title: Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer.
abstract: 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 porcine α-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.
authors.affiliation: College of Animal Sciences, School of Medicine, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Bao
authors.forename: HaiDe
authors.initials: H
authors.lastname: Chen
authors.forename: UiMyong
authors.initials: U
authors.lastname: Jong
authors.forename: CholHo
authors.initials: C
authors.lastname: Rim
authors.forename: WenLing
authors.initials: W
authors.lastname: Li
authors.forename: XiJuan
authors.initials: X
authors.lastname: Lin
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.forename: Qiong
authors.initials: Q
authors.lastname: Luo
authors.forename: Chun
authors.initials: C
authors.lastname: Cui
authors.forename: HeFeng
authors.initials: H
authors.lastname: Huang
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Lei
authors.initials: L
authors.lastname: Xiao
authors.forename: ZhiXin
authors.initials: Z
authors.lastname: Fu
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004720:Endonucleases / Q000378:metabolism*; D005700:Galactosyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D053652:Nuclear Transfer Techniques; D013552:Swine; D016335:Zinc Fingers
chemical_list: D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine; D004720:Endonucleases