title: Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy.
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.
authors.affiliation: Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom.
authors.forename: Paul A
authors.initials: PA
authors.lastname: De Sousa
authors.forename: John R
authors.initials: JR
authors.lastname: Dobrinsky
authors.forename: Jie
authors.initials: J
authors.lastname: Zhu
authors.forename: Alan L
authors.initials: AL
authors.lastname: Archibald
authors.forename: Alison
authors.initials: A
authors.lastname: Ainslie
authors.forename: Wim
authors.initials: W
authors.lastname: Bosma
authors.forename: June
authors.initials: J
authors.lastname: Bowering
authors.forename: John
authors.initials: J
authors.lastname: Bracken
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Ferrier
authors.forename: Judy
authors.initials: J
authors.lastname: Fletcher
authors.forename: Bianca
authors.initials: B
authors.lastname: Gasparrini
authors.forename: Linda
authors.initials: L
authors.lastname: Harkness
authors.forename: Paul
authors.initials: P
authors.lastname: Johnston
authors.forename: Marjorie
authors.initials: M
authors.lastname: Ritchie
authors.forename: William A
authors.initials: WA
authors.lastname: Ritchie
authors.forename: Ailsa
authors.initials: A
authors.lastname: Travers
authors.forename: David
authors.initials: D
authors.lastname: Albertini
authors.forename: Andras
authors.initials: A
authors.lastname: Dinnyes
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: King
authors.forename: Ian
authors.initials: I
authors.lastname: Wilmut
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology / Q000648:ultrastructure; D019976:Cloning, Organism; D003571:Cytochalasin B / Q000494:pharmacology; D004558:Electric Stimulation; D004624:Embryo Transfer; D005260:Female; D008870:Microtubules / Q000187:drug effects; D015739:Nocodazole / Q000494:pharmacology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology* / Q000648:ultrastructure; D010312:Parthenogenesis; D011247:Pregnancy; D013552:Swine
chemical_list: D003571:Cytochalasin B; D015739:Nocodazole