Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs.Conventional gene targeting in pig somatic cells is extremely inefficient.Zinc-finger nuclease(ZFN)technology has been shown to be a powerful tool for efficiently inducing mutations in the genome.However,ZFN-mediated targeting in pigs has rarely been achieved.Here,we used ZFNs to knock out the porcineα-1,3-galactosyl-transferase(GGTA1)gene,which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation.Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1.Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4%and 5.2%,respectively.The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer(SCNT).Three GGTA1 null piglets were born,and one knockout primary fibroblast cell line was established from a cloned fetus.Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane.Functionally,GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum.This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs.GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.
