Somatic Cell Nuclear Transfer in the Pig: Control of Pronuclear Formation and Integration with Improved Methods for Activation and Maintenance of Pregnancy1
| Autor: | Andras Dinnyes, Ailsa Travers, Ian Wilmut, W. A. Ritchie, Judy Fletcher, J.R. Dobrinsky, Wim Bosma, Alison Ainslie, Alan Archibald, Paul A. De Sousa, David F. Albertini, John Bracken, Linda Harkness, June Bowering, Bianca Gasparrini, Paul Johnston, Tim King, Marjorie Ritchie, Jie Zhu, Patricia M. Ferrier |
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| Rok vydání: | 2002 |
| Předmět: |
Pronucleus
Somatic cell Cell Biology General Medicine Reproductive technology Biology Molecular biology Cytoplast Embryo transfer Cell biology chemistry.chemical_compound medicine.anatomical_structure Reproductive Medicine chemistry embryonic structures medicine Somatic cell nuclear transfer Blastocyst Cytochalasin B |
| Zdroj: | Biology of Reproduction. 66:642-650 |
| ISSN: | 1529-7268 0006-3363 |
| DOI: | 10.1095/biolreprod66.3.642 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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