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

Biol Reprod. 2002 Mar;66(3):642-50. doi: 10.1095/biolreprod66.3.642.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blastocyst / physiology
  • Blastocyst / ultrastructure
  • Cloning, Organism*
  • Cytochalasin B / pharmacology
  • Electric Stimulation
  • Embryo Transfer
  • Female
  • Microtubules / drug effects
  • Nocodazole / pharmacology
  • Nuclear Transfer Techniques*
  • Oocytes / physiology*
  • Oocytes / ultrastructure
  • Parthenogenesis
  • Pregnancy
  • Swine*

Substances

  • Cytochalasin B
  • Nocodazole