WO2005083087A1 - Transgennic animal allowing inductive deletion of osteocytes - Google Patents
Transgennic animal allowing inductive deletion of osteocytes Download PDFInfo
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- WO2005083087A1 WO2005083087A1 PCT/JP2005/003467 JP2005003467W WO2005083087A1 WO 2005083087 A1 WO2005083087 A1 WO 2005083087A1 JP 2005003467 W JP2005003467 W JP 2005003467W WO 2005083087 A1 WO2005083087 A1 WO 2005083087A1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
Definitions
- Transgenic animals capable of inductively deleting bone cells.
- the present invention relates to transgenic animals, and more particularly, to transgenic animals capable of inductively deleting bone cells.
- osteoclasts As bone-related cells, osteoclasts, osteoblasts and osteocytes are known. Several proteins produced by osteoclasts and osteoblasts are known, and some of them are used in the clinical field as markers of bone metabolism. On the other hand, for osteocytes, no specific gene products are known, and their functions in vivo have not been elucidated. Osteocytes are cells obtained by terminally dividing osteoblasts. Osteocytes do not proliferate during the rest period and are buried in hard tissue. Therefore, it is extremely difficult to isolate bone cells based on morphological features such as cell projections. In addition, since the gene expression pattern in vitro is generally different from that in vivo, it is difficult to analyze the biological characteristics of bone cells in vivo, even if bone cells can be isolated. It is ru.
- a method of analyzing the biological characteristics of specific cells in vivo a method of deleting target cells in an animal body and examining the effects thereof can be considered.
- a transgenic mouse in which hepatocytes are selectively destroyed by administration of diphtheria toxin has been developed (WO98Z33899 pamphlet; Saito M. et al.). , Nature Biotechnology 19, 746-750, 2001).
- the transgenic mouse contains the albumin gene, henno, Nsa-Z, which is specifically expressed in hepatic parenchymal cells.
- a DNA construct in which a human diphtheria toxin receptor gene is linked downstream of the promoter has been introduced. Therefore, when diphtheria toxin is administered to the transgenic mouse, hepatocytes expressing the diphtheria toxin receptor are specifically destroyed.
- the DMP1 (dentine matrix protein 1) gene is known as a gene expressed in bone cells! (Toyosawa S. et al., J. Bone Miner. Res. 16, 2017- 2026, 2001). It has also been reported that in the rat DMP1 gene promoter, the sequence that regulates the specificity of the expression of the gene in the rat DMP1 gene also has a transcription initiation point force within a region up to 2. Okb upstream (Thotakura SR et al.). ., J. Biol. Chem. 275, 10272-10277, 2000). Summary of the Invention
- the present inventors By using a DNA construct that expresses the receptor of the cytotoxic conjugate under the control of the DMP1 gene promoter, the present inventors have found that in a bone cell of a transgenic animal into which the DNA construct has been introduced, It is possible to specifically express the receptor, and further, by using a compound that is essentially non-toxic to a host as the cytotoxic compound, the compound to the transgenic animal can be expressed. Was found to be able to delete bone cells in the animal by the administration of. The present invention is based on these findings.
- the DNA construct according to the present invention is a DNA construct capable of deleting a bone cell in a host by administering a compound having essentially no toxicity to the host.
- the transgenic animal according to the present invention is a transgenic non-human animal comprising the DNA construct according to the present invention or a vector containing the same.
- osteocytes can be inducibly deleted by administering a compound.
- a transgenic animal is provided. By using this transgenic animal, it becomes possible to analyze gene expression and protein production in bone cells by comparison with natural animals, and thus to analyze the function of bone cells in vivo. .
- the findings obtained from such an analysis are extremely useful in diagnosing or treating bone-related diseases.
- FIG. 1 is a diagram showing the structure of a mouse DMP1 gene promoter.
- Fig. 2 is a bar graph showing the results of luciferase Atsusei showing the transcriptional activity of mouse DMP1 gene promoter having various nucleotide lengths in MC3T3-E1 cells.
- FIG. 3 is a view showing a structure of a DNA construct for expressing a diphtheria toxin human receptor (human HB-EGF).
- FIG. 4 is a graph showing protein synthesis inhibition by diphtheria toxin in MC3T3-E1 cells transfected with pDTR9.6 kb.
- FIG. 5 is a graph showing the inhibition of protein synthesis by diphtheria toxin in MC3T3-E1 cells transfected with 1.5 kb of pDTR.
- FIG. 6 is a graph showing the inhibition of protein synthesis by diphtheria toxin in ST-2 cells transfected with 1.5 kb or 9.6 kb pDTR.
- the DNA construct according to the present invention is a DNA construct that enables deletion of bone cells in a host by administering a compound that exhibits essentially no toxicity to the host.
- the DNA construct comprises a DNA encoding a receptor that binds to the compound, and a DMP1 gene promoter operably linked to the DNA, wherein the DMP1 gene promoter is operably linked to the compound.
- the killing power S of the cells expressing the receptor is provided.
- DMP1 gene promoter refers to a functional region that drives the transcription of DMP1 (dentine matrix protein 1) gene into mRNA in the body of a natural organism. It is located on the 5 'side (upstream) of the transcription start point of the gene. is there.
- DMP1 gene promoter can be easily produced or isolated by those skilled in the art.
- the DMP1 gene promoter can be isolated from a genomic library of an organism having the DMP1 gene using a probe designed based on the nucleotide sequence of the DMP1 gene.
- SEQ ID NO: 1 shows the promoter region of the mouse DMP1 gene up to 9614 bp upstream of the transcription start site (residues 1 to 9614), the first exon (residues 9615 to 9701), and the first intron (residue 9702).
- the DMP1 gene promoter is preferably derived from the host animal into which the DNA construct according to the present invention is introduced.
- the DMP1 gene promoter comprises at least a 1.5 kbp promoter region adjacent to the 5 ′ side of the transcription start site in the DMP1 gene. More preferably, it comprises at least a 9.6 kbp promoter region adjacent to the 5 'side of the transcription start point.
- the term "compound which shows essentially no toxicity to a host” refers to a compound that does not show toxicity to a host animal when the animal is not artificially engineered. Compound. Such compounds can be easily selected by those skilled in the art depending on the species of the animal to be used as a host.
- the receptor that binds to the compound is not particularly limited as long as it binds to the compound and kills the cell that expresses the receptor. Such receptors and the DNA encoding them are readily available to one of skill in the art, for example, in animals where the compound is toxic. Can be isolated. Examples of such a combination of a compound and a receptor include those that inhibit intracellular protein synthesis by their binding and those that cause apoptosis.
- the compound is diphtheria toxin
- the receptor is a diphtheria toxin receptor.
- Diphtheria toxin is an A-B toxin consisting of fragment A, the active site of the enzyme, and fragment B. Fragment A has ADP-ribose transferase activity and exerts toxicity by inactivating peptide elongation factor EF-2, which is essential for eukaryotic protein synthesis. Diphtheria toxin enters a cell via a diphtheria toxin receptor present on the cell surface, and kills the cell by inhibiting protein synthesis in the cell.
- the diphtheria toxin receptor is also known as the heparin-binding epidermal growth factor HB-EGF membrane-bound precursor (HB-EGF).
- HB-EGF heparin-binding epidermal growth factor
- Examples of the diphtheria toxin receptor and the DNA encoding the same include human-derived HB-EGF and genomic DNA, cDNA, and synthetic DNA encoding the same, for example, represented by SEQ ID NO: 5.
- Examples include a protein containing an amino acid sequence and a cDNA containing a nucleotide sequence represented by SEQ ID NO: 4 (NCBI database access number: NM-001945). Therefore, as the DNA encoding the diphtheria toxin receptor, a DNA containing the nucleotide sequence represented by residues 262 to 888 in SEQ ID NO: 4 can be used.
- the DNA encoding the receptor and the DMP1 gene promoter are operably linked.
- operably linked means that the DNAs are linked so that the DNA can express the receptor under the control of the DMP1 gene promoter. Such a connection can be made according to standard methods well-known in the art.
- the DNA construct according to the present invention comprises an intron between the DNA encoding the receptor and the DMP1 gene promoter, more preferably the DMP1 gene Of the first intron.
- the DNA construct according to the present invention can be used in a form incorporated in a vector upon introduction into a host (host cell or host animal).
- a vector comprising a DNA construct according to the present invention.
- the vector according to the present invention is not limited as long as it stably retains the DNA construct according to the present invention in a host animal or its cells and enables expression of the receptor under the control of the DMP1 gene promoter. Good. Such vectors are appropriately produced by those skilled in the art according to the method of gene transfer into the host to be used.
- the DNA construct according to the present invention and the vector according to the present invention can be introduced into host cells.
- a host cell comprising a DNA construct according to the present invention or a vector according to the present invention.
- the host cell various animal cells, preferably mammalian cells, can be used.
- the host cell is preferably a non-human animal cell, more preferably a cell from an animal for use in a biological experiment, such as a mouse, a rat, a rabbit, a dog, a monkey, or the like, and more preferably a cell.
- a biological experiment such as a mouse, a rat, a rabbit, a dog, a monkey, or the like, and more preferably a cell.
- the host cell according to the present invention can be produced by various methods that can be used for introducing a DNA construct or vector into a cell. Examples of such a method include a calcium phosphate method, a DEAE-dextran method, a lipofection method, a ribosome method, a mouth injection method, a method using a gene gun, and an electoration method. And a method using a recombinant virus vector such as an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector. Further, the host cell according to the present invention preferably contains the DNA construct according to the present invention in its genome.
- Methods for introducing a DNA construct into the genome of a cell include a method for retaining the DNA construct in the cell for a long time, and a method using a protein that recognizes a specific nucleotide sequence and an integrase. (Science 267, 1443-1444, 1995).
- the DNA construct according to the present invention and the vector according to the present invention can be introduced into a host animal, whereby a transgenic animal can be produced.
- a transgenic non-human animal comprising a DNA construct according to the present invention or a vector according to the present invention.
- the transgenic non-human animal according to the invention preferably comprises the DNA construct according to the invention in its genome.
- the host animal is not particularly limited as long as it is an animal other than human (non-human animal), Preferably, it is a non-human mammal, more preferably, an animal for use in biological experiments, for example, a mouse, a rat, a rabbit, a dog, a monkey, and the like, and further preferably, a mouse.
- the transgenic non-human animal according to the present invention can be produced by introducing the DNA construct according to the present invention or the vector according to the present invention into a desired non-human animal (host animal). More specifically, the transgenic non-human animal can be obtained by, for example, directly injecting the DNA construct into the nucleus of a pronuclear egg using a micropipette (microinjection method: Proc. Natl. Acad. Sci. USA 77, 7380-7384, 1980), a method using a recombinant retrovirus vector (Proc. Natl. Acad. Sci. USA 82, 6148—6152, 1985; Proc. Natl. Acad. Sci. USA 82, Proc. Natl. Acad.
- mice can do.
- the method for producing transgenic mice is widely known (Hogan B. et al., "Manual for Manipulating Mouse Embryos", Kyushu Shuppan, Tokyo, 1989; Tatsuji Nomura et al., “Developmental Engineering Experiment Manual” , Kodansha, Tokyo, 1987; Kenichi Yamamura, "Molecular medicine from the viewpoint of mice", Nankodo, Tokyo, 1993), and these methods can be applied to other animals.
- the DNA construct or the vector is introduced into a non-human animal of interest by injecting the DNA construct or the vector into a fertilized egg from the non-human animal. It is performed by raising fertilized eggs ⁇ !
- This method involves, for example, collecting fertilized eggs of the desired non-human animal power, injecting the DNA construct into the pronucleus of the fertilized eggs using a micromanipulator, and transferring the obtained fertilized eggs to the oviduct. Can be done by
- the DMP1 gene promoter causes the receptor of a compound that is essentially non-toxic to the host (non-human animal) to be specifically expressed in bone cells. ing. Therefore, by administering the compound to this transgenic non-human animal, bone cells can be specifically killed.
- the dose and timing of administration of the compound are not particularly limited.
- the route of administration of the compound is not particularly limited, and may be any route, such as intraperitoneal or intramedullary cavity.
- the route of administration of the compound should be close to the site of interest to kill bone cells. For example, when bone cells in the femur are to be killed, administration into the medullary cavity of the femur can be performed.
- Example 1 Preparation of a DNA construct that is a human form of diphtheria toxin under the control of the mouse DMP1 gene promoter
- a mouse genome library was screened for a clone having a sequence 5 ′ to the transcription start point of the gene. Specifically, targeting 1.2 ⁇ 10 ° plaques from the Mouse genomic library Lambda FIX II (Stratagene), 3500 nucleotides-3000 nucleotides (500 nucleotides length: attached to nucleotides) of the mouse DMP1 gene (The number indicates the position of the nucleotide residue when the adenine residue in the translation initiation codon of the gene is 1). As a result, 14 positive clones were obtained.
- the region required for transcriptional activity was examined by the luciferase atsay method using a mouse osteoblast cell line (MC 3T3-E1 cells) expressing the endogenous DMP1 gene.
- a mouse osteoblast cell line (MC 3T3-E1 cells) expressing the endogenous DMP1 gene.
- DMP1 gene mRNA was detected in the cells.
- a mouse bone marrow-derived stromal cell line ST-2 cell
- TRIzol Reagent Invitorogen: “TRIzol” was purchased from Invitorogen. (Registered trademark)
- the contaminated genomic DNA was digested by DNasel treatment.
- the presence or absence of mRNA from the DMP1 gene was examined by RT-PCR using these total RNAs as type II.
- DMP1-F 5, -agagggtagaggaatcgcat-3 ′: SEQ ID NO: 6
- DMP1-R 5′-cggtctatactggcctctgtcg-3 ′: SEQ ID NO: 7
- the size of the amplification product expected to indicate the presence of DMP1 gene mRNA is 278 bp.
- luciferase assay was performed. Specifically, the promoter region up to about 1. Okbp, 1.5 kbp, and 1.7 kbp upstream of the transcription start point can be added to the predicted transcription start point 230 bp downstream of the transcription start point by the multi-cloning of pGL3-Lusiferase basic vector (Promega). It was incorporated into the Xhol site in the single site.
- the DNA construct was prepared as follows. First, 1.5 kbp, located upstream of the transcription start site of the mouse DMP1 gene, which is expected to act as a promoter specific to the target osteocytes, is inserted into the pBluescript II SK (+) plasmid vector multiclone. And the human HB-EGF cDNA and j8-globin ZSV40 poly (A) signal sequence were ligated downstream of the second exon, second intron and third exon sequence of
- Fig. 1 which was introduced into the NotlZXbal (Nhel) site in the multicloning site of the pBluescript II SK (+) plasmid vector, and further downstream of human HB-EGF cDNA.
- a DNA fragment ligated with the j8-globin ZSV40 poly (A) signal sequence was introduced into the SpelZEcoRV site (the resulting plasmid vector was called "pDTR9.6kb").
- Fig. 3 shows the structure of the HB-EGF expression cassette contained in pDTR1.5kb and pDTR9.6kb.
- the prepared plasmid vector (pDTR1.5 kb or pDTR9.6 kb) and pDsRed2-Nuc (CLONTECH) containing the neomycin resistance gene were mixed at a ratio of 5: 1 with 4 ml of a culture solution (DMEM, 10% FBS).
- DMEM fetal calf serum
- Genes were transfected into MC3T3-E1 cells expressing the 60% confluent mouse DMP1 gene and ST-2 cells not expressing this in a 6 cm dish containing the same by the lipofection method.
- the obtained cells are placed in a CO incubator at 37 ° C for 48 hours.
- the cells are washed twice with PBS, then dissolved in a 0.1N aqueous sodium hydroxide solution, and the protein in the cell lysate is precipitated using 10% trichloroacetic acid (TCA). Whatman). After washing the glass filter three times with PBS, it was dried, and the scintillation solution was dried and the radioactivity of [ 35 S] methionine Z cysteine used for protein synthesis was measured using a liquid scintillation counter. Then, the inhibition rate of protein synthesis was evaluated.
- TCA trichloroacetic acid
- the plasmid vectors pDTR1.5kb and pDTR9.6kb were digested with the restriction enzyme SacIlZClal, and agarose gel extraction was performed to isolate DNA constructs DTR1.5kb and DTR9.6kb (FIG. 3). Subsequently, one of these DNA constructs was injected into fertilized mouse eggs
- mice (3 males and 6 females) were successfully weaned, of which the presence of DTR 9.6 kb was confirmed in 2 mice (1 male and 1 female).
- mice three males and three females were successfully weaned from 133 fertilized eggs, of which one mouse (one female) had a DTR of 9.6 kb. Presence confirmed.
- mice nine mice were successfully weaned from 236 fertilized eggs, and the presence of DTR9.6kb was confirmed in one mouse (one male).
- DTR1.5kb 5 transgenic mice (4 males and 1 female) were produced, and for transfection of DTR9.6kb, 4 transgenic mice (2 males and Two female transgenic mice were created. Among these, three males were stable for DTR 1.5 kb, and two male and two female for DTR 9.6 kb.
- the mouse into which DTR1.5kb was introduced was named “DTR1.5Tg”, and the mouse into which DTR9.6kb was introduced was named “DTR9.6Tg”.
- HB-EGF-2F The presence or absence of HB-EGF expression was determined for one of three DTR1.5Tg mice (DTR1.5 # 1) and one of four DTR9.6Tg mice (DTR9.6 # 1). confirmed. Specifically, various major organs were extracted from DTR1.5 # 1 and DTR9.6 # 1 mice, Total RNA was extracted using TRIzol Reagent (Invitorogen: “TRIzol” is a registered trademark of Invitorogen), and the contaminated genomic DNA was digested by DNasel treatment. Next, the presence or absence of HB-EGF mRNA expression was examined by RT-PCR using these total RNAs as type II. In RT PCR, for DTR1.5 # 1 mice, HB-EGF-2F:
- DMP1E1-F 5'-cgagaacttcgctgagg-3 '(SEQ ID NO: 14) and HB-EGF-2R: 5'-gagggtccgggttgctggttcc-3' (SEQ ID NO: 11) was used.
- mice received diphtheria toxin (Sigma) intraperitoneally or were administered to the bone marrow cavity of the joint force femur.
- the dose was 50 gZ kg or 500 gZ kg, and the number of doses was once or twice.
- the tibia and femur were taken out, immersed in a demineralized solution (10% EDTA, pH 7.4) for 2 weeks to demineralize, dehydrated, and prepared as paraffin-embedded slices.
- the resulting bone specimen was stained with HE and observed under a microscope to determine whether or not the bone cells had died.
- the DTR9.6Tg mouse has higher sensitivity to diphtheria toxin in bone than the DTR1.5Tg mouse.
- diphtheria toxin was directly injected into the medullary cavity of the femur because almost no bone cell death was observed.
- Administration As a result, complete killing of bone cells was observed over a wide area in the femur on the medullary cavity side of both transgenic mice.
- Direct administration of diphtheria toxin to the medullary cavity of the femur is a preferable and preferred method of administration, in that the effect of the toxin on other organs is small.
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Abstract
It is intended to provide a DNA construct which is usable in producing a transgenic animal allowing the inductive deletion of osteocytes. Namely, it is intended to provide a DNA constrct enabling the inductive deletion of osteocytes in a host by administering a compound essentially showing no toxicity to the host. This DNA construct comprises a DNA encoding a receptor binding to the above compound and a DMP1 gene promoter attached in such a manner that it can exert its function on the DNA. Due to the binding of the compound to the receptor, cells expressing the receptor are exterminated.
Description
明 細 書 Specification
誘導的に骨細胞を欠失させることのできるトランスジエニック動物 関連出願の参照 Transgenic animals capable of inductively deleting bone cells.
[0001] 本特許出願は、先に出願された日本国における特許出願である特願 2004— 5799 8号（出願日： 2004年 3月 2日）に基づく優先権の主張を伴うものである。この先の特 許出願における全開示内容は、引用することにより本明細書の一部とされる。 [0001] This patent application is accompanied by a priority claim based on Japanese Patent Application No. 2004-57998 (filed on March 2, 2004), which was a previously filed patent application in Japan. The entire disclosure content of the subsequent patent application is incorporated herein by reference.
発明の背景 Background of the Invention
[0002] 発明の分野 [0002] Field of the Invention
本発明は、トランスジエニック動物に関するものであり、より詳細には、誘導的に骨細 胞を欠失させることのできるトランスジエニック動物に関するものである。 The present invention relates to transgenic animals, and more particularly, to transgenic animals capable of inductively deleting bone cells.
[0003] 普晋 術 [0003] Jujinju
骨に関連する細胞としては、破骨細胞、骨芽細胞および骨細胞が知られている。破 骨細胞や骨芽細胞の産生するタンパク質についてはいくつか知られており、そのうち の一部は骨代謝のマーカーとして臨床分野において利用されている。一方で、骨細 胞（osteocyte)については、特異的な遺伝子産物は知られておらず、 in vivoでの機 能も解明されていない。骨細胞は、骨芽細胞が終末分ィ匕した細胞である。骨細胞は 、休止期にあって増殖せず、硬組織に埋没している。そのため、細胞突起などの形 態学的な特徴に基づいて骨細胞を単離することは可能と思われる力 その単離は極 めて困難である。また、一般に in vitroでの遺伝子の発現パターンは in vivoとは異な るため、骨細胞の単離が可能であったとしても、 in vivoにおける骨細胞の生物学的特 性を分析することは困難とされて 、る。 As bone-related cells, osteoclasts, osteoblasts and osteocytes are known. Several proteins produced by osteoclasts and osteoblasts are known, and some of them are used in the clinical field as markers of bone metabolism. On the other hand, for osteocytes, no specific gene products are known, and their functions in vivo have not been elucidated. Osteocytes are cells obtained by terminally dividing osteoblasts. Osteocytes do not proliferate during the rest period and are buried in hard tissue. Therefore, it is extremely difficult to isolate bone cells based on morphological features such as cell projections. In addition, since the gene expression pattern in vitro is generally different from that in vivo, it is difficult to analyze the biological characteristics of bone cells in vivo, even if bone cells can be isolated. It is ru.
[0004] 特定の細胞の in vivoにおける生物学的特性を分析する方法として、動物の生体内 において目的の細胞を欠失させ、その影響を検討する方法が考えられる。このような 方法に有用な実験動物として、ジフテリア毒素の投与により肝細胞が選択的に破壊 されるトランスジヱニックマウスが開発されている（国際公開第 98Z33899号パンフレ ット； Saito M. et al, Nature Biotechnology 19, 746—750, 2001)。このトランスジェ-ッ クマウスには、肝実質細胞に特異的に発現するアルブミン遺伝子のェンノ、ンサ一 Z
プロモーターの下流にヒトジフテリア毒素受容体遺伝子を連結した DNA構築物が導 入されている。従って、このトランスジエニックマウスにジフテリア毒素を投与すると、ジ フテリア毒素受容体を発現している肝細胞が特異的に破壊される。しかし、骨細胞を 特異的に欠失させることのできるトランスジエニック動物は知られていない。 [0004] As a method of analyzing the biological characteristics of specific cells in vivo, a method of deleting target cells in an animal body and examining the effects thereof can be considered. As an experimental animal useful for such a method, a transgenic mouse in which hepatocytes are selectively destroyed by administration of diphtheria toxin has been developed (WO98Z33899 pamphlet; Saito M. et al.). , Nature Biotechnology 19, 746-750, 2001). The transgenic mouse contains the albumin gene, henno, Nsa-Z, which is specifically expressed in hepatic parenchymal cells. A DNA construct in which a human diphtheria toxin receptor gene is linked downstream of the promoter has been introduced. Therefore, when diphtheria toxin is administered to the transgenic mouse, hepatocytes expressing the diphtheria toxin receptor are specifically destroyed. However, there is no known transgenic animal capable of specifically deleting bone cells.
[0005] 一方で、骨細胞にお!、て発現する遺伝子として、 DMP1 (dentine matrix protein 1 )遺伝子が知られている（Toyosawa S. et al.， J. Bone Miner. Res. 16, 2017-2026, 2001)。また、ラット DMP1遺伝子プロモーターにおいて、該遺伝子の発現の糸且織特 異性を調節する配列は、転写開始点力もその 2. Okb上流までの領域内にあることが 報告されている（Thotakura S. R. et al.， J. Biol. Chem. 275, 10272-10277, 2000)。 発明の概要 [0005] On the other hand, the DMP1 (dentine matrix protein 1) gene is known as a gene expressed in bone cells! (Toyosawa S. et al., J. Bone Miner. Res. 16, 2017- 2026, 2001). It has also been reported that in the rat DMP1 gene promoter, the sequence that regulates the specificity of the expression of the gene in the rat DMP1 gene also has a transcription initiation point force within a region up to 2. Okb upstream (Thotakura SR et al.). ., J. Biol. Chem. 275, 10272-10277, 2000). Summary of the Invention
[0006] 本発明者らは、 DMP1遺伝子プロモーターの制御下において細胞毒性ィ匕合物の 受容体を発現する DNA構築物を用いることにより、該 DNA構築物を導入されたトラ ンスジ ニック動物の骨細胞において特異的に前記受容体を発現させることが可能 となり、さらには、宿主に対して本質的に毒性を示さない化合物を前記細胞毒性化合 物として用いることにより、前記トランスジヱニック動物への前記化合物の投与によつ て該動物における骨細胞を欠失させることが可能となることを見出した。本発明はこ れらの知見に基づくものである。 [0006] By using a DNA construct that expresses the receptor of the cytotoxic conjugate under the control of the DMP1 gene promoter, the present inventors have found that in a bone cell of a transgenic animal into which the DNA construct has been introduced, It is possible to specifically express the receptor, and further, by using a compound that is essentially non-toxic to a host as the cytotoxic compound, the compound to the transgenic animal can be expressed. Was found to be able to delete bone cells in the animal by the administration of. The present invention is based on these findings.
[0007] 従って、本発明の目的は、誘導的に骨細胞を欠失させることのできるトランスジェ- ック動物、およびその製造に用いることのできる DNA構築物を提供することにある。 [0007] Accordingly, it is an object of the present invention to provide a transgenic animal capable of inductively deleting bone cells, and a DNA construct that can be used for the production thereof.
[0008] そして、本発明による DNA構築物は、宿主に対して本質的に毒性を示さない化合 物を投与することにより、該宿主において骨細胞を欠失させることを可能とする DNA 構築物であって、前記化合物に結合する受容体をコードする DNA、および該 DNA に機能しうる形で連結された DMP1遺伝子プロモーターを含んでなり、前記化合物と 前記受容体との結合により該受容体を発現している細胞の死滅力 Sもたらされる、 DN A構築物である。 [0008] The DNA construct according to the present invention is a DNA construct capable of deleting a bone cell in a host by administering a compound having essentially no toxicity to the host. A DNA encoding a receptor that binds to the compound, and a DMP1 gene promoter operably linked to the DNA, and expressing the receptor by binding the compound to the receptor. Is a DNA construct that results in cell killing.
[0009] さらに、本発明によるトランスジエニック動物は、本発明による DNA構築物またはこ れを含むベクターを含んでなる、トランスジエニック非ヒト動物である。 [0009] Further, the transgenic animal according to the present invention is a transgenic non-human animal comprising the DNA construct according to the present invention or a vector containing the same.
[0010] 本発明によれば、化合物の投与によって誘導的に骨細胞を欠失させることのできる
トランスジエニック動物が提供される。このトランスジエニック動物を用いることにより、 天然の動物との比較による骨細胞での遺伝子発現およびタンパク質産生の分析が 可能となり、従って、骨細胞の in vivoでの機能を分析することが可能となる。このよう な分析により得られる知見は、骨に関する疾病の診断または治療において極めて有 用である。 [0010] According to the present invention, osteocytes can be inducibly deleted by administering a compound. A transgenic animal is provided. By using this transgenic animal, it becomes possible to analyze gene expression and protein production in bone cells by comparison with natural animals, and thus to analyze the function of bone cells in vivo. . The findings obtained from such an analysis are extremely useful in diagnosing or treating bone-related diseases.
図面の簡単な説明 Brief Description of Drawings
[0011] [図 1]図 1は、マウス DMP1遺伝子プロモーターの構造を示す図である。 FIG. 1 is a diagram showing the structure of a mouse DMP1 gene promoter.
[図 2]図 2は、様々なヌクレオチド長を有するマウス DMP1遺伝子プロモーターの MC 3T3— E1細胞における転写活性を示すルシフェラーゼアツセィの結果を示す棒ダラ フである。 [Fig. 2] Fig. 2 is a bar graph showing the results of luciferase Atsusei showing the transcriptional activity of mouse DMP1 gene promoter having various nucleotide lengths in MC3T3-E1 cells.
[図 3]図 3は、ジフテリア毒素ヒト型受容体 (ヒト HB— EGF)を発現させるための DNA 構築物の構造を示す図である。 FIG. 3 is a view showing a structure of a DNA construct for expressing a diphtheria toxin human receptor (human HB-EGF).
[図 4]図 4は、 pDTR9.6kbでトランスフエタトした MC3T3— E1細胞における、ジフテリ ァ毒素によるタンパク質合成阻害を示すグラフである。 FIG. 4 is a graph showing protein synthesis inhibition by diphtheria toxin in MC3T3-E1 cells transfected with pDTR9.6 kb.
[図 5]図 5は、 pDTR1.5kbでトランスフエタトした MC3T3— E1細胞における、ジフテリ ァ毒素によるタンパク質合成阻害を示すグラフである。 FIG. 5 is a graph showing the inhibition of protein synthesis by diphtheria toxin in MC3T3-E1 cells transfected with 1.5 kb of pDTR.
[図 6]図 6は、 pDTR1.5kbまたは pDTR9.6kbでトランスフエタトした ST— 2細胞における 、ジフテリア毒素によるタンパク質合成阻害を示すグラフである。 FIG. 6 is a graph showing the inhibition of protein synthesis by diphtheria toxin in ST-2 cells transfected with 1.5 kb or 9.6 kb pDTR.
発明の具体的説明 Detailed description of the invention
[0012] 本発明による DNA構築物は、宿主に対して本質的に毒性を示さない化合物を投 与することにより、該宿主において骨細胞を欠失させることを可能とする DNA構築物 である。この DNA構築物は、前記化合物に結合する受容体をコードする DNA、およ び該 DNAに機能しうる形で連結された DMP1遺伝子プロモーターを含んでなり、前 記化合物と前記受容体との結合により該受容体を発現している細胞の死滅力 Sもたら される。 [0012] The DNA construct according to the present invention is a DNA construct that enables deletion of bone cells in a host by administering a compound that exhibits essentially no toxicity to the host. The DNA construct comprises a DNA encoding a receptor that binds to the compound, and a DMP1 gene promoter operably linked to the DNA, wherein the DMP1 gene promoter is operably linked to the compound. The killing power S of the cells expressing the receptor is provided.
[0013] 本明細書において「DMP1遺伝子プロモーター」とは、天然の生物の体内におい て DMP1 (dentine matrix protein 1)遺伝子の mRNAへの転写を駆動する機能性領 域を意味し、典型的には該遺伝子の転写開始点の 5'側（上流側）に位置するもので
ある。このような DMP1遺伝子プロモーターは、当業者であれば容易に製造または 単離することができる。例えば、この DMP1遺伝子プロモーターは、 DMP1遺伝子を 有する生物のゲノムライブラリーから、 DMP1遺伝子のヌクレオチド配列に基づいて 設計されたプローブを用いて単離することが可能である。 DMP1遺伝子のヌクレオチ ド配列およびこれによりコードされるアミノ酸配列としては様々な生物に由来するもの が知られており、例えば、マウス DMP1遺伝子については、配列番号 1で表されるゲ ノム上のヌクレオチド配列、ならびに配列番号 2で表される cDNA配列および配列番 号 3で表されるアミノ酸配列が挙げられる。配列番号 1は、マウス DMP1遺伝子にお ける、転写開始点の 9614bp上流までのプロモーター領域 (第 1一 9614残基）、第 1 ェクソン (第 9615— 9701残基）、第 1イントロン (第 9702— 14064残基）、第 2ェクソ ン（第 14065— 14139残基）、第 2イントロン (第 14140— 14578残基）、第 3ェクソ ン（第 14579— 14626残基）、第 3イントロン (第 14627— 16886残基）、第 4ェクソ ン（第 16887— 16919残基）、第 4イントロン (第 16920— 17090残基）、第 5ェクソ ン（第 17091— 17135残基）、第 5イントロン (第 17136— 18627残基）および第 6ェ クソン (第 18628残基一第 21100残基付近)を含んでおり、該遺伝子の転写開始点 は第 9615残基である。 DMP1遺伝子プロモーターは、本発明による DNA構築物を 導入する対象となる宿主動物に由来するものとすることが好ま 、。 [0013] As used herein, the term "DMP1 gene promoter" refers to a functional region that drives the transcription of DMP1 (dentine matrix protein 1) gene into mRNA in the body of a natural organism. It is located on the 5 'side (upstream) of the transcription start point of the gene. is there. Such a DMP1 gene promoter can be easily produced or isolated by those skilled in the art. For example, the DMP1 gene promoter can be isolated from a genomic library of an organism having the DMP1 gene using a probe designed based on the nucleotide sequence of the DMP1 gene. As the nucleotide sequence of the DMP1 gene and the amino acid sequence encoded thereby, those derived from various organisms are known.For example, for the mouse DMP1 gene, the nucleotide sequence on the genome represented by SEQ ID NO: 1 And the cDNA sequence represented by SEQ ID NO: 2 and the amino acid sequence represented by SEQ ID NO: 3. SEQ ID NO: 1 shows the promoter region of the mouse DMP1 gene up to 9614 bp upstream of the transcription start site (residues 1 to 9614), the first exon (residues 9615 to 9701), and the first intron (residue 9702). 14064 residues), 2nd exon (14014-14139 residues), 2nd intron (14140-14578 residues), 3rd exoon (14579-14626 residues), 3rd intron (14627-residue) 4886 (residues 16887-16919), intron 4 (residues 16920-17090), exoon 5 (residues 17091-17135), and intron 5 (residue 17136) 18627) and the sixth exon (residue 18628-121100), and the transcription start point of the gene is 9615. The DMP1 gene promoter is preferably derived from the host animal into which the DNA construct according to the present invention is introduced.
[0014] 本発明の好ましい実施態様によれば、前記 DMP1遺伝子プロモーターは、 DMP1 遺伝子にお 、て転写開始点の 5 '側に隣接する少なくとも 1. 5kbpのプロモーター領 域を含んでなるものとされ、より好ましくは前記転写開始点の 5'側に隣接する少なくと も 9. 6kbpのプロモーター領域を含んでなるものとされる。 According to a preferred embodiment of the present invention, the DMP1 gene promoter comprises at least a 1.5 kbp promoter region adjacent to the 5 ′ side of the transcription start site in the DMP1 gene. More preferably, it comprises at least a 9.6 kbp promoter region adjacent to the 5 'side of the transcription start point.
[0015] 本明細書において「宿主に対して本質的に毒性を示さない化合物」とは、宿主とな る動物が人工的に遺伝子操作されていない場合に、その動物に対して毒性を示さな い化合物を意味する。このような化合物は、宿主となる動物の種に応じて、当業者で あれば容易に選択することができる。また、前記化合物に結合する受容体としては、 前記化合物との結合により該受容体を発現している細胞が死滅するものであればよ ぐ特に制限されない。このような受容体およびこれをコードする DNAは、当業者で あれば容易に利用することが可能であり、例えば、前記化合物が毒性を示す動物か
ら単離することができる。このような化合物と受容体との組み合わせとしては、例えば 、それらの結合によって細胞内のタンパク質合成を抑制するもの、アポトーシスを起こ すもの等が挙げられる。 [0015] As used herein, the term "compound which shows essentially no toxicity to a host" refers to a compound that does not show toxicity to a host animal when the animal is not artificially engineered. Compound. Such compounds can be easily selected by those skilled in the art depending on the species of the animal to be used as a host. The receptor that binds to the compound is not particularly limited as long as it binds to the compound and kills the cell that expresses the receptor. Such receptors and the DNA encoding them are readily available to one of skill in the art, for example, in animals where the compound is toxic. Can be isolated. Examples of such a combination of a compound and a receptor include those that inhibit intracellular protein synthesis by their binding and those that cause apoptosis.
[0016] 本発明の好ましい実施態様によれば、前記化合物はジフテリア毒素とされ、前記受 容体はジフテリア毒素受容体とされる。ジフテリア毒素は酵素活性部位であるフラグメ ント Aとフラグメント Bカゝらなる A— B型毒素である。フラグメント Aには ADPリボース転 移酵素活性があり、真核細胞のタンパク質合成に必須なペプチド伸張因子 EF— 2を 不活性ィ匕することにより毒性を発揮する。ジフテリア毒素は、細胞表面に存在するジ フテリア毒素受容体を介して細胞内に侵入し、その細胞におけるタンパク質合成を阻 害することにより該細胞を死滅させる。ジフテリア毒素受容体は、へパリン結合性上皮 細胞増殖因子 HB— EGF膜結合型前駆体 (HB— EGF)としても知られている。ジフテ リア毒素受容体およびこれをコードする DNAとしては、ヒトに由来する HB— EGFおよ びこれをコードするゲノム DNA、 cDNA、合成 DNA等が挙げられ、例えば、配列番 号 5で表されるアミノ酸配列を含むタンパク質および配列番号 4で表されるヌクレオチ ド配列を含む cDNA (NCBIデータベースアクセス番号： NM— 001945)が挙げられ る。従って、ジフテリア毒素受容体をコードする DNAとしては、配列番号 4中の第 26 2— 888残基で表されるヌクレオチド配列を含む DNAを用いることができる。 According to a preferred embodiment of the present invention, the compound is diphtheria toxin, and the receptor is a diphtheria toxin receptor. Diphtheria toxin is an A-B toxin consisting of fragment A, the active site of the enzyme, and fragment B. Fragment A has ADP-ribose transferase activity and exerts toxicity by inactivating peptide elongation factor EF-2, which is essential for eukaryotic protein synthesis. Diphtheria toxin enters a cell via a diphtheria toxin receptor present on the cell surface, and kills the cell by inhibiting protein synthesis in the cell. The diphtheria toxin receptor is also known as the heparin-binding epidermal growth factor HB-EGF membrane-bound precursor (HB-EGF). Examples of the diphtheria toxin receptor and the DNA encoding the same include human-derived HB-EGF and genomic DNA, cDNA, and synthetic DNA encoding the same, for example, represented by SEQ ID NO: 5. Examples include a protein containing an amino acid sequence and a cDNA containing a nucleotide sequence represented by SEQ ID NO: 4 (NCBI database access number: NM-001945). Therefore, as the DNA encoding the diphtheria toxin receptor, a DNA containing the nucleotide sequence represented by residues 262 to 888 in SEQ ID NO: 4 can be used.
[0017] 本発明による DNA構築物においては、前記受容体をコードする DNAと DMP1遺 伝子プロモーターとが機能しうる形で連結されている。ここで「機能しうる形で連結さ れ」とは、前記 DNAが DMP1遺伝子プロモーターの制御下において前記受容体を 発現しうるようにこれらが連結されることを意味する。このような連結は、当技術分野に おいて周知の標準的な方法に従って行なうことができる。 In the DNA construct according to the present invention, the DNA encoding the receptor and the DMP1 gene promoter are operably linked. Here, "operably linked" means that the DNAs are linked so that the DNA can express the receptor under the control of the DMP1 gene promoter. Such a connection can be made according to standard methods well-known in the art.
[0018] 本発明の好ましい実施態様によれば、本発明による DNA構築物は、前記受容体 をコードする DNAと前記 DMP1遺伝子プロモーターとの間にイントロンを含んでなる ものとされ、より好ましくは DMP1遺伝子の第 1イントロンを含んでなるものとされる。 According to a preferred embodiment of the present invention, the DNA construct according to the present invention comprises an intron between the DNA encoding the receptor and the DMP1 gene promoter, more preferably the DMP1 gene Of the first intron.
[0019] 本発明による DNA構築物は、宿主 (宿主細胞または宿主動物）への導入の際に、 ベクター中に組み込まれた形態で用いることができる。従って、本発明によれば、本 発明による DNA構築物を含んでなるベクターが提供される。
[0020] 本発明によるベクターは、宿主動物またはその細胞において、本発明による DNA 構築物を安定して保持し、前記 DMP1遺伝子プロモーターの制御下での前記受容 体の発現を可能とするものであればよい。このようなベクターは、利用される宿主への 遺伝子導入法に応じて、当業者によって適宜製造される。 [0019] The DNA construct according to the present invention can be used in a form incorporated in a vector upon introduction into a host (host cell or host animal). Thus, according to the present invention there is provided a vector comprising a DNA construct according to the present invention. [0020] The vector according to the present invention is not limited as long as it stably retains the DNA construct according to the present invention in a host animal or its cells and enables expression of the receptor under the control of the DMP1 gene promoter. Good. Such vectors are appropriately produced by those skilled in the art according to the method of gene transfer into the host to be used.
[0021] 本発明による DNA構築物および本発明によるベクターは、宿主細胞に導入するこ とができる。従って、本発明によれば、本発明による DNA構築物または本発明による ベクターを含んでなる宿主細胞が提供される。 [0021] The DNA construct according to the present invention and the vector according to the present invention can be introduced into host cells. Thus, according to the present invention there is provided a host cell comprising a DNA construct according to the present invention or a vector according to the present invention.
[0022] 前記宿主細胞としては、様々な動物細胞、好ましくは哺乳動物細胞を用いることが できる。また、前記宿主細胞は、好ましくは非ヒト動物細胞、より好ましくは生物実験に 用いるための動物からの細胞、例えば、マウス、ラット、ゥサギ、ィヌ、サル等力もの細 胞とされ、さらに好ましくはマウスからの細胞とされる。 [0022] As the host cell, various animal cells, preferably mammalian cells, can be used. The host cell is preferably a non-human animal cell, more preferably a cell from an animal for use in a biological experiment, such as a mouse, a rat, a rabbit, a dog, a monkey, or the like, and more preferably a cell. Are cells from mice.
[0023] 本発明による宿主細胞は、 DNA構築物またはベクターの細胞への導入に用いるこ とのできる様々な方法によって製造することができる。このような方法としては、例えば 、リン酸カルシウム法、 DEAE—デキストラン法、リポフエクシヨン法、リボソーム法、マ イク口インジェクション法、遺伝子銃を用いる方法、エレクト口ポレーシヨン法などを挙 げることができ、さらには、アデノウイルスベクター、アデノ随伴ウィルスベクター、レト ロウィルスベクターなどの糸且換えウィルスベクターを用いる方法を挙げることができる 。また、本発明による宿主細胞は、本発明による DNA構築物をゲノム中に含んでい ることが好ましい。 DNA構築物を細胞のゲノム中に導入する方法としては、その DN A構築物を細胞中に長時間保持する方法が挙げられ、さらには、特定のヌクレオチド 配列を認識するタンパク質とインテグラーゼとを利用する方法 (Science 267, 1443-1444, 1995)が挙げられる。 [0023] The host cell according to the present invention can be produced by various methods that can be used for introducing a DNA construct or vector into a cell. Examples of such a method include a calcium phosphate method, a DEAE-dextran method, a lipofection method, a ribosome method, a mouth injection method, a method using a gene gun, and an electoration method. And a method using a recombinant virus vector such as an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector. Further, the host cell according to the present invention preferably contains the DNA construct according to the present invention in its genome. Methods for introducing a DNA construct into the genome of a cell include a method for retaining the DNA construct in the cell for a long time, and a method using a protein that recognizes a specific nucleotide sequence and an integrase. (Science 267, 1443-1444, 1995).
[0024] 本発明による DNA構築物および本発明によるベクターは、宿主動物に導入するこ とができ、これによりトランスジエニック動物を製造することができる。従って、本発明に よれば、本発明による DNA構築物または本発明によるベクターを含んでなるトランス ジェニック非ヒト動物が提供される。本発明によるトランスジエニック非ヒト動物は、その ゲノム中に本発明による DNA構築物を含んでなるものとすることが好ま 、。 [0024] The DNA construct according to the present invention and the vector according to the present invention can be introduced into a host animal, whereby a transgenic animal can be produced. Thus, according to the present invention there is provided a transgenic non-human animal comprising a DNA construct according to the present invention or a vector according to the present invention. The transgenic non-human animal according to the invention preferably comprises the DNA construct according to the invention in its genome.
[0025] 前記宿主動物は、ヒト以外の動物（非ヒト動物）であればよぐ特に制限されないが、
好ましくは非ヒト哺乳動物とされ、より好ましくは生物実験に用いるための動物、例え ば、マウス、ラット、ゥサギ、ィヌ、サル等とされ、さらに好ましくはマウスとされる。 The host animal is not particularly limited as long as it is an animal other than human (non-human animal), Preferably, it is a non-human mammal, more preferably, an animal for use in biological experiments, for example, a mouse, a rat, a rabbit, a dog, a monkey, and the like, and further preferably, a mouse.
[0026] 本発明によるトランスジエニック非ヒト動物は、 目的とする非ヒト動物（宿主動物）に本 発明による DNA構築物または本発明によるベクターを導入することによって製造す ることができる。より具体的には、前記トランスジ ニック非ヒト動物は、例えば、微小ピ ペットを用いて前核期卵の核に DNA構築物を直接注入する方法 (マイクロインジエタ シヨン法： Proc. Natl. Acad. Sci. USA 77, 7380-7384, 1980)、組換えレトロウイルスべ クタ一を用いる方法（Proc. Natl. Acad. Sci. USA 82, 6148—6152, 1985 ; Proc. Natl. Acad. Sci. USA 82, 6927—6931, 1985 ; Proc. Natl. Acad. Sci. USA 82, 8587—8591, 1985)、胚性幹細胞を用いる方法（Proc. Natl. Acad. Sci. USA 83, 9065-9069, 1986 )によって製造することができる。特に、トランスジヱニックマウスの製造法は広く知ら れており（Hogan B.ら、「マウス胚の操作マ-ユアル」、近代出版、東京、 1989 ;野村 達次ら、「発生工学実験マニュアル」、講談社、東京、 1987 ;山村研一、「マウスから 見た分子医学」、南江堂、東京、 1993)、これらの方法を他の動物に応用することも 可能である。 [0026] The transgenic non-human animal according to the present invention can be produced by introducing the DNA construct according to the present invention or the vector according to the present invention into a desired non-human animal (host animal). More specifically, the transgenic non-human animal can be obtained by, for example, directly injecting the DNA construct into the nucleus of a pronuclear egg using a micropipette (microinjection method: Proc. Natl. Acad. Sci. USA 77, 7380-7384, 1980), a method using a recombinant retrovirus vector (Proc. Natl. Acad. Sci. USA 82, 6148—6152, 1985; Proc. Natl. Acad. Sci. USA 82, Proc. Natl. Acad. Sci. USA 82, 8587-8591, 1985), manufactured using embryonic stem cells (Proc. Natl. Acad. Sci. USA 83, 9065-9069, 1986). can do. In particular, the method for producing transgenic mice is widely known (Hogan B. et al., "Manual for Manipulating Mouse Embryos", Kyushu Shuppan, Tokyo, 1989; Tatsuji Nomura et al., "Developmental Engineering Experiment Manual" , Kodansha, Tokyo, 1987; Kenichi Yamamura, "Molecular medicine from the viewpoint of mice", Nankodo, Tokyo, 1993), and these methods can be applied to other animals.
[0027] 本発明の好ましい実施態様によれば、 目的とする非ヒト動物への前記 DNA構築物 または前記ベクターの導入は、該非ヒト動物からの受精卵に該 DNA構築物または該 ベクターを注入し、得られた受精卵^!司育することによって行なわれる。この方法は、 例えば、 目的とする非ヒト動物力も受精卵を採取し、マイクロマ-ュピレーターを用い て DNA構築物を受精卵の前核中に注入し、得られた受精卵を卵管に移入すること によって行なうことができる。 According to a preferred embodiment of the present invention, the DNA construct or the vector is introduced into a non-human animal of interest by injecting the DNA construct or the vector into a fertilized egg from the non-human animal. It is performed by raising fertilized eggs ^! This method involves, for example, collecting fertilized eggs of the desired non-human animal power, injecting the DNA construct into the pronucleus of the fertilized eggs using a micromanipulator, and transferring the obtained fertilized eggs to the oviduct. Can be done by
[0028] 本発明によるトランスジエニック非ヒト動物においては、 DMP1遺伝子プロモーター により、宿主 (非ヒト動物）に対して本質的に毒性を示さない化合物の受容体が骨細 胞に特異的に発現している。従って、このトランスジ ニック非ヒト動物に対して前記 化合物を投与することにより、骨細胞を特異的に死滅させることが可能である。化合 物の投与量および投与の時期は特に制限されるものではない。また、化合物の投与 経路も特に制限されるものではなぐ腹腔内、骨髄腔内など、いずれの経路であって もよい。化合物の投与経路は、骨細胞を死滅させようとする目的の部位の近位への
局所投与としてもよぐ例えば、大腿骨における骨細胞を死滅させる場合には、大腿 骨骨髄腔への投与とすることができる。 [0028] In the transgenic non-human animal according to the present invention, the DMP1 gene promoter causes the receptor of a compound that is essentially non-toxic to the host (non-human animal) to be specifically expressed in bone cells. ing. Therefore, by administering the compound to this transgenic non-human animal, bone cells can be specifically killed. The dose and timing of administration of the compound are not particularly limited. The route of administration of the compound is not particularly limited, and may be any route, such as intraperitoneal or intramedullary cavity. The route of administration of the compound should be close to the site of interest to kill bone cells. For example, when bone cells in the femur are to be killed, administration into the medullary cavity of the femur can be performed.
実施例 Example
[0029] 以下、本発明を実施例により具体的に説明するが、これら実施例は本発明を限定 するものではない。 Hereinafter, the present invention will be described specifically with reference to Examples, but these Examples do not limit the present invention.
[0030] 例 1 :マウス DMP1遣伝子プロモーターの制御下においてジフテリア毒素のヒト型^ 容体 する DNA構签物の作製 Example 1: Preparation of a DNA construct that is a human form of diphtheria toxin under the control of the mouse DMP1 gene promoter
1.1マウス DMP1遣伝子のプロモーター領城のクローニング 1.1 Cloning of mouse DMP1 gene promoter
マウス DMP1遺伝子プロモーターを単離するため、マウスゲノムライブラリ一力 該 遺伝子の転写開始点よりも 5'側の配列を有するクローンをスクリーニングした。具体 的には、 Mouse genomic library Lambda FIX II (Stratagene社）からの 1.2 X 10°個のプ ラークを対象として、マウス DMP1遺伝子の第— 3500—— 3000ヌクレオチド（500ヌ クレオチド長：ヌクレオチドに付された番号は同遺伝子の翻訳開始コドン中のアデ- ン残基を 1とした場合におけるそのヌクレオチド残基の位置を示す)をプローブとして スクリーニングを行ない、その結果として 14個の陽性クローンを得た。次いで、これら 陽性クローンの全てのファージ DNAを精製し、 Notl消化により得られた DNA断片 のヌクレオチド配列を決定した。その結果、最も長いプロモーター領域が含まれてい る 2つのクローンを同定した。これらのクローンは、翻訳開始点 (翻訳開始コドン中の アデニン残基）の 300bp上流力も転写開始点の 9614bp上流までを含む同一のクロ ーンであることがわ力つた（図 1を参照)。 In order to isolate the mouse DMP1 gene promoter, a mouse genome library was screened for a clone having a sequence 5 ′ to the transcription start point of the gene. Specifically, targeting 1.2 × 10 ° plaques from the Mouse genomic library Lambda FIX II (Stratagene), 3500 nucleotides-3000 nucleotides (500 nucleotides length: attached to nucleotides) of the mouse DMP1 gene (The number indicates the position of the nucleotide residue when the adenine residue in the translation initiation codon of the gene is 1). As a result, 14 positive clones were obtained. Next, all the phage DNAs of these positive clones were purified, and the nucleotide sequence of the DNA fragment obtained by Notl digestion was determined. As a result, two clones containing the longest promoter region were identified. These clones were found to be identical clones containing up to 30014 bp upstream of the translation initiation site (adenine residue in the translation initiation codon) up to 9614 bp upstream of the transcription initiation site (see FIG. 1).
[0031] 1.2 DMP1遣伝子プロモーターの機能解析 [0031] 1.2 Functional analysis of DMP1 transgene promoter
上述のスクリーニングにより得られた DMP1遺伝子プロモーター配列について、転 写活性に必要な領域を、内在性の DMP1遺伝子を発現するマウス骨芽細胞株 (MC 3T3— E1細胞）を用いるルシフェラーゼアツセィ法によって調べた。 For the DMP1 gene promoter sequence obtained by the above-mentioned screening, the region required for transcriptional activity was examined by the luciferase atsay method using a mouse osteoblast cell line (MC 3T3-E1 cells) expressing the endogenous DMP1 gene. Was.
[0032] まず、 MC3T3— E1細胞において DMP1遺伝子が発現することを確認するため、 該細胞における DMP1遺伝子 mRNAの検出を行なった。陰性対照としては、マウス 骨髄由来ストローマ細胞株（ST— 2細胞）を用いた。具体的には、 MC3T3— E1細胞 および ST— 2細胞から、 TRIzol Reagent (Invitorogen社：「TRIzol」は Invitorogen社の
登録商標である）を用いて全 RNAを抽出し、 DNasel処理により混入ゲノム DNAを 消化した。次に、これらの全 RNAを铸型とする RT— PCRにより、 DMP1遺伝子から の mRNAの有無を調べた。 RT— PCRには、 DMP1- F (5，- agagggtagaggaatcgcat- 3'： 配列番号 6)および DMP1-R (5'-cggtctatactggcctctgtcg-3'：配列番号 7)力 なるプラ イマ一ペアを用いた。 DMP1遺伝子 mRNAの存在を示すものとして予想される増幅 産物のサイズは 278bpである。その結果、 ST— 2細胞では DMP1遺伝子が発現して Vヽな 、のに対し、 MC3T3— E1細胞では DMP1遺伝子が発現して!/、ることが確認さ れた。 [0032] First, in order to confirm that the DMP1 gene was expressed in MC3T3-E1 cells, DMP1 gene mRNA was detected in the cells. As a negative control, a mouse bone marrow-derived stromal cell line (ST-2 cell) was used. Specifically, from MC3T3-E1 cells and ST-2 cells, TRIzol Reagent (Invitorogen: “TRIzol” was purchased from Invitorogen. (Registered trademark), and the contaminated genomic DNA was digested by DNasel treatment. Next, the presence or absence of mRNA from the DMP1 gene was examined by RT-PCR using these total RNAs as type II. For RT-PCR, a primer pair of DMP1-F (5, -agagggtagaggaatcgcat-3 ′: SEQ ID NO: 6) and DMP1-R (5′-cggtctatactggcctctgtcg-3 ′: SEQ ID NO: 7) was used. The size of the amplification product expected to indicate the presence of DMP1 gene mRNA is 278 bp. As a result, it was confirmed that the DMP1 gene was expressed in the ST-2 cells and the DMP1 gene was expressed in the MC3T3-E1 cells, whereas the DMP1 gene was expressed! /.
[0033] 次!、で、様々なヌクレオチド長を有するマウス DMP1遺伝子プロモーターの転写活 性を評価するため、ルシフェラーゼアツセィを行なった。具体的には、予想される転 写開始点の 230bp下流力も転写開始点の約 1. Okbp、 1. 5kbpおよび 1. 7kbp上流 までのプロモーター領域を、 pGL3— Lusiferase basic vector (Promega社）のマルチクロ 一-ングサイト内にある Xhol部位に組み込んだ。得られた 3種のベクターにおいては 、ホタルルシフェラーゼ遺伝子の cDNAの上流に上述の各種プロモーター領域が連 結されている（これらの DNA構築物を、それぞれ「1.0kb/F」、「1.5kb/F」および「 1.7kb/F」という）。それぞれのベクターを、 DMP1遺伝子を発現する MC3T3— E1細 胞に導入し、その 48時間後にルシフェラーゼ活性を測定することにより転写活性を 調べた。この実験では、 DMP1遺伝子をほとんど発現しない ST— 2細胞を陰性対照 とした。 Next, in order to evaluate the transcriptional activity of the mouse DMP1 gene promoter having various nucleotide lengths, luciferase assay was performed. Specifically, the promoter region up to about 1. Okbp, 1.5 kbp, and 1.7 kbp upstream of the transcription start point can be added to the predicted transcription start point 230 bp downstream of the transcription start point by the multi-cloning of pGL3-Lusiferase basic vector (Promega). It was incorporated into the Xhol site in the single site. In the three types of obtained vectors, the above-mentioned various promoter regions were linked upstream of the cDNA of the firefly luciferase gene (these DNA constructs were referred to as “1.0 kb / F” and “1.5 kb / F”, respectively). And "1.7kb / F"). Each vector was introduced into MC3T3-E1 cells expressing the DMP1 gene, and 48 hours later, luciferase activity was measured to examine the transcription activity. In this experiment, ST-2 cells that hardly expressed the DMP1 gene were used as a negative control.
[0034] その結果、図 2に示すように、転写開始点から 1. 5kbおよび 1. 7kb上流までの配 列を含む DNA構築物（それぞれ 1.5kb/Fおよび 1.7kb/F)について顕著に高い転写 活性が見られた。さらに、転写開始点から 1. Okb未満までの配列を含む DNA構築 物においては、転写活性が著しく低下した（図 2中の「Promoter less」）。また、 ST— 2 細胞では、いずれの DNA構築物についても転写活性が見られなかった。これらの結 果から、マウス MC3T3— E1細胞において転写活性を示すのに必要なマウス DMP1 遺伝子プロモーター領域は、転写開始点の 5'側の 1. 5kb以内に存在することが明 らかとなつた。 [0034] As a result, as shown in Fig. 2, remarkably high transcription was observed for DNA constructs containing 1.5 kb and 1.7 kb upstream from the transcription start site (1.5 kb / F and 1.7 kb / F, respectively). Activity was seen. Furthermore, in the DNA construct containing a sequence from the transcription start site to less than 1. Okb, the transcription activity was significantly reduced (“Promoter less” in FIG. 2). In ST-2 cells, no transcription activity was observed for any of the DNA constructs. These results revealed that the mouse DMP1 gene promoter region required to exhibit transcriptional activity in mouse MC3T3-E1 cells was located within 1.5 kb 5 ′ to the transcription start site.
[0035] 1.3 DNA構築物の作製およびその効果の確認
マウス DMP1遺伝子プロモーターの制御下においてジフテリア毒素のヒト型受容体 を発現する DNA構築物を作製し、これにより付与されるジフテリア毒素感受性を確 した 0 1.3 Preparation of DNA Construct and Confirmation of Its Effect To prepare a DNA construct expressing human receptor of diphtheria toxin under the control of the mouse DMP1 gene promoter, was sure diphtheria toxin-sensitive imparted by this 0
[0036] DNA構築物は、次のようにして作製した。まず、標的とする骨細胞に特異的なプロ モーターとして働くことが予想されたマウス DMP1遺伝子の転写開始点の上流に位 置する 1. 5kbpを、 pBluescript II SK(+)プラスミドベクターのマルチクロー-ングサイト 内にある Notl部位に導入し、さらに、 |8—グロビンの第 2ェクソン 第 2イントロン 第 3 ェクソン配列の下流にヒト HB— EGFcDNAおよび j8—グロビン ZSV40ポリ（A)シグ ナル配列を連結した DNA断片を、マルチクロー-ングサイト内にある BamHlZEco RV部位に導入した (得られたプラスミドベクターを 0丁尺1.5¾」という）。また、マウス DMP1遺伝子の転写開始点の 9. 614kbp上流力も翻訳開始点 (翻訳開始コドン中 の A)の 3bp上流までを含んだ DNA断片（マウス DMP1遺伝子の第 1ェクソン、第 1 イントロンおよび第 2ェクソンを含む）を作製し（図 1)、これを pBluescript II SK(+)プラ スミドベクターのマルチクロー-ングサイト内にある NotlZXbal (Nhel)部位に導入 し、さらに、ヒト HB— EGFcDNAの下流に j8—グロビン ZSV40ポリ（A)シグナル配列 を連結した DNA断片を、 SpelZEcoRV部位に導入した (得られたプラスミドベクタ 一を「pDTR9.6kb」という）。 pDTR1.5kbおよび pDTR9.6kbに含まれる HB— EGF発現 カセットの構造を図 3に示す。 [0036] The DNA construct was prepared as follows. First, 1.5 kbp, located upstream of the transcription start site of the mouse DMP1 gene, which is expected to act as a promoter specific to the target osteocytes, is inserted into the pBluescript II SK (+) plasmid vector multiclone. And the human HB-EGF cDNA and j8-globin ZSV40 poly (A) signal sequence were ligated downstream of the second exon, second intron and third exon sequence of | 8-globin. The DNA fragment was introduced into the BamHlZEco RV site in the multi-cloning site (the resulting plasmid vector was referred to as "1.5"). In addition, 9.614 kbp upstream of the transcription start point of the mouse DMP1 gene was determined to be up to 3 bp upstream of the translation start site (A in the translation initiation codon) (the first exon, the first intron and the second intron of the mouse DMP1 gene). Exon) (Fig. 1), which was introduced into the NotlZXbal (Nhel) site in the multicloning site of the pBluescript II SK (+) plasmid vector, and further downstream of human HB-EGF cDNA. A DNA fragment ligated with the j8-globin ZSV40 poly (A) signal sequence was introduced into the SpelZEcoRV site (the resulting plasmid vector was called "pDTR9.6kb"). Fig. 3 shows the structure of the HB-EGF expression cassette contained in pDTR1.5kb and pDTR9.6kb.
[0037] 作製したプラスミドベクター（pDTRl .5kbまたは pDTR9.6kb)とネオマイシン耐性遺伝 子を含む pDsRed2- Nuc (CLONTECH社）とを 5： 1の割合で、培養液（DMEM, 10% FBS) 4mlを含む 6cmディッシュにおいて、 60%コンフルェントのマウス DMP1遺伝 子を発現する MC3T3— E1細胞およびこれを発現しない ST— 2細胞へリポフエクショ ン法により遺伝子導入した。得られた細胞を、 COインキュベータ一中、 37°Cで 48時 The prepared plasmid vector (pDTR1.5 kb or pDTR9.6 kb) and pDsRed2-Nuc (CLONTECH) containing the neomycin resistance gene were mixed at a ratio of 5: 1 with 4 ml of a culture solution (DMEM, 10% FBS). Genes were transfected into MC3T3-E1 cells expressing the 60% confluent mouse DMP1 gene and ST-2 cells not expressing this in a 6 cm dish containing the same by the lipofection method. The obtained cells are placed in a CO incubator at 37 ° C for 48 hours.
2 2
間培養し、培養液中に G418を添加し、 G418に耐性を示す細胞のみを選択した。 得られた細胞を、 10mlの培養液（DMEM, 10%FBS)を含む 10cmディッシュに播 き直した後、 70%コンフルェントになった時点で全 RNAを抽出してノーザンブロット 分析を行なった。その結果、得られた MC3T3— E1細胞において、遺伝子導入され た HB— EGF遺伝子の mRNAの発現が確認された。
[0038] 次いで、得られた細胞におけるジフテリア毒素への感受性を調べるため、毒素によ るタンパク質合成阻害効率を測定した。具体的には、上述のようにネオマイシンにより 選択された細胞を、 1ゥエルあたり培養液 (DMEM, 10% 3) 500 1ぉょび1 10 5個の細胞が含まれるように 24ゥエルプレートに播種し、その 12時間後に、各ゥエル にジフテリア毒素（Sigma社)を用量依存的に（0、 0. 1、 1. 0、または gZml)添 加し、 6時間インキュベートした。 6時間後、得られた細胞を、メチォニンおよびシステ インをともに含まない DMEM (Life Technologies社）に 5 μ CiZmlの [3 ]メチォニン Zシスティンを加えた培養液中で、 COインキュベータ一中、 37°Cで 1時間培養した During the incubation, G418 was added to the culture solution, and only cells showing resistance to G418 were selected. The obtained cells were replated on a 10-cm dish containing 10 ml of a culture solution (DMEM, 10% FBS). When the cells became 70% confluent, total RNA was extracted and Northern blot analysis was performed. As a result, in the obtained MC3T3-E1 cells, mRNA expression of the transfected HB-EGF gene was confirmed. Next, in order to examine the sensitivity of the obtained cells to diphtheria toxin, the efficiency of protein synthesis inhibition by the toxin was measured. Specifically, cells selected by neomycin as described above were placed in a 24-well plate so that 500 1 and 1 105 cells were contained per 1 ml of culture medium (DMEM, 10% 3). After seeding, 12 hours later, each well was added with diphtheria toxin (Sigma) in a dose-dependent manner (0, 0.1, 1.0, or gZml) and incubated for 6 hours. After 6 hours, the obtained cells are placed in a culture solution containing 5 μCiZml of [ 3 ] methionine Z cysteine in DMEM (Life Technologies) containing neither methionine nor cysteine and incubated at 37 ° C. in a CO incubator. C for 1 hour
2 2
。次に、細胞を PBSで 2回洗浄した後、 0. 1N水酸ィ匕ナトリウム水溶液に溶解させ、 細胞溶解液中のタンパク質を 10%トリクロ口酢酸 (TCA)を用いて沈澱させ、ガラスフ ィルター（Whatman社）に吸着させた。このガラスフィルターを PBSで 3回洗浄した後、 これを乾燥させ、シンチレーシヨン溶液をカ卩え、液体シンチレーシヨンカウンタ一にて タンパク質合成に用いられた [35S]メチォニン Zシスティンの放射活性を測定し、タン パク質合成阻害率を評価した。 . Next, the cells are washed twice with PBS, then dissolved in a 0.1N aqueous sodium hydroxide solution, and the protein in the cell lysate is precipitated using 10% trichloroacetic acid (TCA). Whatman). After washing the glass filter three times with PBS, it was dried, and the scintillation solution was dried and the radioactivity of [ 35 S] methionine Z cysteine used for protein synthesis was measured using a liquid scintillation counter. Then, the inhibition rate of protein synthesis was evaluated.
[0039] 図 4および図 5に示すように、 MC3T3— E1細胞では、 pDTR1.5kbを導入した細胞 および pDTR9.6kbを導入した細胞の双方にぉ 、て、遺伝子導入して!/ヽな 、細胞に比 ベて最大で 70%のタンパク質合成阻害が見られた。一方で、図 6に示すように、 ST- 2細胞では、顕著なタンパク質合成阻害は見られな力つた。以上の結果から、構築さ れた二種類の DNA構築物は、 DMP1遺伝子を発現する細胞においてのみジフテリ ァ毒素受容体を発現し、このジフテリア毒素受容体が発現した細胞においては、ジフ テリア毒素の作用によりタンパク質合成が阻害されることが明ら力となった。 As shown in FIG. 4 and FIG. 5, in MC3T3-E1 cells, the gene was transfected into both the cells transfected with pDTR1.5kb and the cells transfected with pDTR9.6kb, and Up to 70% inhibition of protein synthesis was seen compared to cells. On the other hand, as shown in FIG. 6, in ST-2 cells, no significant inhibition of protein synthesis was observed. Based on the above results, the two types of constructed DNA constructs express the diphtheria toxin receptor only in the cells expressing the DMP1 gene, and the effect of diphtheria toxin on the cells expressing the diphtheria toxin receptor. It became clear that the inhibition of protein synthesis by.
[0040] 例 2：トランスジエニックマウスの作製 Example 2: Preparation of transgenic mouse
2.1 DNA構築物を含むマウスの作出 2.1 Generation of mice containing DNA constructs
プラスミドベクター pDTR1.5kbおよび pDTR9.6kbを制限酵素 SacIlZClalで消化し、 ァガロースゲル抽出を行なうことにより、 DNA構築物 DTR1.5kbおよび DTR9.6kb (図 3)を単離した。その後、これら DNA構築物のいずれかを、マウス受精卵へ注入した The plasmid vectors pDTR1.5kb and pDTR9.6kb were digested with the restriction enzyme SacIlZClal, and agarose gel extraction was performed to isolate DNA constructs DTR1.5kb and DTR9.6kb (FIG. 3). Subsequently, one of these DNA constructs was injected into fertilized mouse eggs
[0041] 各マウスにおける DNA構築物の存否は、離乳後、尻尾力 抽出した DNAを铸型
とする PCR法によって確認した。 DTR1.5kbの導入確認用プライマーとしては、 β - globinE3- F: 5し ctgggcaacgtgctgg- 3' (配列番号 8)および HB- EGF- 1R: [0041] The presence or absence of the DNA construct in each mouse is determined by determining the tail force of the extracted DNA after weaning. It was confirmed by the PCR method. As primers for confirming the introduction of DTR1.5kb, β-globinE3-F: 5 and ctgggcaacgtgctgg-3 ′ (SEQ ID NO: 8) and HB-EGF-1R:
5 ' -cgccagtcaccagtgccgag-3 ' (配列番号 9)からなるプライマーペアを用い、 DTR9.6kb の導入確認用プライマーとしては、 DMP1E2- F: 5，- gtatcatgaagtgaaacatc- 3' (配列番 号 10)および HB-EGF-2R: 5'- gagggtccgggttgctggttcc- 3' (配列番号 11)からなるプラ イマ一ペアを用いた。 Using a primer pair consisting of 5'-cgccagtcaccagtgccgag-3 '(SEQ ID NO: 9), DMP1E2-F: 5, -gtatcatgaagtgaaacatc-3' (SEQ ID NO: 10) and HB- A primer pair consisting of EGF-2R: 5'-gagggtccgggttgctggttcc-3 '(SEQ ID NO: 11) was used.
[0042] DTR1.5kbの導入については、 1回目の実験では、受精卵 215個より、 6匹のマウス [0042] Regarding the introduction of DTR1.5kb, in the first experiment, 6 mice were used from 215 fertilized eggs.
(雄 4匹および雌 2匹）の離乳に成功した。このうち、 3匹のマウス (雄 2匹および雌 1匹 )において DTR1.5kbの存在が確認された。 2回目の実験では、受精卵 105個より、 1 1匹のマウス（雄 8匹および雌 3匹）の離乳に成功し、このうち、 2匹のマウス（雄 2匹） にお 、て DTR1.5kbの存在が確認された。 (4 males and 2 females) were successfully weaned. Of these, the presence of 1.5 kb of DTR was confirmed in three mice (two males and one female). In the second experiment, we succeeded in weaning 1 mouse (8 males and 3 females) from 105 fertilized eggs, of which 2 mice (2 males) had DTR1. The presence of 5 kb was confirmed.
[0043] DTR9.6kbの導入については、 1回目の実験では、受精卵 216個より、 9匹のマウス [0043] Regarding the introduction of DTR 9.6kb, in the first experiment, 9 mice were used from 216 fertilized eggs.
(雄 3匹および雌 6匹）の離乳に成功し、このうち、 2匹のマウス (雄 1匹および雌 1匹） において DTR9.6kbの存在が確認された。 2回目の実験では、受精卵 133個より、 6匹 のマウス（雄 3匹および雌 3匹）の離乳に成功し、このうち、 1匹のマウス（雌 1匹）にお いて DTR9.6kbの存在が確認された。 3回目の実験では、受精卵 236個より、 9匹のマ ウスの離乳に成功し、このうち、 1匹のマウス（雄 1匹）において DTR9.6kbの存在が確 f*i¾ れ 。 (3 males and 6 females) were successfully weaned, of which the presence of DTR 9.6 kb was confirmed in 2 mice (1 male and 1 female). In the second experiment, six mice (three males and three females) were successfully weaned from 133 fertilized eggs, of which one mouse (one female) had a DTR of 9.6 kb. Presence confirmed. In the third experiment, nine mice were successfully weaned from 236 fertilized eggs, and the presence of DTR9.6kb was confirmed in one mouse (one male).
[0044] すなわち、 DTR1.5kbの導入については 5匹（雄 4匹および雌 1匹）のトランスジェ- ックマウスを作出し、 DTR9.6kbの導入につ!、ては 4匹（雄 2匹および雌 2匹）のトランス ジェニックマウスを作出した。このうち、系統として安定したものは、 DTR1.5kbについ ては雄 3匹であり、 DTR9.6kbについては雄 2匹および雌 2匹であった。 DTR1.5kbを導 入したマウスを「DTR1.5Tg」と命名し、 DTR9.6kbを導入したマウスを「DTR9.6Tg」と命 名した。 [0044] That is, for transfection of DTR1.5kb, 5 transgenic mice (4 males and 1 female) were produced, and for transfection of DTR9.6kb, 4 transgenic mice (2 males and Two female transgenic mice were created. Among these, three males were stable for DTR 1.5 kb, and two male and two female for DTR 9.6 kb. The mouse into which DTR1.5kb was introduced was named “DTR1.5Tg”, and the mouse into which DTR9.6kb was introduced was named “DTR9.6Tg”.
[0045] 2.2トランスジエニックマウスにおける HB— EGFの発現 [0045] 2.2 Expression of HB-EGF in transgenic mice
3系統の DTR1.5Tgマウスのうちの 1系統（DTR1.5#1)および 4系統の DTR9.6Tgマウ スのうちの 1系統（DTR9.6#1)について、 HB— EGFの発現の有無を確認した。具体 的には、 DTR1.5#1マウスおよび DTR9.6#1マウスから各種主要臓器を取り出し、
TRIzol Reagent (Invitorogen社：「TRIzol」は Invitorogen社の登録商標である）を用い て全 RNAを抽出し、 DNasel処理により混入ゲノム DNAを消化した。次に、これらの 全 RNAを铸型とする RT— PCRにより、 HB— EGFの mRNA発現の有無を調べた。 R T PCRにおいて、 DTR1.5#1マウスについては、 HB- EGF- 2F: The presence or absence of HB-EGF expression was determined for one of three DTR1.5Tg mice (DTR1.5 # 1) and one of four DTR9.6Tg mice (DTR9.6 # 1). confirmed. Specifically, various major organs were extracted from DTR1.5 # 1 and DTR9.6 # 1 mice, Total RNA was extracted using TRIzol Reagent (Invitorogen: “TRIzol” is a registered trademark of Invitorogen), and the contaminated genomic DNA was digested by DNasel treatment. Next, the presence or absence of HB-EGF mRNA expression was examined by RT-PCR using these total RNAs as type II. In RT PCR, for DTR1.5 # 1 mice, HB-EGF-2F:
5 ' -gtatccacggaccagctgctac-3 ' (配列番号 12)および HB- EGF- 3R: 5'-gtatccacggaccagctgctac-3 '(SEQ ID NO: 12) and HB-EGF-3R:
5 ' -gggtccctcttcttccctagc-3 ' (配列番号 13)からなるプライマーペアを用い、 Using a primer pair consisting of 5'-gggtccctcttcttccctagc-3 '(SEQ ID NO: 13),
DTR9.6#1マウスについては、 DMP1E1-F: 5'- cgagaacttcgctgagg- 3' (配列番号 14)お よび HB- EGF- 2R: 5'- gagggtccgggttgctggttcc- 3' (配列番号 11)力 なるプライマーべ ァを用いた。 For DTR9.6 # 1 mouse, DMP1E1-F: 5'-cgagaacttcgctgagg-3 '(SEQ ID NO: 14) and HB-EGF-2R: 5'-gagggtccgggttgctggttcc-3' (SEQ ID NO: 11) Was used.
[0046] DTR1.5#1マウスでは、脳、心臓、肺、肝臓、脾臓および骨において HB— EGFの m RNAの発現が確認され、一方で、腎臓、骨髄および歯牙においては前記 mRNAの 発現は確認されなかった。これに対し、 DTR9.6#1マウスでは、骨および歯牙にのみ 前記 mRNAの発現が確認され、一方で、脳、心臓、肺、肝臓、脾臓、腎臓、卵巣およ び骨髄においては前記 mRNAの発現は確認されな力つた。この結果から、約 9. 6k bpの DMP1遺伝子プロモーターおよび DMP1遺伝子の第 1イントロン中のサイレン サ一の機能により、骨に対する組織特異性が高まるものと考えられる。 [0046] In DTR1.5 # 1 mice, HB-EGF mRNA expression was confirmed in the brain, heart, lung, liver, spleen, and bone, while expression of the mRNA in kidney, bone marrow, and teeth was confirmed. Not confirmed. In contrast, in the DTR9.6 # 1 mouse, the expression of the mRNA was confirmed only in bone and teeth, while the expression of the mRNA was confirmed in the brain, heart, lung, liver, spleen, kidney, ovary, and bone marrow. Expression was unconfirmed. From this result, it is considered that the function of the DMP1 gene promoter of about 9.6 kbp and the silencer in the first intron of the DMP1 gene enhance the tissue specificity to bone.
[0047] 2.3トランスジエニックマウスへのジフテリア毒素投与による骨細胞の死減 [0047] 2.3 Administration of diphtheria toxin to transgenic mice reduces bone cell death
DTR1.5#1マウスおよび DTR9.6#1マウスに、ジフテリア毒素（Sigma社）を腹腔内投 与するか、または関節力 大腿骨骨髄腔に投与した。投与量は 50 gZ体重 kgまた は 500 gZ体重 kgとし、投与回数は 1回または 2回とした。最終投与の 2日後に脛 骨および大腿骨を取り出し、脱灰液（10%EDTA, pH7. 4)に 2週間浸して脱灰し、 脱水した後にパラフィン包埋薄切標本を作製した。得られた骨標本を HE染色し、顕 微鏡で観察することにより、骨細胞の死滅の有無を確認した。 DTR1.5 # 1 and DTR9.6 # 1 mice received diphtheria toxin (Sigma) intraperitoneally or were administered to the bone marrow cavity of the joint force femur. The dose was 50 gZ kg or 500 gZ kg, and the number of doses was once or twice. Two days after the final administration, the tibia and femur were taken out, immersed in a demineralized solution (10% EDTA, pH 7.4) for 2 weeks to demineralize, dehydrated, and prepared as paraffin-embedded slices. The resulting bone specimen was stained with HE and observed under a microscope to determine whether or not the bone cells had died.
[0048] その結果として、両方のトランスジエニックマウスからの骨標本において、ジフテリア 毒素投与後に骨細胞の死滅が確認された。骨細胞は、脛骨において特に顕著に死 滅しており、また、骨細胞の死滅は、海綿骨に比べて皮質骨で多く見られた。同濃度 の毒素（50 gZ体重 kg)投与による骨細胞の死滅の割合は、 DTR1.5 マウスに比 ベて DTR9.6Wマウスの方が高力つた。毒素を大腿骨に直接投与した動物では、大
腿骨の皮質骨における骨細胞の死滅が観察された。 [0048] As a result, it was confirmed that bone cells were killed after administration of diphtheria toxin in bone samples from both transgenic mice. Osteocytes are particularly prominently killed in the tibia, and bone cell death is more common in cortical bone than in trabecular bone. The rate of bone cell killing by administration of the same concentration of toxin (50 gZ kg body weight) was higher in DTR9.6W mice than in DTR1.5 mice. In animals that received the toxin directly into the femur, large Death of bone cells in the cortical bone of the femur was observed.
[0049] より詳細には、 DTR1.5#1マウスの腹腔へ gZ体重 kgのジフテリア毒素を一回 投与した場合においては、変形した脛骨骨細胞の像は確認されたが、脛骨骨細胞が 完全に死滅して空になった骨小腔の割合は少な力つた。なお、野生型マウスに同様 に毒素を投与した場合には、このような変形した脛骨骨細胞および空になった骨小 腔はほとんど認められなかった。 DTR1.5#1マウスの腹腔へ 500 /z gZ体重 kgのジフ テリア毒素を一回投与した場合においては、脛骨骨細胞が死滅して空になった骨小 腔の像が広範囲にわたって確認された。 [0049] More specifically, when a single dose of diphtheria toxin with a gZ body weight of kg was administered to the abdominal cavity of DTR1.5 # 1 mouse once, an image of deformed tibial bone cells was confirmed, but the tibial bone cells were completely transformed. The percentage of bone lacuna that died and became empty was less vigorous. When the toxin was similarly administered to wild-type mice, such deformed tibial bone cells and empty bone lacuna were scarcely observed. When a single dose of 500 / z gZ body weight of diphtheria toxin was administered to the peritoneal cavity of DTR1.5 # 1 mice, a widespread image of the empty bone lacuna with dead tibial bone cells was observed. .
[0050] DTR9.6#1マウスの腹腔へ 50 μ gZ体重 kgのジフテリア毒素を一回投与した場合に おいては、脛骨骨細胞が死滅して空になった骨小腔の像が確認された。さらに、同 マウスの腹腔へ 50 μ gZ体重 kgのジフテリア毒素を二回投与した場合においては、 著しく変形した脛骨骨細胞が確認され、また、脛骨骨細胞の完全な死滅によって空 になった骨小腔が広範囲にわたって確認された。 [0050] In the case of a single administration of 50 µgZ body weight of diphtheria toxin to the abdominal cavity of DTR9.6 # 1 mouse, an image of an empty bone cavity in which the tibial osteocytes had died was confirmed. Was. Furthermore, when 50 μgZ body weight kg of diphtheria toxin was administered twice to the abdominal cavity of the same mouse, markedly deformed tibial bone cells were confirmed, and bone bone emptied due to complete death of the tibial bone cells. The cavity was extensively confirmed.
[0051] これらの結果から、 DTR9.6Tgマウスは、 DTR1.5Tgマウスに比べて、骨におけるジフ テリア毒素に対する感受性が高いものと考えられる。 From these results, it is considered that the DTR9.6Tg mouse has higher sensitivity to diphtheria toxin in bone than the DTR1.5Tg mouse.
[0052] 腹腔内にジフテリア毒素を投与した DTR1.5#1マウスおよび DTR9.6#1マウスの大腿 骨では骨細胞の死滅がほとんど確認できな力つたため、大腿骨骨髄腔にジフテリア 毒素を直接投与した。その結果、両トランスジエニックマウスの骨髄腔側の大腿骨に おいて、骨細胞の完全な死滅が広範囲にわたって見られた。ジフテリア毒素の大腿 骨骨髄腔への直接投与は、他の臓器への毒素の影響が少な 、と 、う点にお 、ても、 好まし 、投与方法であると思われる。
[0052] In the femurs of DTR1.5 # 1 and DTR9.6 # 1 mice to which diphtheria toxin was intraperitoneally administered, diphtheria toxin was directly injected into the medullary cavity of the femur because almost no bone cell death was observed. Administration. As a result, complete killing of bone cells was observed over a wide area in the femur on the medullary cavity side of both transgenic mice. Direct administration of diphtheria toxin to the medullary cavity of the femur is a preferable and preferred method of administration, in that the effect of the toxin on other organs is small.
Claims
請求の範囲 The scope of the claims
[I] 宿主に対して本質的に毒性を示さない化合物を投与することにより、該宿主におい て骨細胞を欠失させることを可能とする DNA構築物であって、 [I] A DNA construct capable of deleting a bone cell in a host by administering a compound having essentially no toxicity to the host,
前記化合物に結合する受容体をコードする DNA、および該 DNAに機能しうる形 で連結された DMP1遺伝子プロモーターを含んでなり、前記化合物と前記受容体と の結合により該受容体を発現している細胞の死滅力 Sもたらされる、 DNA構築物。 A DNA encoding a receptor that binds to the compound, and a DMP1 gene promoter operably linked to the DNA, wherein the receptor is expressed by binding the compound to the receptor. Cell killing S DNA DNA constructs.
[2] 前記 DMP1遺伝子プロモーターが、 DMP1遺伝子において転写開始点の 5'側に 隣接する少なくとも 1. 5kbpのプロモーター領域を含んでなるものである、請求項 1に 記載の DNA構築物。 [2] The DNA construct according to [1], wherein the DMP1 gene promoter comprises at least a 1.5 kbp promoter region adjacent to the 5 'side of the transcription start site in the DMP1 gene.
[3] 前記 DMP1遺伝子プロモーターが、 DMP1遺伝子において転写開始点の 5'側に 隣接する少なくとも 9. 6kbpのプロモーター領域を含んでなるものである、請求項 1に 記載の DNA構築物。 [3] The DNA construct according to claim 1, wherein the DMP1 gene promoter comprises at least a 9.6 kbp promoter region adjacent to the 5 'side of the transcription start site in the DMP1 gene.
[4] 前記化合物がジフテリア毒素であり、前記受容体がジフテリア毒素受容体である、 請求項 1に記載の DNA構築物。 [4] The DNA construct according to claim 1, wherein the compound is diphtheria toxin, and the receptor is a diphtheria toxin receptor.
[5] 前記受容体をコードする DNAと前記 DMP1遺伝子プロモーターとの間に、 DMP1 遺伝子の第 1イントロンを含んでなるものである、請求項 1に記載の DNA構築物。 [5] The DNA construct according to claim 1, wherein the DNA construct comprises a first intron of the DMP1 gene between the DNA encoding the receptor and the DMP1 gene promoter.
[6] 請求項 1一 5のいずれか一項に記載の DNA構築物を含んでなる、ベクター。 [6] A vector comprising the DNA construct according to any one of claims 115.
[7] 請求項 1一 5のいずれか一項に記載の DNA構築物または請求項 6に記載のベクタ 一を含んでなる、宿主細胞。 [7] A host cell comprising the DNA construct according to any one of claims 11 to 5 or the vector according to claim 6.
[8] 請求項 1一 5のいずれか一項に記載の DNA構築物または請求項 6に記載のベクタ 一を含んでなる、トランスジエニック非ヒト動物。 [8] A transgenic non-human animal comprising the DNA construct according to any one of claims 11 to 5 or the vector according to claim 6.
[9] 前記 DNA構築物がゲノム中に含まれている、請求項 8に記載のトランスジエニック 非ヒト動物。 [9] The transgenic non-human animal according to claim 8, wherein the DNA construct is contained in a genome.
[10] 前記非ヒト動物が生物実験に用いるための動物である、請求項 8に記載のトランス ジエニック非ヒト動物。 [10] The transgenic non-human animal according to claim 8, wherein the non-human animal is an animal for use in biological experiments.
[II] 請求項 8— 10のいずれか一項に記載のトランスジエニック非ヒト動物を製造する方 法であって、目的とする非ヒト動物に請求項 1一 5のいずれか一項に記載の DNA構 築物または請求項 6に記載のベクターを導入することを含んでなる、方法。
目的とする非ヒト動物への前記 DNA構築物の導入が、該非ヒト動物からの受精卵 に該 DNA構築物を注入し、得られた受精卵を飼育することによって行なわれる、請 求項 11に記載の方法。
[II] A method for producing the transgenic non-human animal according to any one of claims 8 to 10, wherein the intended non-human animal is described in any one of claims 115. A method comprising introducing the DNA construct of claim 1 or the vector of claim 6. The method according to claim 11, wherein the introduction of the DNA construct into a target non-human animal is performed by injecting the DNA construct into a fertilized egg from the non-human animal and breeding the obtained fertilized egg. Method.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08322427A (en) * | 1995-01-19 | 1996-12-10 | Fujita Gakuen | Method wherein marked cell and tissue are made target in transgenic animal |
| WO1998033899A1 (en) * | 1997-01-31 | 1998-08-06 | Dnavec Research Inc. | Knockout animals |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08322427A (en) * | 1995-01-19 | 1996-12-10 | Fujita Gakuen | Method wherein marked cell and tissue are made target in transgenic animal |
| WO1998033899A1 (en) * | 1997-01-31 | 1998-08-06 | Dnavec Research Inc. | Knockout animals |
Non-Patent Citations (5)
| Title |
|---|
| MACDOUGALL M. ET AL: "Identification of a novel isoform of mouse dentin matrix protein 1: spatial expression in mineralized tissues.", J.BONE MINER.RES., vol. 13, no. 3, 1998, pages 422 - 431, XP002992163 * |
| MITAMURA T. ET AL: "Diphteria Toxin Binds to the Epidermal Growth Factor(EGF)-like Domain of human Heparin-binding EGF-like Growth Factor/Diphteria Toxin Receptor and Inhibits Specifically Its Mitogenic Activity.", J.BIOL.CHEM., vol. 270, no. 3, 20 January 1995 (1995-01-20), pages 1015 - 1019, XP002912324 * |
| TADA Y. ET AL: "Rat Keikotsu no Shoki Hassei Katei ni Okeru dentin matrix protein 1 Oyobi type I collagen no Ideshi Hatsugen Pattern.", NICHIDAI SHIKAGU., vol. 76, 2002, pages 267 - 276, XP002992164 * |
| TATSUMI S. ET AL: "Kotsusaibo o Yudotekini ablate suru Transgenic Mouse no Kaihatsu.", THE MOLECULAR BIOLOGY SOCIETY OF JAPAN NENKAI PROGRAM., 25 November 2004 (2004-11-25), pages 701, XP002992161 * |
| THOTAKURA S.R. ET AL: "Cloning and characterization of rat dentin matrix protein.", J.BIOL.CHEM., vol. 275, no. 14, 2000, pages 10272 - 10277, XP002992162 * |
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