＩとＮｈｅＩで処理した断片を連結してヒトａＰ２－０／ｐＧＬ３を作製した。次にこのプラスミドをＮｈｅＩとＸｈｏＩで処理し、配列番号２８を含む増幅断片をＮｈｅＩとＸｈｏＩで処理した断片を連結してヒトａＰ２－３／ｐＧＬ３を作製した。 Production of human aP2-0, human aP2-11, human aP2-3 / pGL3 The sequence shown in SEQ ID NOs: 21, 22, and 23 with respect to about 10 kb upstream from the transcription start site of the human aP2 gene. The homologous regions were respectively searched to obtain the sequences shown in SEQ ID NOs: 27, 28, and 29. In order to clone these regions, using human genomic DNA as a template, the following primer pair (primer consisting of the base sequence of SEQ ID NO: 30 and primer consisting of the base sequence of SEQ ID NO: 31), (from the base sequence of SEQ ID NO: 32) And a primer consisting of the base sequence of SEQ ID NO: 33) and (a primer consisting of the base sequence of SEQ ID NO: 34 and a primer consisting of the base sequence of SEQ ID NO: 35). And NheI, NheI and XhoI, and BglII and HindIII restriction enzyme sites were obtained. The amplified fragment containing SEQ ID NO: 29 was treated with BglII and HindIII, and ligated to the BglII and HindIII sites of pGL3-basic (Promega) to obtain human aP2-11 / pGL3. Next, this plasmid was treated with SacI and NheI, and the amplified fragment containing SEQ ID NO: 27 was ligated with the fragment treated with SacI and NheI to prepare human aP2-0 / pGL3. Next, this plasmid was treated with NheI and XhoI, and the amplified fragment containing SEQ ID NO: 28 was ligated with the fragment treated with NheI and XhoI to prepare human aP2-3 / pGL3.
培養細胞における各プロモーターの反応
３Ｔ３－Ｌ１脂肪分化細胞におけるトランジェント・アッセイを用い、ヒトａＰ２プロモーター由来の各コンストラクトの脂肪細胞における転写活性を調べた。比較のために、分化誘導していない３Ｔ３－Ｌ１細胞、ＨｅＬａ細胞、２９３細胞を用いた。１０％ＦＣＳ（ウシ胎仔血清）を含有するＤＭＥＭを用い、５％ＣＯ２、３７℃の条件下で３Ｔ３－Ｌ１細胞を培養し、コンフルエント２日後、インスリン、デキサメタゾン、ＩＢＭＸを含む培地で分化誘導を開始した。トランスフェクションはエレクトロポレーションにて行った。また、未分化の３Ｔ３－Ｌ１細胞、ＨｅＬａ細胞、２９３細胞のトランスフェクションはＦｕＧｅｎｅ６（ロシュ社）を用い、添付のプロトコールに従った。内部コントロールレポーターとしてウミシイタケルシフェラーゼを発現するベクターであるｐＲＬ－Ｔｋベクターを導入した。トランスフェクションを行ってから２４時間後、それぞれの細胞を溶解し、Ｄｕａｌ－Ｌｕｃｉｆｅｒａｓｅ　Ｒｅｐｏｒｔｅｒ　Ａｓｓａｙ　Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社）を用いて、発光強度を測った。結果を図３の上段に示し、比較対象にマウスの結果を下段に示した。この図においては、未分化の３Ｔ３－Ｌ１細胞にａＰ２－１１を導入したときの発光強度（図中ではＬｕｃｉｆｅｒａｓｅ活性と示す）を１として他のコンストラクトを用いたときの発光強度の相対値を示した。 Reaction of each promoter in cultured cells Using a transient assay in 3T3-L1 adipose differentiated cells, the transcriptional activity of each construct derived from the human aP2 promoter in adipocytes was examined. For comparison, 3T3-L1 cells, HeLa cells, and 293 cells that were not induced to differentiate were used. Using DMEM containing 10% FCS (fetal calf serum), 3T3-L1 cells are cultured under conditions of 5% CO 2 and 37 ° C. After 2 days of confluence, differentiation induction is performed in a medium containing insulin, dexamethasone and IBMX. Started. Transfection was performed by electroporation. For transfection of undifferentiated 3T3-L1 cells, HeLa cells, and 293 cells, FuGene6 (Roche) was used and the attached protocol was followed. As an internal control reporter, the pRL-Tk vector, which is a vector for expressing Renilla luciferase, was introduced. Twenty-four hours after transfection, each cell was lysed and the luminescence intensity was measured using a Dual-Luciferase Reporter Assay System (Promega). The results are shown in the upper part of FIG. 3, and the results of the mouse are shown in the lower part for comparison. This figure shows the relative value of the luminescence intensity when other constructs were used with the luminescence intensity when aP2-11 was introduced into undifferentiated 3T3-L1 cells (indicated as Luciferase activity in the figure) as 1. It was.
この結果、ａＰ２－１１に対してａＰ２－０、ａＰ２－３を導入した場合、マウスの場合と同様に、ヒトにおいても３Ｔ３－Ｌ１脂肪分化細胞でのみ転写活性の上昇が認められた。このことから、ヒト由来の配列でもａＰ２プロモーターの相同領域には生体内で脂肪組織及び膵臓組織に特異的に導入遺伝子を発現させるために必要なエレメントが含まれることが予想される。　 As a result, when aP2-0 and aP2-3 were introduced into aP2-11, as in the case of mice, an increase in transcriptional activity was observed only in 3T3-L1 adipose differentiated cells in humans. From this, it is expected that even in the human-derived sequence, the homologous region of the aP2 promoter contains elements necessary for specifically expressing the transgene in adipose tissue and pancreatic tissue in vivo. *

Claims (17)

  1. 配列番号：２２に示される塩基配列を少なくとも有するＤＮＡであって、かつ脂肪組織および／もしくは膵臓において特異的なプロモーター活性を有する、単離されたＤＮＡ。　 An isolated DNA having at least the base sequence shown in SEQ ID NO: 22 and having specific promoter activity in adipose tissue and / or pancreas. *
  2. （ａ）配列番号：２２に示される塩基配列において、少なくとも１塩基の置換、欠失、挿入又は付加を有するＤＮＡ、（ｂ）配列番号：２２に示される塩基配列からなるＤＮＡの相補鎖とストリンジェントな条件下でハイブリダイズしうるＤＮＡ、及び（ｃ）配列番号：２２に示される塩基配列からなるＤＮＡに少なくとも９０％の配列同一性を有するＤＮＡからなる群より選択されたＤＮＡであって、かつ脂肪組織および／または膵臓組織において特異的なプロモーター活性を有する、単離されたＤＮＡ。 (A) DNA having at least one base substitution, deletion, insertion or addition in the base sequence shown in SEQ ID NO: 22; (b) a complementary strand and string of DNA consisting of the base sequence shown in SEQ ID NO: 22 DNA selected from the group consisting of DNA that can hybridize under a gentle condition, and (c) DNA having at least 90% sequence identity to DNA consisting of the base sequence shown in SEQ ID NO: 22, And isolated DNA having specific promoter activity in adipose tissue and / or pancreatic tissue.
  3. さらに配列番号：２１に示される塩基配列を有するＤＮＡである、請求項１又は２記載の単離されたＤＮＡ。　 Furthermore, the isolated DNA of Claim 1 or 2 which is DNA which has a base sequence shown by sequence number: 21. *
  4. さらに配列番号：２３に示される塩基配列を有するＤＮＡである、請求項１～３のいずれかに記載の単離されたＤＮＡ。 The isolated DNA according to any one of claims 1 to 3, which is a DNA having the base sequence represented by SEQ ID NO: 23.
  5. 配列番号：２８に示される塩基配列を少なくとも有するＤＮＡであって、かつ脂肪組織および／もしくは膵臓において特異的なプロモーター活性を有する、単離されたＤＮＡ。　 An isolated DNA having at least the base sequence shown in SEQ ID NO: 28 and having specific promoter activity in adipose tissue and / or pancreas. *
  6. （ａ）配列番号：２８に示される塩基配列において、少なくとも１塩基の置換、欠失、挿入又は付加を有するＤＮＡ、（ｂ）配列番号：２８に示される塩基配列からなるＤＮＡの相補鎖とストリンジェントな条件下でハイブリダイズしうるＤＮＡ、及び（ｃ）配列番号：２８に示される塩基配列からなるＤＮＡに少なくとも９０％の配列同一性を有するＤＮＡからなる群より選択されたＤＮＡであって、かつ脂肪組織および／または膵臓組織において特異的なプロモーター活性を有する、単離されたＤＮＡ。 (A) a DNA having at least one base substitution, deletion, insertion or addition in the base sequence shown in SEQ ID NO: 28; (b) a complementary strand and string of DNA consisting of the base sequence shown in SEQ ID NO: 28 DNA selected from the group consisting of DNA that can hybridize under a gentle condition, and (c) DNA having at least 90% sequence identity to DNA consisting of the base sequence shown in SEQ ID NO: 28, And isolated DNA having specific promoter activity in adipose tissue and / or pancreatic tissue.
  7. さらに配列番号：２７に示される塩基配列を有するＤＮＡである、請求項５又は６記載の単離されたＤＮＡ。　 Furthermore, the isolated DNA of Claim 5 or 6 which is DNA which has a base sequence shown by sequence number: 27. *
  8. さらに配列番号：２９に示される塩基配列を有するＤＮＡである、請求項５～７のいずれかに記載の単離されたＤＮＡ。 The isolated DNA according to any one of claims 5 to 7, which is further DNA having the base sequence represented by SEQ ID NO: 29.
  9. 請求項１～８のいずれかに記載の単離されたＤＮＡに外来遺伝子が連結されたＤＮＡ。 A DNA obtained by linking a foreign gene to the isolated DNA according to any one of claims 1 to 8.
  10. 請求項１～８のいずれかに記載の単離されたＤＮＡと外来遺伝子が作動可能に連結されてなる発現ベクター。 An expression vector comprising the isolated DNA according to any one of claims 1 to 8 and a foreign gene operably linked.
  11. 外来遺伝子が、対象疾患を治療するためのタンパク質をコードする遺伝子である、請求項１０に記載の発現ベクター。 The expression vector according to claim 10, wherein the foreign gene is a gene encoding a protein for treating the target disease.
  12. 外来遺伝子が、対象疾患を治療するための核酸である、請求項１０に記載の発現ベクター。 The expression vector according to claim 10, wherein the foreign gene is a nucleic acid for treating the target disease.
  13. 請求項１１または１２のいずれかに記載の発現ベクターを有効成分として含有してなる治療剤。 A therapeutic agent comprising the expression vector according to claim 11 as an active ingredient.
  14. 請求項１０～１３のいずれかに記載の発現ベクターを保持してなるトランスジェニック非ヒト哺乳動物。 A transgenic non-human mammal comprising the expression vector according to any one of claims 10 to 13.
  15. 発現ベクターが哺乳動物の染色体中に組み込まれてなる、請求項１４記載のトランスジェニック非ヒト哺乳動物。 The transgenic non-human mammal according to claim 14, wherein the expression vector is integrated into the chromosome of the mammal.
  16. 請求項１４または請求項１５のいずれかに記載されたトランスジェニック非ヒト哺乳動物に由来する細胞 A cell derived from the transgenic non-human mammal according to any one of claims 14 and 15.
  17. 請求項１４または１５のいずれかに記載のトランスジェニック非ヒト哺乳動物を用いることを特徴とする、スクリーニング方法。 A screening method, comprising using the transgenic non-human mammal according to claim 14.
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US11001857B2 (en) 2010-07-12 2021-05-11 Universitat Autonoma De Barcelona Gene therapy composition for use in diabetes treatment
US10711281B2 (en) 2012-08-02 2020-07-14 Universitat Autónoma De Barcelona Adeno-associated viral (AAV) vectors useful for transducing adipose tissue
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CN105916990A (en) * 2013-08-02 2016-08-31 巴塞罗那自治大学 Adeno-associated viral (AAV) vectors useful for trasducing adipose tissue
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