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Development of an efficient transgenic method bu using GFP-reporter gene and nuclear transfer

Research Project

Project/Area Number 10556065
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Applied animal science
Research InstitutionOKAYAMA UNIVERSITY

Principal Investigator

FUNAHASHI Hiroaki  Okayama University, Graduate School of Natural Science and Technology, Associate Professor, 大学院・自然科学研究科, 助教授 (50284089)

Co-Investigator(Kenkyū-buntansha) AOYAGI Yoshito  JA-Zen-Noh, ET Center, Manager, 飼料畜産中央研究所ETセンター, 所長(研究職)
NIWA Koji  Okayama Univ., Faculty of Agriculture, Professor, 農学部, 教授 (40089115)
OKABE Masaru  Osaka Univ., Institute of Gene Information, Professor, 遺伝子情報実験施設, 教授 (30089875)
Project Period (FY) 1998 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥12,300,000 (Direct Cost: ¥12,300,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1999: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥6,700,000 (Direct Cost: ¥6,700,000)
Keywordscattle / transgenic / GFP / nuclear transfer / early development / in vitro fertilization / swine / polyspermy
Research Abstract

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12-24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to difficulty in visualizing the pronucleus, the incidence of successful injection of linear DNA into pronucleus was higher when injected from 20 to 24 h, as compared with an early period from 12 to 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP-expression rate were not different among the variously-timed microinjections. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5 % of morulae that developed from the NT-eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of non-integrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.
Efficient methods to produce porcine zygotes in vitro for the microinjection of DNA constructs were also developed. Replacement of caffeine with adenosine or fertilization promoting peptide, which induced capacitation but prevented acrosome reaction of boar spermatozoa, reduced the incidence of polyspermic penetration of porcine oocytes without any reduction in penetration.

Report

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

    (17 results)

All Other

All Publications (17 results)

  • [Publications] Hiroaki FUNAHASHI: "Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes"Biology of Reproduction. 63. 1157-1163 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Hiroaki FUNAHASHI: "Sperm selection by a climbing-over-a-wall IVF method redices the incidence of polysper-mic penetration of porcine oocytes"Journal of Reproduction and Development. 46. 319-324 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Hiroaki FUNAHASHI: "Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle"Cloning and Stem Cells. 3. 241-248 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Hiroaki FUNAHASHI: "Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine"Molecular Reproduction and Development. 58. 424-431 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] A.Ideta: "An efficient method to produce transgenic cattle by using microinjection with an EGFP-reporter and nuclear transfer"Theriogenology. 55. 522 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 出田篤司: "マイクロインジェクション法と核移植技術を組み合わせた形質転換牛胚の効率的生産手法の開発"北海道牛受精卵移植研究会会報. (印刷中). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] H. Funahashi, A. Ideta, M. Konishi, M. Urakawa, K. Uruno, Y. Aoyagi, M. Okabe, K. Niwa: "Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle"Cloning and Stem Cells. 3(4). 241-248 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] H. Funahashi, T. Nagai: "Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine"Molecular Reproduction and Development. 58(4). 424-431 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] A. Ideta, M. Konishi, M. Urakawa, K. Uruno, Y. Aoyagi, M. Okabe, H. Funahashi: "An efficient method to produce transgenic cattle by using microinjection with an EGFP-reporter and nuclear transfer"Theriogenology. 55(1). 522 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] H. Funahashi, T. Fujiwara, T. Nagai: "Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes"Biology of Reproduction. 63(4). 1157-1163 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] H. Funahashi, T. Nagai: "Sperm selection by a climbing-over-a-wall IVF method reduces the incidence of polyspermic penetration of porcine oocytes"Journal of Reproduction and Development. 46(5). 319-324 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Hiroaki FUNAHASHI: "Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle"Cloning and Stem Cells. 3. 241-248 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Hiroaki FUNAHASHI: "Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine"Molecular Reproduction and Development. 58. 424-431 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] A.Ideta: "An efficient method to produce transgenic cattle by using microinjection with an EGFPreporter and nuclear transfer"Theriogenology. 55. 522 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 出田篤司: "マイクロインジェクション法と核移植技術を組み合わせた形質転換牛胚の効率的生産手法の開発"北海道牛受精卵移植研究会会報. (印刷中). (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Hiroaki FUNAHASHI: "Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes"Biology of Reproduction. 63. 1157-1163 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Hiroaki FUNAHASHI: "Sperm selection by a climbing-over-a-wall IVF method reduces the incidence of polyspermic penetration of porcine oocytes"Journal of Reproduction and Development. 46. 319-324 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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