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Somatic Cell Nuclear Transfer in the Pig: Control of Pronuclear Formation and Integration with Improved Methods for Activation and Maintenance of Pregnancy¹

^a Departments of Gene Expression and Development ^b Genomics and Bioinformatics, Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom ^c Germplasm and Gamete Physiology Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705 ^d Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111

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.

First decision: 25 July 2001.

¹ This work was funded by Geron Bio-Med.

² Correspondence. FAX: 44 0131 527 4493; ian.wilmut{at}bbsrc.ac.uk

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