An efficient method for production α(1,3)-galactosyltransferase gene knockout pigs
Date
2004
Authors
Harrison, S.
Boquest, A.
Grupen, C.
Faast, R.
Guildolin, A.
Giannakis, C.
Crocker, L.
McIlfatrick, S.
Ashman, R.
Wengle, J.
Editors
Advisors
Journal Title
Journal ISSN
Volume Title
Type:
Journal article
Citation
Cellular Reprogramming, 2004; 6(4):327-331
Statement of Responsibility
Sharon Harrison, Andrew Boquest, Christopher Grupen, Renate Faast, Angelo Guildolin, Christopher Giannakis, Lesley Crocker, Stephen McIlfatrick, Rodney Ashman, James Wengle, Ian Lyons, Paul Tolstoshev, Peter Cowan, Allan Robins, Philip O'Connell, Anthony J.F. D'Apice and Mark Nottle
Conference Name
Abstract
We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.