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

Abstract

We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the α(1,3)-galactosyltransferase (α1,3-GT) gene in porcine fetal fibroblasts using a non-isogenic promoterless construct approach. Here we report the production of α1,3-GT gene knockout pigs using these procedures. Seven α1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neoR) 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 ..