title: An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs.
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.
authors.affiliation: BresaGen Limited, Adelaide, South Australia, Australia.
authors.forename: Sharon
authors.initials: S
authors.lastname: Harrison
authors.forename: Andrew
authors.initials: A
authors.lastname: Boquest
authors.forename: Christopher
authors.initials: C
authors.lastname: Grupen
authors.forename: Renate
authors.initials: R
authors.lastname: Faast
authors.forename: Angelo
authors.initials: A
authors.lastname: Guildolin
authors.forename: Christopher
authors.initials: C
authors.lastname: Giannakis
authors.forename: Lesley
authors.initials: L
authors.lastname: Crocker
authors.forename: Stephen
authors.initials: S
authors.lastname: McIlfatrick
authors.forename: Rodney
authors.initials: R
authors.lastname: Ashman
authors.forename: James
authors.initials: J
authors.lastname: Wengle
authors.forename: Ian
authors.initials: I
authors.lastname: Lyons
authors.forename: Paul
authors.initials: P
authors.lastname: Tolstoshev
authors.forename: Peter
authors.initials: P
authors.lastname: Cowan
authors.forename: Allan
authors.initials: A
authors.lastname: Robins
authors.forename: Philip
authors.initials: P
authors.lastname: O'Connell
authors.forename: Anthony J F
authors.initials: AJ
authors.lastname: D'Apice
authors.forename: Mark
authors.initials: M
authors.lastname: Nottle
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D005260:Female; D005347:Fibroblasts; D005700:Galactosyltransferases / Q000235:genetics*; D017353:Gene Deletion; D053652:Nuclear Transfer Techniques; D009865:Oocytes; D011247:Pregnancy; D013552:Swine / Q000235:genetics*
chemical_list: D005700:Galactosyltransferases