Knockout of GGTA1 Gene by Promoter-Trap Strategy in Wuzhishan Miniature Pig

Objective: To disrupt GGTA1 gene in Wuzhishan miniature pig fetus and develop a basic platform for xenotransplantation research.Methods: The promoterless gene knockout vector which contained neo positive selection marker was generated.The construct was linearized and electroporated into the porcine fetal fibroblasts cells.The drug resistant cell clones were screened by PCR after G418 selection.Reconstructed embryos established with positive cells were transferred into surrogate sows.Results: G418 resistant cell of 176 clones were screened by PCR,and homologous recombination was found in two cell clones.The GGTA1+/-cells were used as donors to perform nuclear transplantation.Reconstructed embryos were transferred into two naturally estrous gilts.One early pregnancy was acquired,and the pregnant sow was killed at day 38 and two fetuses were got.The two fetuses which were identified by PCR and Southern blotting were one GGTA1 allelic disrupted.Conclusion: The promoterless vector targeting the exon 4 locus of GGTA1 gene was constructed successfully.One GGTA1 allelic knockout fetus were produced and a basic platform for developing GGTA1 null pigs has been established