An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs.
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.
Harrison
Sharon
S
BresaGen Limited, Adelaide, South Australia, Australia.
Boquest
Andrew
A
Grupen
Christopher
C
Faast
Renate
R
Guildolin
Angelo
A
Giannakis
Christopher
C
Crocker
Lesley
L
McIlfatrick
Stephen
S
Ashman
Rodney
R
Wengle
James
J
Lyons
Ian
I
Tolstoshev
Paul
P
Cowan
Peter
P
Robins
Allan
A
O'Connell
Philip
P
D'Apice
Anthony J F
AJ
Nottle
Mark
M
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*
D005700:Galactosyltransferases