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Studies for Production of Porcine Somatic Cell Clone Animals using Molecular Genetics

Research Project

Project/Area Number 11556051
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Applied animal science
Research InstitutionTHE UNIVERSITY OF TOKYO

Principal Investigator

NAITO Kunihiko  Graduate School of Agricultural and Life Sciences, THE UNIVERSITY OF TOKYO, Associate Professor, 大学院・農学生命科学研究科, 助教授 (20188858)

Co-Investigator(Kenkyū-buntansha) KIKUCHI Kazuhiro  National Institute of Agrobiological Sciences, Senior Researcher, 主任研究官
YAMANOUCHI Keitaro  Graduate School of Agricultural and Life Sciences, THE UNIVERSITY OF TOKYO, Associate Professor, 大学院・農学生命科学研究科, 助教授 (70272440)
TOJO Hideaki  Graduate School ofAgricultural and Life Sciences, THE UNIVERSITY OF TOKYO, Professor, 大学院・農学生命科学研究科, 教授 (20041668)
菊地 和弘  農水省, 農業生物資源研究所, 主任研究官
Project Period (FY) 1999 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥11,600,000 (Direct Cost: ¥11,600,000)
Fiscal Year 2001: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1999: ¥4,800,000 (Direct Cost: ¥4,800,000)
Keywordsporcine oocytes / somatic cell clone / enucleation / nuclear transfer / MPF / MRPK / protein synthesis / Rb
Research Abstract

The initial purpose of the present study is the production of porcine somatic cell clones. In 2000, however, the porcine somatic cell clones have been produced in other laboratories. Therefore I analyzed thereafter the physiological mechanisms and developmental regulation factors in oocytes and early embryos, which are the materials for the clone animal, in order to improve the efficiency of the clone animal production.
(1) At First, I examined the possibility of the maturation promoting factor (MPF) and MAP kinase (MAPK) removal by the procedure for clone production, those are the removal of the spindle and the exchange of somatic nucleus. I was able to show that the MPF removal was very small and might have no effect on the embryo development, but that about 20 % of MAPK was removed by the process and might affect the embryo development.
(2) Next, I studied whether these factors localized on the replaced somatic chromosomes in the reconstitute oocytes. I found that MPF and MAPK localiz … More ed only on the tubulin-localized-spindles but not on the tubulin-non localized-spindle. In addition, as the frequency of the tubulin-localized-spindle was well agreed with the frequency of pronucleus formation, I suggested that the oocytes having a tubulin-localized-spindle could form a pronucleus.
(3) I analyzed next the factors regulating the protein-synthesis-pattern exchange, which occurred around meiotic resumption. I postulated MPF and MAPK as the factor and their mRNAs or antisense RNAs were injected into the oocytes in order to change their activity artificially. The results showed that the protein-synthesis-pattern exchange was regulated by MPF but not affected by MAPK acitvity.
(4) Lastly, I investigated the regulator of the early embryo specific cell cycle, which has no gap phases and repeats only S phase and M phase rapidly. I postulated the absence ofRb protein, inhibitory regulator of S phase, as the factor and examined its expression levels during early embryo development at mRNA and protein levels. 1 found that Rb was actually absent between 4-cell stage and blastocyst stage, and its involvement was confirmed by the inhibition of development by the injection of Rb expression vector into embryos.
These results are the first reports not only mammals but also all species, and might be useful to improve the efficiency of the clone animal production as basic data. Less

Report

(4 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • Research Products

    (23 results)

All Other

All Publications (23 results)

  • [Publications] Kikuchi Kazuhiro: "Maturation/M-phase preomoting factor : A regulator of aging in porcine oocytes"Biol. Reprod.. 63. 715-722 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sato Eimei: "Molecular basis of oocyte maturation in the pig"J. Reprod. Dev. Suppl. 47. S63-S69 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Goto Seitaro: "Localization of Cyclin B and MAP kinase around chromosomes of cumulus nuclei transferred into porcine oocytes"J. Mamm. Ova Res.. 18. 33-38 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ohashi Satoshi: "Expression of exogenous proteins in porcine maturing oocytes after mRNA injection: kinetic analysis and oocyte selection using EGFP mRNA"J. Reprod. Dev.. 47. 351-357 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sugiura Koji: "Studies the germinal vesicle requirement for MAP kinase activities in porcine oocyte maturation"Mol. Reprod. Dev.. 59. 215-220 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sugiura Koji: "Activation of ribosomal S6 kinase (RSK) during porcine oocyte maturation"Zygote. (In press). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 内藤 邦彦: "妊娠の生物学(分担執筆:卵の構造と成熟制御機構)"中山徹也・牧野恒久・高橋迪雄監修、塩田邦夫・松林秀彦編、永井書店. 396 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kikuchi K, Naito K, Noguchi J, Shimada A, Kaneko H, Yamashita M., Aoki F, Tojo H, Toyoda Y: "Maturation/M-phase preomoting factor : A regulator of aging in porcine oocytes"Biol Reprod. 63. 715-722 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sato E, Kimura N, Yokoo M, Sasada H, Naito K: "Molecular basis of oocyte maturation in the pig"J. Reprod. Dev. suppl.. 47. S63-S69 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Goto S, Naito K, Sugiura K, Tojo H: "Localization of Cyclin B and MAP kinase around chromosomes of cumulus nuclei transferred into porcine oocytes"J Mamm Ova Res. 18. 33-38 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ohashi S, Naito K, Liu J, Sheng Y, Yamanouchi K, Tojo H: "Experssion of exogenous proteins in porcine maturing oocytes after mRNA injection : kinetic analysis and oocyte selection using EGFP mRNA"J Reprod Dev. 47. 351-357 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sugiura K, Naito K, Iwamori N, Kagii H, Yamashita M, Goto S, Ohashi S, Yamanouchi K, Tojo H: "Studies on the germinal vesicle erquirement for MAP kinase activities in porcine oocyte maturation"Mol Reprod Dev. 59. 215-220 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sugiura K, Naito K, Iwamori N, Kagii H, Goto S, Ohashi S, Narita H, Yada E, Yamaguchi K, Tojo H: "Activation of ribosomal S6 kinase (RSK) during porcine oocyte maturation"Zygote. (in press). (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sugiura Koji: "Studies on the germinal vesicle requirement for MAP kinase activities in porcine oocyte maturation"Mol. Reprod. Dev.. 59. 215-220 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Sato Eimei: "Molecular basis of oocyte maturation in the pig"J. Reprod. Dev. Suppl. 47. S63-S69 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Goto Seitaro: "Localization of Cyclin B and MAP kinase around chromosomes of cumulus nuclei transferred into porcine oocytes"J. Mamm. Ova Res.. 18. 33-38 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Ohashi Satoshi: "Expression of exogenous proteins in porcine maturing oocytes after mRNA injection : kinetic analysis and oocyte selection using EGFP mRNA"J, Reprod. Dev.. 47. 351-357 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Sugiura Koji: "Activation of ribosomal S6 kinase (RSK) during porcine oocyte maturation"Zygote. (In press). (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 内藤 邦彦: "妊娠の生物学(分担執筆:卵の構造と成熟制御機構)"中山徹也・牧野恒久・高橋迪雄監修、塩田邦夫・松林秀彦編、永井書店. 396 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kagii Hireyuki: "Requirement of mitogen-activated protein kinase activation for the meiotic resumption of porcine oocytes."J.Reprod.Dev.. 46. 249-256 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kikuchi Kazuhiro: "Maturation/M-phase preomoting factor : A regulator of aging in porcine oocytes."Biol.Reprod.. 63. 715-722 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Koji Sugiura: "Analysis of the germinal vesicle requirement for the activation of MPF in maturation of porcine oocytes"J.Mamm.Ova Res.. 16・3. 130-134 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kunihiko Naito: "Establishment of a small-scale western blotting system named as "micro-western blotting" for mammalian ova analysis"J.Mamm.Ova Res.. 16・3. 154-157 (1999)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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