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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
- Source :
- Biology of Reproduction. 66:642-650
- Publication Year :
- 2002
- Publisher :
- Oxford University Press (OUP), 2002.
-
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.
- Subjects :
- 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
Subjects
Details
- ISSN :
- 15297268 and 00063363
- Volume :
- 66
- Database :
- OpenAIRE
- Journal :
- Biology of Reproduction
- Accession number :
- edsair.doi...........9cdeaf28b5d982c46b13cb019032e5ce