摘要
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.
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.
基金
supported by grants from Ministry of Science and Technology of China(2011CBA01001,2012AA020503)
the National Science Fund for Distinguished Young Scholars(31025016)
the National Natural Science Foundation of China(31271577)
Novel Agricultural Variety Breeding Project of Zhejiang Province(2012C12906-8)
the Fundamental Research Funds for the Central Universities
the Key Construction Pro-gram of the National"985"Project(118000+193411801/006)
the Research Fund for the Doctoral Program of Higher Education of China(20120101110089)
