WO2007046398A1 - Promoteur de la nucleostemine specifique d'une cellule souche - Google Patents

Promoteur de la nucleostemine specifique d'une cellule souche Download PDF

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WO2007046398A1
WO2007046398A1 PCT/JP2006/320706 JP2006320706W WO2007046398A1 WO 2007046398 A1 WO2007046398 A1 WO 2007046398A1 JP 2006320706 W JP2006320706 W JP 2006320706W WO 2007046398 A1 WO2007046398 A1 WO 2007046398A1
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gene
dna
stem cell
seq
egfp
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PCT/JP2006/320706
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Japanese (ja)
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Toshio Suda
Atsushi Hirao
Kazuhiro Sakurada
Masahiko Ishihara
Hiromasa Miyaji
Norihiko Shiraishi
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Kyowa Hakko Kogyo Co., Ltd.
Keio University
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Priority to JP2007540995A priority Critical patent/JPWO2007046398A1/ja
Publication of WO2007046398A1 publication Critical patent/WO2007046398A1/fr

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    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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; AVICULTURE; APICULTURE; PISCICULTURE; 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)
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates to DNA having stem cell-specific promoter activity, a vector containing the DNA, and a transgenic non-human mammal and transformed cell containing the vector.
  • organ transplantation is performed as a radical treatment for tissue degeneration such as hematopoietic failure, autoimmune disease, heart failure, liver failure, renal failure, skin injury, neurodegenerative disease and the like.
  • tissue degeneration such as hematopoietic failure, autoimmune disease, heart failure, liver failure, renal failure, skin injury, neurodegenerative disease and the like.
  • donor-provided organ donation only satisfies about 5% of transplant needs (non- Patent Document 1).
  • organ transplantation has problems such as the risk of suffering from donor-derived infections because it is necessary to continue to take immunosuppressants for life.
  • Stem cells are cells that have the ability to replicate daughter cells that are identical in nature and inheritance (self-renewal ability) and that can supply multiple differentiated tissue constituent cells (multipotency). is there. Stem cells are roughly classified into embryonic stem cells (ES cells) and adult stem cells.
  • ES cells embryonic stem cells
  • ES cells theoretically proliferate indefinitely and have the ability to be distributed to all the cells that make up adult tissue, by inducing differentiation after increasing the number of ES cells in large quantities It is expected to be able to supply the tissues or cells necessary for organ transplantation.
  • ES cells are established from human fertilized eggs more than cells that are in the process of ontogeny, there is an ethical problem and there is a risk of forming a tumor when ES cells are transplanted.
  • stem cells that supply functional cells of tissues such as hematopoietic stem cells, neural stem cells, and mesenchymal stem cells.
  • Hematopoietic stem cells have the ability to reconstitute the hematopoietic system, and include autologous or allogeneic bone marrow and umbilical cord blood -Derived hematopoietic stem cells have already been clinically applied (Patent Documents 1 to 7, Non-Patent Document 2)
  • Neural stem cells have the ability to be divided into neurons, glial cells, and the like, and are expected to be used for radical therapy for neurodegenerative diseases such as Parkinson's disease.
  • Mesenchymal stem cells have the ability to differentiate into bone cells, chondrocytes, skeletal muscle cells, adipocytes, fibroblasts, etc., and treat diseases in bone, cartilage, tendon, skeletal muscle, ligament tissue, and adipose tissue. It is expected to be used for wound healing of skin (Patent Document 8).
  • Non-Patent Document 3 Recently, the presence of adult pluripotent stem cells having the ability to dissociate into almost all cells in the adult thread and weave has been shown (Patent Documents 9 and 10, Non-Patent Document 3).
  • Adult pluripotent stem cells have not been reported to form tumors, unlike ES cells.
  • adult pluripotent stem cells can acquire the bone marrow, skeletal muscle, skin, brain, and other forces of postnatal mammals including humans, there are no ethical problems, and if the stem cells acquired with self-organization power are used for transplantation treatment There is no worry about immunity rejection.
  • adult pluripotent stem cells are currently attracting attention as a medical tool for tissue degenerative diseases that can solve ethical problems in addition to their pluripotency and safety.
  • Known methods for isolating various tissue stem cells include methods that utilize surface antigens expressed in stem cells and their characteristics, and methods that use a reporter vector having a promoter of a tissue stem cell-specific gene, and an enzyme. It has been.
  • CD34 ⁇ / c-kit + / Scal + / Lineage (Lin) ⁇ (non-patent document 4) in mouse bone marrow, and CD34 + / CD38 ⁇ / Lin ⁇ (non-patent document 5, 6) or CD34 + / KDR + (Non-patent document 7), human bone marrow, CD34- / Lin- (non-patent document 8), human cord blood !
  • CD34- / Lin- / CD133 + / CD7 -It is known that a cell fraction enriched with hematopoietic stem cells can be separated using (Non-patent Document 9) as a marker.
  • Non-patent Document 10 a method for separating hematopoietic stem cells using the jS-catenin gene as a marker is known (Patent Documents 11 and 12).
  • Non-Patent Documents 11, 12, and 13 As separation methods, Sox (Patent Document 13;), Nestin or Musashi (Patent Document 14), and AC133 (Patent Document 15) are used as markers. The method is known.
  • separation methods include SH2 +, SH4 +, CD29 +, CD44 +, CD71 +, CD90 +, CD106 +, CD120a +, CD124 +, CD14-, A method using CD34-, CD45- as a marker is known.
  • separation methods include C-Met + / c-kit- / CD49f + or low / CD29 + / CD45- / Terll9- A method of using as a marker is known.
  • Oct-3 / 4 is known as a gene that is specifically expressed in embryonic germ cells (EG cells) in an undifferentiated state.
  • Oct-3 / 4 is expressed in the fetus before gastrulation, in the early stage of division, in the inner cell mass of blastocysts and in embryonic carcinoma, and decreases when differentiated. It is clear that Oct-3 / 4 is necessary for maintenance of undivided cells such as ES cells.
  • Oct-3 / 4 binds to Rox-1 and activates transcription of Rex-1, a Zn finger protein necessary for maintaining undifferentiated ES (Non-patent Document 24).
  • a technique for identifying adult pluripotent stem cells in adult tissues using Oct-3 / 4 gene expression as an indicator has been reported (Patent Document 9).
  • Non-Patent Documents 25 and 26 It is a cell that has a cell surface marker of CD45- / Sca-l + / c-kit-/ CD34 + / Thy-1 + in skeletal muscle tissue. There are also reports (Non-Patent Documents 25 and 26).
  • Patent Document 23 Also known is a method for isolating pluripotent stem cells using j8 2-microglobulin as an index (Patent Document 23), but identifying negative pluripotent stem cells from ⁇ 2-microglobulin-positive cell populations in tissues It ’s not easy.
  • Patent Document 1 US5,635,387
  • Patent Document 2 US5,460,964
  • Patent Document 3 US5,677,136
  • Patent Document 4 US5, 750,397
  • Patent Document 5 US5, 759,793
  • Patent Document 6 US5,681,599
  • Patent Document 7 US5,716,827
  • Patent Document 8 US5,486,359
  • Patent Document 9 WO2001 / 11011
  • Patent Document 10 WO2001 / 21767
  • Patent Document 11 US 6,465,249
  • Patent Document 12 WO03 / 102215
  • Patent Document 13 US2002 / 0135539
  • Patent Document 14 JP 2002-034580
  • Patent Document 15 US6,468,794
  • Patent Document 16 US5,827,735
  • Patent Document 17 US 5,811,094
  • Patent Document 18 US5, 736,396
  • Patent Document 19 US 5,837,539
  • Patent Document 20 US5,837,670
  • Patent Document 21 US5,827,740
  • Patent Document 22 WO01 / 48149
  • Patent Document 23 US2002 / 0119564
  • Patent Document 24 WO2004 / 031731
  • Non-patent document 1 J Am Med Assoc, 267, 239-246 (1992)
  • Non-patent document 2 Exp Hematol, 24, 936-943 (1996)
  • Non-patent document 3 Nature, 418, 41-49 (2002)
  • Non-Patent Document 4 Science, 273, 242-245 (1996)
  • Non-Patent Document 5 Blood, 89, 3919-24 (1997)
  • Non Patent Literature 6 Proc Natl Acad Sci USA, 94, 5320-5 (1997)
  • Non Patent Literature 7 Science, 285, 1553-8 (1999)
  • Non-Patent Document 8 Nature Medicine, 4, 1038-45 (1998)
  • Non-Patent Document 9 Blood, 95, 2813-20 (2000)
  • Non-Patent Document 10 J Exp Medicine, 183, 1797-806 (1996)
  • Non-Patent Document 11 Science, 287, 1433-1438 (2000)
  • Non-Patent Document 12 Brain Pathol, 9, 499-513 (1999)
  • Non-Patent Document 13 Mech Dev, 59, 89-102 (1996)
  • Non-Patent Document 14 J Cell Biochem, 64, 295-312 (1997)
  • Non-Patent Document 15 J Bone Miner Res, 11,1264-73 (1996)
  • Non-Patent Document 16 J Bone Joint Surg Am, 80, 1745-57 (1998)
  • Non-Patent Document 17 Exp Cell Res, 238, 265-72 (1998)
  • Non-Patent Document 18 Blood, 84, 4164-73 (1994)
  • Non-Patent Document 19 J Clin Invest, 103, 697-705 (1999)
  • Non-patent literature 20 Philos Trans R Soc Lond B Biol Sci, 353, 821-30 (1998)
  • Non-patent literature 21 Philos Trans R Soc Lond B Biol Sci, 353, 831-7 (1998)
  • Non-Patent Document 22 J Hepatol, 29, 676-82 (1998)
  • Non-Patent Document 23 Molecular Medicine, 40, 169-175 (2003)
  • Non-Patent Document 24 Molecular & Cellular Biology, 18, 1866-78 (1998)
  • Non-Patent Document 25 Blood, 98, 2615-1625 (2001)
  • Non-Patent Document 26 Nature, 418, 41-49 (2002)
  • Non-Patent Document 27 Genes & Development, 16, 2991-3003 (2002)
  • nucleostemin is a marker suitable for the isolation and identification of stem cells and pluripotent stem cells present in adult tissues, as well as necessary for maintaining stem cell sternness due to its expression pattern. Is expected to be.
  • nucleostemin is localized in the nucleolus, so it detects nucleostemin expressed in cells. To do so, cell fixation is required, and cells expressing nucleostamine cannot be isolated in a living state. In addition, there has been no report so far on the expression control region such as the promoter of the nucleostemin gene.
  • the present invention provides a DNA having a stem cell-specific promoter activity, a vector containing the DNA, or a transgenic non-human mammal or a transformed cell containing the vector. Objective.
  • the present invention relates to the following (1) to (15).
  • a DNA having a nucleotide sequence ability represented by SEQ ID NO: 1 or SEQ ID NO: 3 and having a stem cell-specific promoter activity is represented by SEQ ID NO: 1 or SEQ ID NO: 3 and having a stem cell-specific promoter activity.
  • a DNA comprising a nucleotide sequence having 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 and having a stem cell-specific promoter activity.
  • Promoter activity specific to a stem cell which is hybridized under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence represented by SEQ ID NO: 1, 2 or 3. Having DNA.
  • a vector for expressing a foreign gene in a stem cell-specific manner wherein the DNA of any one of (1) to (6) above is linked upstream of the foreign gene sequence.
  • Foreign gene is / 3 Galactosidase gene, Alkaline phosphatase gene, Chloramphe-coal acetyltransferase gene, Growth hormone gene, Luciferase gene, Green fluorescent protein gene, Blue fluorescent protein gene, Yellow fluorescent protein gene, Red fluorescent protein
  • the vector according to (7) which is a gene selected from the group consisting of a gene, a j8-lactamase gene, and derivatives thereof.
  • the present invention provides DNA having stem cell-specific promoter activity, a vector containing the DNA, or a transgene non-human mammal or transformed cell containing the vector.
  • FIG. 1 is a view showing the expression level of nucleostemin (NS) gene in human tissues.
  • the vertical axis represents the expression level of NS gene as a relative amount (%) with respect to the GAPDH gene.
  • the horizontal axis represents the organization: 1. liver, 2. lung, 3. brain, 4. heart, 5. stomach, 6. spleen, 7. spinal cord, 8. small intestine, 9. skeletal muscle, 10. uterus, 11 .Represents trachea, 12. Thyroid, 13. Thymus, 14. Testis, 15. Salivary gland, 16. Prostate, 17. Placenta, 18. Lymph node, 19. Spleen, 20. Large intestine, 21. Peripheral blood, 22. Represents kidney .
  • FIG. 2 shows the expression level of human NS gene in adult and fetal tissues.
  • the vertical axis represents the expression level of NS gene as a relative amount (%) with respect to the GAPDH gene.
  • the horizontal axis represents tissue, the rocolumn represents fetal tissue, the country column represents adult tissue, 1. Brain, 2. Kidney, 3. Liver, 4. Lung.
  • FIG. 3 shows a restriction map of pPLEmNSgKSB10.
  • FIG. 4 ES cell line stably transfected with pPLEmNSgKSB10 using F reporter activity as an indicator.
  • the cells were separated into EGFP high-expressing cell population (EGFP ++) and EGFP-low-expressing cell population (EGFP +), and the expression levels of endogenous NS gene (Rocolum) and EGFP gene (country column) in each were measured by RT-PCR. It is the figure which showed the result compared.
  • the vertical axis represents the relative expression level of the gene.
  • FIG. 5 shows a restriction map of pNS Promoter7-EGFP.
  • FIG. 6 A shows the results of FACS analysis of EGFP expression in bone marrow mononuclear cells of transgenic genotypes (Tg) mice introduced with a sequence combining approximately 7 kb upstream of the NS gene and EGFP. It is.
  • FIG. B shows the results of FACS analysis of the expression of EGFP, lineage marker, and stem cell marker in bone marrow mononuclear cells of the Tg mouse.
  • FIG. 7 A shows the result of comparison of EGFP expression intensity and colony-forming ability of bone marrow mononuclear cells of transgeneic (Tg) mice introduced with an approximately 7 kb genomic sequence upstream of the NS gene and an EGFP-binding sequence.
  • FIG. The vertical axis represents the number of colonies per 300 cells.
  • FIG. B is a graph showing the results of comparing the EGFP expression intensity and bone marrow remodeling ability of bone marrow mononuclear cells of the Tg mice.
  • the vertical axis represents the ratio (%) of donor-derived cells per 1000 cells in peripheral blood.
  • FIG. 8 shows the results of FACS analysis of EGFP expression in EpCAM-positive cells in the seminiferous tubules of transgenic mice introduced with an approximately 7 kb genomic sequence upstream of the NS gene and an EGFP-binding sequence.
  • the vertical axis represents the count number.
  • the horizontal axis shows the fluorescence intensity of EGFP.
  • a stem cell refers to a cell that has self-replication ability, possibly having the ability to produce stem cells.
  • any stem cell can be used as long as it has the above-mentioned properties.
  • Preferred examples include embryonic stem cells (ES cells), adult stem cells, and more preferred adult stem cells.
  • Examples of adult stem cells include various tissue stem cells and adult pluripotent stem cells.
  • tissue stem cells examples include hematopoietic stem cells, neural stem cells, mesenchymal stem cells, Examples thereof include a vaginal tissue stem cell and a reproductive tissue stem cell.
  • a promoter includes an expression regulatory region capable of associating with RNA polymerase in a cell, and a region having a function of initiating transcription of a coding sequence existing downstream (toward the 3 ′ end) of the region. Refers to the upper area.
  • promoters having the above properties are included! /
  • And misregulated promoters are included, but the efficiency of transcription is controlled! /
  • And promoters including the so-called enhancer region are preferable! /.
  • Promoter activity refers to the activity of transcribing a coding sequence existing downstream of the promoter.
  • the stem cell-specific promoter activity means that the promoter activity in stem cells is high compared to other cells and tissues.
  • Examples of the DNA of the present invention include a DNA having a base sequence ability represented by SEQ ID NO: 1 or 3, and a DNA having a stem cell-specific promoter activity.
  • Examples of the DNA containing the DNA having the base sequence ability represented by SEQ ID NO: 1 include the DNA comprising the base sequence represented by SEQ ID NO: 2, and the like.
  • the base sequence represented by SEQ ID NO: 1 is from about 7 kb from the 6th g downstream of the translation start point (Is t atg) of the mouse nucleostemin gene to the 7078th g upstream of the translation start point. Is an array of regions.
  • the nucleotide sequence represented by SEQ ID NO: 2 is a region having a force of about 10 kb from the 6th g downstream of the mouse nucleostemin gene translation start point (Istatg) to the 10058th g upstream of the translation start point. Is an array of
  • the base sequence represented by SEQ ID NO: 3 has a force of about 10 kb from the third a downstream of the translation start point (1st a tg) of the human nucleostemin gene to the 9998th a upstream of the translation start point.
  • the sequence of the mouse nucleostemin gene can be referred to by Genbank Accession Number N W — 000091, and the sequence of the human nucleostemin gene can be referred to by Genbank Accession Number NT — 00595986.
  • the method for measuring the promoter activity of the DNA of the present invention may be any measurement method as long as the promoter activity of the DNA can be measured.
  • the reporter gene assay system may be used. Can be measured. Specifically, it can be performed by the following steps (1) to (3).
  • luciferase gene Downstream of the promoter to be tested, for example, luciferase gene, anolecalciphosphatase gene, chloramphee-chol resistance gene, ⁇ -galactosidase gene, green fluorescent protein gene, blue fluorescent protein gene, yellow fluorescent protein gene, red fluorescent protein gene
  • a step of preparing a plasmid for activity measurement comprising a sequence operably linked to a reporter gene such as a j8-lactamase gene or a derivative thereof;
  • the promoter to be tested can be identified as a stem cell-specific promoter, and the expression intensity of the reporter gene Becomes the strength of promoter activity.
  • the DNA of the present invention is obtained from a nucleotide sequence in which one or more of the nucleotide groups in the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 has been deleted, substituted, or added (hereinafter sometimes simply referred to as “mutation”). And having a stem cell-specific promoter activity.
  • the number of mutations and the position of introduction are determined based on the stem cell-specific promoter activity of the DNA after mutation. If you talk! Examples of the method for measuring the stem cell-specific promoter activity include the above-mentioned reporter gene assembly system.
  • One or more bases are deleted, substituted or added in the DNA of the present invention when one or more bases are deleted, substituted or added at any position in one or more base sequences in the same sequence. Deletions, substitutions or additions may occur simultaneously.
  • the number of bases to be deleted, substituted or added is not particularly limited, but is a number that can be deleted, substituted or added by a known method such as the above site-specific mutation method, : LOOO, preferably 1 to 500, more preferably 1 to 100, particularly preferably 1 to 50.
  • SEQ ID NO: 1 It is preferred to have at least 60% or more, usually 80% or more, particularly 95% or more homology with the base sequence represented by 2 or 3.
  • the homology of the base sequence is determined using the algorithm BLAST [Pro. Natl. Ac ad. Sci. USA, 90, 5873 (1993)] by FA and [Methods EnzymoL, 183, 63 (1990)] Can be determined.
  • BLAST Pro. Natl. Ac ad. Sci. USA, 90, 5873 (1993)
  • BLASTN a program called BLASTN
  • the DNA of the present invention is stringent under conditions that are stringent under conditions that are complementary to DNA having a nucleotide sequence complementary to the DNA represented by SEQ ID NO: 1, 2, or 3 and specific to a stem cell. DNA having a typical promoter activity is included.
  • DNA that can be hybridized under stringent conditions refers to DNA having the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 as a probe, and is used as a probe. It means DNA obtained by using the session method or the Southern blot hybridization method.
  • a filter immobilizing DNA derived from rainbow trout 0.7-1.
  • Omol / 1 sodium chloride in the presence of 65 ° C noisy hybridization 0.1-2 fold concentration
  • SSC solution concentration of 1x SSC solution is 150 mmol / l sodium chloride, 15 mmol / l sodium quenate
  • the DNA capable of hybridizing is a DNA having at least 60% homology with the base sequence represented by SEQ ID NO: 1, 2, or 3 when calculated using the above-mentioned BLAST or the like, preferably Examples thereof include DNA having 80% or more homology, more preferably DNA having 95% or more homology.
  • the DNA of the present invention is linked upstream of the foreign gene sequence, and the foreign gene can be expressed specifically in stem cells.
  • Examples of the foreign gene include j8-galactosidase gene, alkaline phosphatase gene, chloramfue-coal acetyltransferase gene, growth hormone gene, luciferase gene, green fluorescent protein gene, blue fluorescent protein gene, yellow fluorescent protein gene, Marker genes such as red fluorescent protein gene, ⁇ -lactamase gene and their derivatives are included.
  • the derivatives include artificially produced mutants.
  • the 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, and the like. Specifically, pNASS ⁇ , pGL3, pCAT3, p ⁇ gal, pEGFP-1, pEGF P-Nl, pEYFP-1 PSEAP2, pGeneBLAzer-TOPO and the like.
  • the transgenic non-human mammal of the present invention is a transgenic non-human mammal containing the vector of the present invention, and the foreign gene in the expression vector is expressed specifically in stem cells.
  • stem cells can be easily separated using the presence or strength of the expression of the gene specific to the stem cell as an index. Can be identified.
  • the vector may be integrated into the chromosome of the mammal.
  • non-human mammals include mammals other than humans, for example, mammals such as mice, rats, rabbits, pigs, nu, hidge and goats. Of these, mice are particularly preferred because rodents represented by mice, rats and the like are preferred from the viewpoints of being used for pharmaceutical research and easy preparation of pathological models.
  • transgenic non-human animal of the present invention examples include mouse embryo manipulation manual (Modern Publishing, 1989), molecular biology protocol (Nanedo, 1994), gene targeting (Yodosha, 1995). ) Etc.
  • a female in which superovulation is induced and a male and female are mated. After approximately 12 hours of mating, remove the female oviduct, collect fertilized eggs at the 1-cell stage (pronuclear stage), and place them in an appropriate culture medium.
  • the vector of the present invention is microinjected into the pronucleus of a fertilized egg.
  • a male that has undergone vasectomy is mated with a female that has reached sexual maturity to produce a pseudopregnant female. After the mouth injection, surviving fertilized eggs are transplanted into the oviduct of the pseudopregnant female.
  • the fetus generated from the fertilized egg transplanted into the fallopian tube is removed by natural delivery or open surgery.
  • Genomic DNA is prepared from a portion of the pup tail. Analyze the resulting DNA to see if the resulting pup is a transgenic animal. Crossing transgenic animals to establish the system. Expression analysis is performed for foreign genes present in vectors in established transgenic animals to confirm stem cell-specific expression. By the above operation, the transgenic non-human animal of the present invention can be obtained.
  • the transformed cell of the present invention is a transformed cell containing the vector of the present invention, and when the transformed cell is a stem cell, the foreign gene in the vector can be specifically expressed. Monkey.
  • the presence or absence and strength of the marker gene is used as an index for transformation.
  • Stem cells can be easily separated and identified from the transformed cells.
  • the transformed cell of the present invention was prepared by a known transformation method, which may be isolated from a transgenic non-human mammal carrying a vector containing the marker gene.
  • a transformation method which may be isolated from a transgenic non-human mammal carrying a vector containing the marker gene.
  • Known transformation methods include, for example, the calcium phosphate method, the DEAE-dextran method, the elect mouth position method, and the like.
  • nucleostemin gene in human tissues was measured by quantitative RT-PCR.
  • Each yarn and weave cDNA was synthesized using 1 ⁇ g poly A RNA (BD Biosciences Clontec h) derived from human normal yarn and weave using Superscript First-strand System for RT-PCR (Invitrogen). did.
  • cDNA Human nucleocapsid Creos Temin gene-specific primers, to prepare a SyberGreen I (Molec ular Probe Inc.), dNTPs mix and DNA Taq polymerase (TaKaRa Ex Taq ' M R- PCR, the reaction solution containing the Ta KaRa3 ⁇ 4 :), ABI PCR reaction was performed using PRISM 7700 Sequence Detection System (Applied Biosystems). The amount of cDNA used was equivalent to 2 ng of poly A RNA, PCR primers were added to a final concentration of 300 nmol / L, and dNTP mix was added to a final concentration of 300 nmol / L.
  • sequences of primers specific for the human nucleostemin gene are shown in SEQ ID NOs: 4 and 5
  • sequences of primers specific for the human dalyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene are shown in SEQ ID NOs: 6 and 7, respectively.
  • Plasmid DNA obtained by cloning the PCR product amplified with each PCR primer into pT7Blue (Novagen) was prepared in advance as a template, and this was serially diluted to create a calibration curve, and the amount of amplification in each sample was quantified. I'm sorry.
  • the expression level of the nucleostemin gene in each tissue was expressed as a relative amount to the expression level of the GAPDH gene.
  • the results are shown in FIG.
  • the expression level of the nucleostemin gene is higher in testis, spleen, lung, etc. than in other tissues, especially in testis. It was. It has been suggested that the presence of stem cells has been reported in these tissues [Kanatsu-Shinohara, M., et al. Biol Reprod, 69, 612-616 (2003), Zulewski, H "et al. Diabetes, 50, 521-533 (2001), Kotton, DN, et al. Experimental Hematology, 32, 340 40-343 (2004)], compared to other tissues with higher nucleostemin gene expression.
  • nucleostemin gene The expression level of nucleostemin gene in various blood cell populations of mice was examined by RT-PCR.
  • Lineage positive cell population (Lin +), which is considered to be ⁇ , considered to contain only a few hematopoietic stem cells Hoechst33342 low-efficiency subgroup (KSL) in negative cell population (Lin-), c-kit positive Sca-1 positive lineage negative cell population (KSL) MP / KSL) and high-efficiency subgroups (SP / KSL) were sorted.
  • an anti-c-kit antibody (BD PROM, 2B8 ), Anti-Sea-1 antibody (BD PROM, D7), anti-CD4 antibody (BD Regngen, L3T4), anti-CD8 antibody (BD Regngen, 53-6.72), anti-B220 antibody (BD Regngen) , RA 3-6B2), anti-TER-119 antibody (BD PARngen), anti-Gr-1 antibody (BD POngen, RB6-8C5) and anti-Mac-1 antibody (BD PARngen, M1 / 70) was used.
  • CD4 and CD8 are T cell markers
  • B220 is a B cell marker
  • TER-119 is a red blood cell marker
  • Gr-1 is a granulocyte marker
  • Mac-1 is a macrophage marker
  • the cells that were positive were regarded as lineage positive cells, and the cells that were all negative were regarded as lineage negative cells.
  • cDNA was prepared by reacting with reverse transcriptase (Superscriptll, Invitrogen).
  • the PCR product was electrophoresed on a 2% agarose gel, stained with ethidium bromide (EtBr, Nacalai), and then visualized and photographed with a UV sample photographing device (FASm, TOYOBO).
  • EtBr ethidium bromide
  • FOSm UV sample photographing device
  • a clone (RP23-84017, Invitrogen) containing a mouse nucleostemin gene (Genbank Accession ⁇ BC037996) was selected from the BAC clone (BACPAC Resources Center) into which the mouse genome was inserted and subjected to the following Southern analysis.
  • the probe for Southern analysis was obtained by using a mouse genomic DNA purified from the tail of a 6-week-old male C57B1 / 6 mouse (CLEA Japan, Inc.) as a cocoon and represented by SEQ ID NOs: 12 and 13, respectively. Amplified using a Temin gene-specific primer and DIG-labeled with PCR DIG Probe Synthesis Kit (Roche) was used.
  • 1 ⁇ 23-84017 contains a nucleostemin gene sequence of a length that is expected to have a mouse genome sequence (Genbank Accession ** NW_000091) force.
  • a library was prepared by cloning the fragment obtained by enzymatic treatment of RP23-84017 with Kpnl into pBlueScriptllSK (-) (Stratagene), and the above probe and DIG High Prime DNA Labeling and Detection Starter Kit II. (Roche) was used to confirm a Kpnl fragment (12.2 kb) subclone containing the upstream sequence of the nucleostemin gene.
  • the cloned fragment contained a sequence containing 10 kb upstream from the translation start point (1st at g) of the nucleostemin gene.
  • a reporter vector in which the promoter, Henno and sensor regions were excised.
  • the Kpnl fragment (12.2kb) restriction enzyme site Kpnl force is also translated
  • the lOkb sequence in Stu located downstream of the start point (atg) is inserted from the restriction enzyme site Kpnl of pPLE into BamHI (smooth ⁇ process), and 10 kb upstream from the translation start position of the nucleostemin gene.
  • An EGFP reporter vector pPLEmNSgKSBlO (Fig. 3) containing the genomic sequence was constructed.
  • the genomic sequence of 1 Okb upstream from the translation start position of the nucleostemin gene contained in pPLEmNSgKSB10 is shown in SEQ ID NO: 2.
  • pPLEmNSgKSBlO was introduced into an embryonic stem cell (ES cell) [Cell, 94, 33 9-352 (1998)] by means of an electoral position (GenePulser, BioRad). By culturing in the presence of 300 g / mL G418 (Geneticin, Invitrogen), a pPLEmNSgKSBlO stably-introduced ES cell line was established.
  • FACS sorter (BD FACSVantage SE System, BD Biosystems) is used to sort high EGFP expression level, cell population (EGFP ++) and low EGFP expression, and cell population (EGF P +), The expression level of EGFP in each cell population was examined.
  • cDNA was synthesized from the sorted cells as described above, and quantitative RT-PCR was performed using ABI PRISM 7700 Sequence Detection System (Applied Biosystems).
  • the sequence of the mouse nucleostemin gene specific primer is shown in SEQ ID NOs: 8 and 9
  • the sequence of the mouse G APDH gene specific primer is shown in SEQ ID NOs: 10 and 11
  • the sequence of the EGFP gene specific primer is shown in SEQ ID NOs: 14 and 15, respectively. Indicated.
  • PCR product amplified with each PCR primer set was prepared by cloning in pT7Blue (TaKaRa), and a standard curve was prepared by serially diluting them and each sample was prepared. The expression level of the gene in the cell was quantified.
  • nucleostemin gene and EGFP gene in each sample was expressed as a relative amount to the expression level of GAPDH gene.
  • SEQ ID NO: 1 shows the base sequence of the approximately 7 kb upstream nucleostemin gene contained in pPLE-Nl mNSgKSBlO AU7078.
  • a Notl cleavage site was inserted into the 5th primer (SEQ ID NO: 16), and a Not Sail cleavage site was inserted into the 3rd primer (SEQ ID NO: 17). After digesting the PCR product with Notl, it was inserted into the Notl site of NS promoter 7-EGFP / pCMV-SPORT6 to prepare pNS Promoter7-EGFP ( Figure 5).
  • the restriction fragment Sail was used to purify the fragment excised from the nucleostemin gene transcription regulatory sequence-EGFP-SV40 PolyA, and injected into the pronuclei of the C57BL / 6-derived fertilized egg. did. Introduced fertilized eggs were transplanted into the oviduct of the foster parent according to a conventional method, and developed into individuals. The resulting first transgenic mice (F0) were crossed with C57BL / 6 mice to establish the strain.
  • Bone marrow fluid was collected from the femur of the transgenic mouse prepared in Example 4, and mononuclear cells were separated by a specific gravity method (Lymphoprep, specific gravity 1.077, Fresenius Kabi Norge AS). As a result of confirming the expression of EGFP by FACS (FACS Vantage, Becton Dickinson), about 90% of the cells were positive for EGFP (A in FIG. 6).
  • the mononuclear cell population separated by the expression intensity of EGFP was further divided into anti-c-kit antibody (BD Regngen, 2B8), anti-Sea-1 antibody (BD Regngen, D7), anti-CD4 Antibody (BD Regngen, L3T4), anti-CD8 antibody (BD Regngen, 53-6.72), anti-B220 antibody (BD Regngen, RA3-6B2), anti-TER-119 antibody (BD PRONgen) ), Anti-Gr-1 antibody (BD Par mingen, RB6-8C5) and anti-Mac-1 antibody (BD poungen, M1 / 70) were analyzed by FACS.
  • nucleostemin protein is high in the EGFP strongly positive cell population (EGFP +++), but weakly positive in EGFP. Low in the population (EGFP +) was evident, indicating that there was a correlation between EGFP expression and endogenous nucleostemin expression.
  • EGFP strong positive cells (EGFP +++), medium positive cells (EGFP ++), and weak positive cells (EGFP +) isolated by FACS were used as stem cell factor (SCF), IL-6, and IL- 3. Cultivated with erythropoietin in methylcellulose medium for 1 week and examined its ability to form a mouth.
  • EGFP strong positive cells (EGFP +++) had higher ability to form colonies than medium to weak positive cells (EGFP ++ to EGFP +). From this result, it is considered that hematopoietic stem cells are included at high frequency in the EGFP strong positive cell population (EGFP +++)! / [0063] Further, in order to examine the stem cell activity of EGFP strong positive cells (EGFP +++) and weak positive cells (EGFP +), bone marrow reconstitution experiments were performed by bone marrow transplantation.
  • C57BL / 6 mice (Ly5.1) were used as recipient mice, irradiated with lethal doses of X-rays (Neogen 9.5 Gy, 0.5 Gy / min), then EGFP strong positive (EGFP +++) or weak from the tail vein Positive cells (EGFP +) were injected and bred for 4 months. At that time, 4 ⁇ 10 5 Ly5.1 mouse-derived bone marrow mononuclear cells were simultaneously injected.
  • Ten-week-old male transgenic mice also prepared testicular tissue frozen sections and seminiferous whole-mount samples, and analyzed the expression of EGFP with a fluorescence microscope (OLYMPUS FV1000, Olympus).
  • EGFP expression was considered to be an undifferentiated germ stem cell! /, And was strongly decreased in type A spermatogonia and spermatocytes strongly differentiated in type A spermatogonia. On the other hand, sperm also showed strong expression. The expression pattern of EGFP was consistent with that of endogenous nucleostemin.
  • EpCAM positive cells three cell populations with different EGFP expression intensities were observed.
  • EGFP strongly positive cell population (EGFP +++: P3) is not c-kit negative A type A spermatogonia
  • EFGP positive cell population (EGFP ++: P2) is a type A spermatogonia
  • EFGP weakly positive cell population (EGFP +: P1) is c- Use kit-positive B-type spermatogonia.
  • each cell population was transplanted into the seminiferous tubule of a WBB6F1 W / Wv mouse (purchased from SLC) with a germ cell differentiation disorder, and engraftment was examined after 3 months.
  • EGFP ++ + cells were found to have engraftment ability, indicating that the cells had stem cell activity.
  • Small intestine tissue specimens from 10-week-old male transgenic mice were prepared and analyzed for EGFP expression with a fluorescence microscope.
  • EGFP expression was observed in cells located in the lower part of the Talibut, where the stem cells are thought to exist, suggesting that gastrointestinal tissue stem cells were labeled. That is, it was revealed that EG FP is expressed specifically in the digestive tract tissue stem cells in the digestive tract tissue of the present transgenic mouse.
  • EGFP central nervous tissue specimens of 10-week-old male transgenic mice were prepared and analyzed for EGFP expression with a fluorescence microscope.
  • EGFP was particularly strongly expressed in the subventricular zone facing the hippocampal dentate gyrus and lateral ventricle, which is known to have stem cells of the central nervous system.
  • tissue immunostaining with an antibody against Glial fibrillary acidic protein (GFAP) resulted in a match between strongly EGFP positive cells and Glial fibrillary acidic protein (GFAP) positive cells. It was thought that neural stem cells were labeled. That is, it was revealed that EGFP force S neural stem cells are specifically expressed in the central nervous tissue of the present transgenic mouse.
  • GFAP Glial fibrillary acidic protein

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Abstract

La présente invention concerne une technique d'isolement et d'identification d'une cellule souche adulte et un modèle animal permettant d'évaluer aisément la fonction de la cellule souche dans le but d'établir un procédé thérapeutique utilisant la cellule souche adulte. L'invention concerne un ADN présentant une activité de promoteur dans une cellule souche spécifique, un vecteur contenant l'ADN et un mammifère transgénique non humain ou une cellule transformée contenant le vecteur.
PCT/JP2006/320706 2005-10-18 2006-10-18 Promoteur de la nucleostemine specifique d'une cellule souche WO2007046398A1 (fr)

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JP2015527083A (ja) * 2012-09-07 2015-09-17 チルドレンズ メディカル センター コーポレーション 造血幹細胞特異的レポーターマウスおよびその使用

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WO2001011011A2 (fr) * 1999-08-05 2001-02-15 Mcl Llc Cellules souches adultes toutes-puissantes et procede d'isolement
WO2003102215A2 (fr) * 2002-05-31 2003-12-11 The Board Of Trustees Of The Leland Stanford Junior University Procedes d'identification et d'isolement de cellules souches et de cellules souches cancereuses

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WO2001011011A2 (fr) * 1999-08-05 2001-02-15 Mcl Llc Cellules souches adultes toutes-puissantes et procede d'isolement
WO2003102215A2 (fr) * 2002-05-31 2003-12-11 The Board Of Trustees Of The Leland Stanford Junior University Procedes d'identification et d'isolement de cellules souches et de cellules souches cancereuses

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015527083A (ja) * 2012-09-07 2015-09-17 チルドレンズ メディカル センター コーポレーション 造血幹細胞特異的レポーターマウスおよびその使用
US10080354B2 (en) 2012-09-07 2018-09-25 Children's Medical Center Corporation Hematopoietic stem cell specific reporter mouse and uses thereof

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