Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy.
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.
De Sousa
Paul A
PA
Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom.
Dobrinsky
John R
JR
Zhu
Jie
J
Archibald
Alan L
AL
Ainslie
Alison
A
Bosma
Wim
W
Bowering
June
J
Bracken
John
J
Ferrier
Patricia M
PM
Fletcher
Judy
J
Gasparrini
Bianca
B
Harkness
Linda
L
Johnston
Paul
P
Ritchie
Marjorie
M
Ritchie
William A
WA
Travers
Ailsa
A
Albertini
David
D
Dinnyes
Andras
A
King
Timothy J
TJ
Wilmut
Ian
I
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
D003571:Cytochalasin B; D015739:Nocodazole