An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs

作者：Harrison S; Boquest A; Grupen C; Faast R; Guildolin A; Giannakis C; Crocker L; McIlfatrick S; Ashman R; Wengle J; Lyons I; Tolstoshev P; Cowan P; Robins A; O'Connell P; D'Apice AJF; Nottle M^*

来源：Cloning and Stem Cells, 2004, 6(4): 327-331.

DOI：10.1089/clo.2004.6.327

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.

• 出版日期2004