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 language | English |
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Pages (from-to) | 642-50 |
Number of pages | 9 |
Journal | Biology of Reproduction |
Volume | 66 |
Issue number | 3 |
Publication status | Published - 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