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ARS Home » Research » Publications at this Location » Publication #129705
Title: SOMATIC CELL NUCLEAR TRANSFER IN THE PIG: CONTROL OF PRONUCLEAR FORMATION AND INTEGRATION WITH IMPROVED METHODS FOR ACTIVATION AND MAINTENANCE OF PREGNANCY

Author
item DE SOUSA, P - ROSLIN INST,EDINBURGH,UK
item Dobrinsky, John
item ZHU, J - ROSLIN INST,EDINBURGH,UK
item ARCHIBALD, A - ROSLIN INST,EDINBURGH,UK
item AINSLIE, A - ROSLIN INST,EDINBURGH
item BOSMA, W - ROSLIN INST,EDINBURGH,UK
item BOWERING, J - ROSLIN INST,EDINBURGH,UK
item BRACKEN, J - ROSLIN INST,EDINBURGH,UK
item FERRIER, P - ROSLIN INST, EDINBURGH,UK
item FLETCHER, J - ROSLIN INST,EDINBURGH,UK

Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/22/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: In our novel approach to cloning pigs by somatic cell nuclear transfer, we confirmed an electrical activation method on ovulated oocytes. Further, we evaluated delayed versus simultaneous activation (DA vs SA) strategies and 2 lines of nuclear donor cells. Delayed activation yielded blastocysts more reliably and with a higher nuclear count than SA. Comparable rates of development using DA were obtained following culture of embryos cloned fro ovulated or in vitro matured cytoplasts and fibroblast or cumulus nuclei. For transfer of cloned embryos to recipient females, we employed the novel co-transfer of cloned embryos with developmentally-terminal parthenogenetic embryos to assist in maternal maintenance of pregnancy. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by co-transfer of parthenogenetic embryos. The combination of novel approaches are a successful system for the production of cloned pigs. Cloning enables the introduction or modificatio of genes in order to make animals resistant to disease, while providing methodology for custom production of animal models for treatment of diseases, medical alternatives for organ transplantation, or for expression and extraction of biopharmaceuticals, and founder animals for livestock production.

Technical Abstract: To clone a pig from somatic cells we first confirmed an electrical activation method on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs SA) strategies, 2 lines of nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei iand 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 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) post-fusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment post-fusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after fusion improved nuclear retention and favored the formation of single pronuclei, although with no improvement in blastocyst development. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by co- transfer of parthenogenetic embryos.