Somatic cell nuclear transfer in the pig: Control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

Paul A. De Sousa, John R. Dobrinsky, Jie Zhu, Alan L. Archibald, Alison Ainslie, Wim Bosma, June Bowering, John Bracken, Patricia M. Ferrier, Judy Fletcher, Bianca Gasparrini, Linda Harkness, Paul Johnston, Marjorie Ritchie, William A. Ritchie, Ailsa Travers, David Albertini, Andras Dinnyes, Timothy J. King, Ian Wilmut

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Abstract

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.

Original languageEnglish
Pages (from-to)642-650
Number of pages9
JournalBiology of Reproduction
Volume66
Issue number3
DOIs
Publication statusPublished - Jan 1 2002

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Keywords

  • Developmental biology
  • Early development
  • Embryo
  • Reproductive technology

ASJC Scopus subject areas

  • Reproductive Medicine
  • Cell Biology

Cite this

De Sousa, P. A., Dobrinsky, J. R., Zhu, J., Archibald, A. L., Ainslie, A., Bosma, W., Bowering, J., Bracken, J., Ferrier, P. M., Fletcher, J., Gasparrini, B., Harkness, L., Johnston, P., Ritchie, M., Ritchie, W. A., Travers, A., Albertini, D., Dinnyes, A., King, T. J., & Wilmut, I. (2002). Somatic cell nuclear transfer in the pig: Control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy. Biology of Reproduction, 66(3), 642-650. https://doi.org/10.1095/biolreprod66.3.642