Development of a general method for obtaining double KO swine cells which will be used as donors for production of cloned homozygous piglets

• Project/Area Number: 21580350
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied animal science
• Research Institution: Kagoshima University
• Principal Investigator: SATO Masahiro 鹿児島大学, フロンティアサイエンス研究推進センター, 教授 (30287099)
• Co-Investigator(Kenkyū-buntansha): YOSHIDA Mitsutoshi 鹿児島大学, 農学部, 教授 (00174954) ; MIYOSHI Kazuchika 鹿児島大学, 農学部, 准教授 (70363611)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2011: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2010: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2009: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: KOブタ / ホモ / gene targeting / 薬剤耐性 / 体細胞核移植 / 胎仔性繊維芽細胞 / 遺伝子導入 / double KO / ホモKOブタ / gene targetin / ホセKOブタ

Research Abstract

This study aims to develop a general method for obtaining double KO swine cels in which the target gene expression is completely blocked. This was successfully performed by selection of heterogenous KO cels under higher concentrations of selective drugs after repeated delivery of a targeting vector(RDTV). These resulting double KO cells will be used for somatic cell nuclear transfer-mediated production of cloned pigs.

Research Products

• [Journal Article] Successful suppression of endogenous α-1, 3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro (2012)
  • Author(s): Chi H, Shinohara M, Yokomine T, Sato M, Takao S, Yoshida M, Miyoshi K.
  • Journal Title: J Reprod Dev Volume: 58(1) Pages: 69-76
  • NAID: 10030407205
  • Peer Reviewed
• [Journal Article] Determination of the Optimal Concentration of Several Selective Drugs Useful for Generating Multi-Transgenic Porcine Embryonic Fibroblasts (2012)
  • Author(s): Sato M, Ohtsuka M, Miura H, Miyoshi K, Watanabe S.
  • Journal Title: Reproduction in Domestic Animals/John Wiley & Sons, Inc. Volume: 47 Issue: 5 Pages: 759-765
  • DOI: 10.1111/j.1439-0531.2011.01964.x
  • Peer Reviewed
• [Journal Article] Expression analysis of a α-1,3-galactosyltransferase, an enzyme that creates xenotransplantation-related α-Gal epitope, in pig preimplantation embryos (2012)
  • Author(s): Chi H, Sato M, Yoshida M, Miyoshi K
  • Journal Title: Animal Science Journal Volume: 83 Pages: 88-93
  • Peer Reviewed
• [Journal Article] Successful suppression of endogenous α-1, 3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro (2012)
  • Author(s): Chi H, Shinohara M, Yokomine T, Sato M, Takao S, Yoshida M, Miyoshi K
  • Journal Title: Journal of Reproduction and Developmen Volume: 58 Pages: 69-76
  • NAID: 10030407205
  • Peer Reviewed
• [Journal Article] Targeted transgenesis through pronuclear injection of improved vectors into in vitro fertilized eggs (2012)
  • Author(s): Ohtsuka M, Miura H, Nakaoka H, Kimura M, Sato M, Inoko H
  • Journal Title: Transgenic Research Volume: 21 Pages: 225-226
  • Peer Reviewed
• [Journal Article] Determination of the optimal concentration of several selective drugs useful for generating multi-transgenic porcine embryonic fibroblasts (2012)
  • Author(s): Sato M, Ohtsuka M, Miura H, Miyoshi K, Watanabe S
  • Journal Title: Reproduction in Domestic Animals Volume: (in press)
  • Peer Reviewed
• [Journal Article] Generation of a set of mouse STO feeder cell lines that confer resistance to several types of selective drugs (2012)
  • Author(s): Saitoh I, Sato M, Iwase Y, Akasaka E, Yamasaki Y, Noguchi H
  • Journal Title: Cell Medicine (Part B of Cell Transplantation) Volume: (in press)
  • Peer Reviewed
• [Journal Article] Targeted Toxin as a Useful Reagent for Enrichment ofα-Gal Epitope-Negative Cells Used for Somatic Cell Nuclear Transfer in Pigs (2011)
  • Author(s): Sato M, Chi H, Miyoshi K
  • Journal Title: Xenotransplantation(InTech Open Acess Book)
  • Peer Reviewed
• [Journal Article] Cultivation with untransfected fibroblasts can stimulate proliferation of a single gene-modified fibroblast derived from a Clawn miniature swine fetus (2011)
  • Author(s): Sato M, Yoshida M, Miyoshi K, Ohtsuka M, Watanabe S
  • Journal Title: Reproduction in Domestic Animals/John Wiley & Sons, Inc. Volume: Vol.46, No.5 Pages: 911-916
  • Peer Reviewed
• [Journal Article] Whole-genome amplification-based GenomiPhi for multiple genomic analysis of individual early porcine embryos (2011)
  • Author(s): Akasaka E, Ozawa A, Mori H, Mizobe Y, Yoshida M, Miyoshi K, Sato M.
  • Journal Title: Theriogenology/Elsevier B. V. Volume: 75(8)
  • Peer Reviewed
• [Journal Article] Whole-genome amplification-based GenomiPhi for multiple genomic analys is of individual early porcine embryos (2011)
  • Author(s): Akasaka E, Ozawa A, Mori H, Mizobe Y, Yoshida M, Miyoshi K, Sato M
  • Journal Title: Theriogenology/Elsevier B.V. Volume: 75 Pages: 1543-1549
  • Peer Reviewed
• [Journal Article] Cultivation with untransfected fibroblasts can stimulate proliferation of a single gene-modified fibroblast derived from a Clawn miniature swine fetus (2011)
  • Author(s): Sato M, Yoshida M, Miyoshi K, Ohtsuka M, Watanabe S
  • Journal Title: Reproduction in Domestic Animals Volume: 46 Pages: 911-916
  • Peer Reviewed
• [Journal Article] Improvement of a phiC31 integrase-based gene delivery system that confers high and continuous transgene expression (2011)
  • Author(s): Watanabe S, Nakamura S, Sakurai T, Akasaka K, Sato M
  • Journal Title: New Biotechnology Volume: 28 Pages: 312-319
  • Peer Reviewed
• [Journal Article] A novel glycosylation signal regulates transforming growth factor receptors as evidenced by endo-β-galactosidase C expression in rodent cells (2011)
  • Author(s): Watanabe S, Misawa M, Matsuzaki T, Sakurai T, Muramatsu T, Sato M
  • Journal Title: Glycobiology Volume: 21 Pages: 482-492
  • Peer Reviewed
• [Journal Article] Simple cloning strategy using GFPuv gene as positive/negative indicator (2011)
  • Author(s): Miura H, Inoko H, Inoue I, Tanaka M, Sato M, Ohtsuka M
  • Journal Title: Analytical Biochemistry Volume: 416 Pages: 237-239
  • Peer Reviewed
• [Journal Article] Innate immune system still works at diapause, a physiological state of dormancy in insects (2011)
  • Author(s): Nakamura A, Takezawa Y, Ohnami N, Sato M, Ono C, Harada Y, Yoshida K, Kawano N, Kanai S, Miyado M, Umezawa A
  • Journal Title: Biochemical and Biophysical Research Communication Volume: 410 Pages: 351-357
  • Peer Reviewed
• [Journal Article] Beta-catenin is a molecular switch that regulates transition of cell-cell adhesion to fusion (2011)
  • Author(s): Takezawa Y, Yoshida K, Miyado K, Sato M, Nakamura A, Kawano N, Sakakibara K, Kondo T, Harada Y, Ohnami N, Kanai S, Miyado M, Saito H, Takahashi Y, Akutsu H, Umezawa A
  • Journal Title: Scientific Reports Volume: 1 Pages: 68-68
  • Peer Reviewed
• [Journal Article] Targeted toxin as a useful reagent for enrichment of α-Gal epitope-negative cells used for somatic cell nuclear transfer in pigs (2011)
  • Author(s): Sato M, Chi H, Miyoshi K
  • Journal Title: Xenotransplantation/In Tech
  • Peer Reviewed
• [Journal Article] Production of genetically modified porcine blastocysts by somatic cell nuclear transfer : preliminary results toward production of xenograft-competent miniature pigs (2010)
  • Author(s): Himaki T, Watanabe S, Chi H, Yoshida M, Miyoshi K, Sato M.
  • Journal Title: Journal of Reproduction and Development Volume: 56巻 Pages: 630-638
  • NAID: 10027730311
  • Peer Reviewed
• [Journal Article] Effects of trichostatin A on in vitro development and transgene function in somatic cell nuclear transfer embryos derived from transgenic Clawn miniature pig cells (2010)
  • Author(s): Himaki T, Yokomine T, Sato M, Miyoshi K, Takao S, Yoshida M.
  • Journal Title: Animal Science Journal Volume: 81巻 Pages: 558-563
  • NAID: 10027889588
  • Peer Reviewed
• [Journal Article] Beneficial effects of reversine on in vitro development of miniature pig somatic cell nuclear transfer embryos (2010)
  • Author(s): Miyoshi K, Mori H, Mizobe Y, Himaki T, Yoshida M, Sato M.
  • Journal Title: Journal of Reproduction and Development Volume: 56巻 Pages: 291-296
  • NAID: 10026324772
  • Peer Reviewed
• [Journal Article] Latrunculin A dramatically improves the developmental capacity of nuclear tramsfer embryos derived from gene-modified Clawn miniature pig cells (2010)
  • Author(s): Himaki T, Mori H, Mizobe Y, Miyoshi K, Sato M, Takao S, Yoshida M.
  • Journal Title: Cellular Reprogramming Volume: 12巻 Pages: 127-131
  • Peer Reviewed
• [Journal Article] Valproic acid enhances in vitro development and Oct-3/4 expression of miniature pig somatic cell nuclear transfer embryos (2010)
  • Author(s): Miyoshi K, Mori H, Mizobe Y, Akasaka E, Ozawa A, Yoshida M, Sato M.
  • Journal Title: Cellular Reprogramming Volume: 12巻 Pages: 67-74
  • Peer Reviewed
• [Journal Article] Usefulness of a non-invasive reporter system for monitoring reprogramming state in pig cells : Results of a cell fusion experiment (2010)
  • Author(s): Ozawa A, Akasaka E, Watanabe S, Yoshida M, Miyoshi K, Sato M.
  • Journal Title: Journal of Reproduction and Development Volume: 56巻 Pages: 363-369
  • NAID: 10026621377
  • Peer Reviewed
• [Journal Article] Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate (2010)
  • Author(s): Akasaka E, Watanabe S, Himaki T, Ohtsuka M, Yoshida M, Miyoshi K, Sato M.
  • Journal Title: Xenotransplantation Volume: 17巻 Pages: 81-89
  • Peer Reviewed
• [Journal Article] Effects of trichostatin A on in vitro development and transgene function in somatic cell nuclear transfer embryos derived from transgenic Clawn miniature pig cells. (2010)
  • Author(s): Himaki T, et al.
  • Journal Title: Animal Science Journal Volume: 81 Pages: 558-563
  • Peer Reviewed
• [Journal Article] Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate. (2010)
  • Author(s): Akasaka E, et al.
  • Journal Title: Xenotransplantation Volume: 17 Pages: 81-89
  • Peer Reviewed
• [Journal Article] Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression. (2010)
  • Author(s): Ohtsuka M, et al.
  • Journal Title: Nucleic Acids Res Volume: 56 Pages: 630-638
  • Peer Reviewed
• [Journal Article] Production of genetically modified porcine blastocysts by somatic cell nuclear transfer : Preliminary results toward production of xenograft-competent miniature pigs. (2010)
  • Author(s): Himaki T, et al.
  • Journal Title: J Reprod Dev Volume: 38
  • Peer Reviewed
• [Journal Article] Valproic acid enhances in vitro development and Oct-3/4 expression of miniature pig somatic cell nuclear transfer embryos (2010)
  • Author(s): Miyoshi K, et al.
  • Journal Title: Cell Reprogram 12 Pages: 67-74
  • Peer Reviewed
• [Journal Article] Development of a noninvasive monitoring system for evaluation of Oct-3/4 promoter status in miniature pig somatic cell nuclear transfer embryos (2009)
  • Author(s): Miyoshi K, Mori H, Mizobe Y, Akasaka E, Ozawa A, Yoshida M, Sato M.
  • Journal Title: Journal of Reproduction and Development Volume: 55巻 Pages: 661-669
  • NAID: 10026322831
  • Peer Reviewed
• [Journal Article] Development of a noninvasive monitoring system for evaluation of Oct-3/4 promoter status in miniature pig somatic cell nuclear transfer embryos (2009)
  • Author(s): Miyoshi K, et al.
  • Journal Title: J Reprod Dev 55 Pages: 661-669
  • NAID: 10026322831
  • Peer Reviewed
• [Journal Article] A novel glycosylation signal regulates transforming growth factor β receptors as evidenced by endo-β-galactosidase C expression in rodent cells.
  • Author(s): Watanabe S, et al.
  • Journal Title: Glycobiology Volume: (in press)
  • Peer Reviewed
• [Journal Article] Latrunculin A dramatically improves the developmental capacity of nuclear transfer embryos derived from gene-modified Clawn miniature pig cells
  • Author(s): Himaki T, et al.
  • Journal Title: Cloning Stem Cells (in press)
  • Peer Reviewed
• [Journal Article] Beneficial effects of reversine on in vitro development of miniaturepig somatic cell nuclear transfer embryos
  • Author(s): Miyoshi K, et al.
  • Journal Title: J Reprod Dev (in press)
  • Peer Reviewed
• [Journal Article] Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate
  • Author(s): Akasaka E, et al.
  • Journal Title: Xenotransplantation (in press)
  • Peer Reviewed
• [Journal Article] Usefulness of a non-invasive reporter system for monitoring reprogramming state in pig cells : Results of a cell fusion experiment
  • Author(s): Ozawa A, et al.
  • Journal Title: J Reprod Dev (in press)
  • Peer Reviewed
• [Presentation] 異種移植関連抗原α-Galエピトープ陰性ブタ体細胞の選択的濃縮のためのtargeted toxinの有効性 (2011)
  • Author(s): 池海英、三好和睦、佐藤正宏
  • Organizer: 第104回日本繁殖生物学会
  • Place of Presentation: いわて県民情報交流センター・アイーナ(岩手県盛岡市)
  • Year and Date: 2011-09-15
• [Presentation] 異種移植関連抗原α-Galエピトープ陰性ブタ体細胞の選択的濃縮のためのtargeted toxinの有効性 (2011)
  • Author(s): 池海英、三好和睦、佐藤正宏
  • Organizer: 第104回日本繁殖生物学会
  • Place of Presentation: いわて県民情報交流センター・アイーナ(岩手県)
• [Presentation] 全ゲノム増幅法による核移植ブタ胚でのgene-targeted allele検出の有効性 (2011)
  • Author(s): 池海英、吉田光敏、三好和睦、佐藤正宏
  • Organizer: 第58回日本実験動物学会総会
  • Place of Presentation: タワーホール船堀(東京都)
• [Presentation] Skt^<Cre>系統を用いたコンディショナルノックアウト法による椎間板形成の解析 (2010)
  • Author(s): 谷河麻耶
  • Organizer: 第33回日本分子生物学会年会/第83回日本生化学会大会合同大会
  • Place of Presentation: 神戸ポートアイランド(神戸市)
  • Year and Date: 2010-12-07
• [Presentation] A novel glycosylation signal regulates transforming growth factor-beta receptors as evidenced by endo-beta-galactosidase C expression in rodent cells (2010)
  • Author(s): 渡部聡
  • Organizer: 第33回日本分子生物学会年会/第83回日本生化学会大会合同大会
  • Place of Presentation: 神戸ポートアイランド(神戸市)
  • Year and Date: 2010-12-07
• [Presentation] A novel glycosylation signal regulates transforming growth factor-beta receptors as evidenced by endo-beta-galactosidase C expression in rodent cells (2010)
  • Author(s): 渡部聡、三澤雅子、松崎貴、桜井敬之、村松喬、佐藤正宏
  • Organizer: 第33回日本分子生物学会年会/第83回日本生化学会大会合同大会
  • Place of Presentation: 神戸ボートアイラントド(神戸市)
• [Presentation] Skt^<Cre>系統を用いたコンディショナルノックアウト法による椎間板形成の解析 (2010)
  • Author(s): 谷河麻耶、荒木喜美、仙波圭、佐藤正宏、檜山明彦、酒井大輔、持田譲治、山村研一、阿部幸一郎
  • Organizer: 第33回日本分子生物学会年会/第83回日本生化学会大会合同大会
  • Place of Presentation: 神戸ボートアイランド(神戸市)
• [Presentation] ミニブタ体細胞の in vitro 脱分化誘導の試み (2009)
  • Author(s): 佐藤正宏
  • Organizer: 第56回日本実験動物学会総会
  • Place of Presentation: 大宮ソニックシティ(埼玉県)
  • Year and Date: 2009-05-15
• [Presentation] 全ゲノム増幅法を用いた核移植由来ブタ胚細胞における外来性遺伝子検出の試み (2009)
  • Author(s): 赤坂恵理、佐藤正宏
  • Organizer: 第56回日本実験動物学会総会
  • Place of Presentation: 大宮ソニックシティ(埼玉県)
• [Presentation] ミニブタ体細胞のin vitro脱分化誘導の試み (2009)
  • Author(s): 佐藤正宏、赤坂恵理、小澤明央
  • Organizer: 第56回日本実験動物学会総会
  • Place of Presentation: 大宮ソニックシティ(埼玉県)
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