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

查看全文 作　　者：BAO [1,2]Lei;CHEN [1]HaiDe;JONG [1,3]UiMyong;RIM [1,3]CholHo;LI [1]WenLing;LIN [1]XiJuan;ZHANG [4,5]Dan;LUO [6]Qiong;CUI [1]Chun;HUANG [4,7,8]HeFeng;ZHANG [9]Yan;XIAO [1]Lei;FU [1,2,9,10]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)》索引2014年第57卷第2期,共6页

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. 关键词：体细胞核移植转基因猪基因敲除核酸酶锌指半乳糖基转移酶免疫排斥反应成纤维细胞系

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

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