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

Research output: Contribution to journalArticlepeer-review

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)642-50
Number of pages9
JournalBiology of Reproduction
Volume66
Issue number3
Publication statusPublished - Mar 2002

Keywords / Materials (for Non-textual outputs)

  • Animals
  • Blastocyst
  • Cloning, Organism
  • Cytochalasin B
  • Electric Stimulation
  • Embryo Transfer
  • Female
  • Microtubules
  • Nocodazole
  • Nuclear Transfer Techniques
  • Oocytes
  • Parthenogenesis
  • Pregnancy
  • Swine

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