Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer  

作　　者：BAO Lei CHEN HaiDe JONG UiMyong RIM CholHo LI WenLing LIN XiJuan ZHANG Dan LUO Qiong CUI Chun HUANG HeFeng ZHANG Yan XIAO Lei FU ZhiXin 

机构地区：[1]College of Animal Sciences, School of Medicine, Zhejiang University [2]Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences [3]Department of Animal Cloning, Cloning Research Center, Biotechnology Branch, Academy of Sciences, Pyongyang, the Democratic People's Republic of Korea [4]Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University [5]Key Laboratory of Reproductive Genetics, Zhejiang University [6]Department of Reproductive Genetics, Women's Hospital, School of Medicine, Zhejiang University [7]Key Laboratory of Reproductive Genetics, Zhejiang University,Ministry of Education [8]Department of Obstetrics, Women's Hospital, School of Medicine, Zhejiang University [9]Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences [10]College of Animal Science and Technology, Hebei Normal University of Science and Technology

出　　处：《Science China(Life Sciences)》2014年第2期263-268,共6页中国科学（生命科学英文版）

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)

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.

关 键 词：PIG XENOTRANSPLANTATION ZFNs GGTA1 biallelic knockout SCNT 

分 类 号：R346[医药卫生—基础医学]