Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle

Hiroaki Funahashi, A. Ideta, M. Konishi, M. Urakawa, K. Uruno, Y. Aoyagi, M. Okabe, K. Niwa

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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 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. 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 nonintegrated 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.

Original languageEnglish
Pages (from-to)183-190
Number of pages8
JournalCloning and Stem Cells
Volume3
Issue number4
Publication statusPublished - 2001

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ASJC Scopus subject areas

  • Biotechnology
  • Developmental Biology
  • Applied Microbiology and Biotechnology

Cite this

Funahashi, H., Ideta, A., Konishi, M., Urakawa, M., Uruno, K., Aoyagi, Y., Okabe, M., & Niwa, K. (2001). Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle. Cloning and Stem Cells, 3(4), 183-190.