title: Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs.
abstract: Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science, Jilin University, Changchun, China ; Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province, Animal Science and Technology College, Yunnan Agricultural University, Kunming, China.
authors.forename: Jige
authors.initials: J
authors.lastname: Xin
authors.affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China.
authors.forename: Huaqiang
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China.
authors.forename: Nana
authors.initials: N
authors.lastname: Fan
authors.affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China.
authors.forename: Bentian
authors.initials: B
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Ouyang
authors.affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China.
authors.forename: Zhaoming
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science, Jilin University, Changchun, China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Song
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science, Jilin University, Changchun, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China.
authors.forename: Qingjian
authors.initials: Q
authors.lastname: Zou
authors.affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China.
authors.forename: Quanmei
authors.initials: Q
authors.lastname: Yan
authors.affiliation: Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province, Animal Science and Technology College, Yunnan Agricultural University, Kunming, China.
authors.forename: Yangzhi
authors.initials: Y
authors.lastname: Zeng
authors.affiliation: Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science, Jilin University, Changchun, China ; Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China ; Key Laboratory of Banna Mini-pig Inbred Line of Yunnan Province, Animal Science and Technology College, Yunnan Agricultural University, Kunming, China.
authors.forename: Liangxue
authors.initials: L
authors.lastname: Lai
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D005347:Fibroblasts / Q000378:metabolism; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics*; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D009154:Mutation; D010641:Phenotype; D013552:Swine; D013556:Swine, Miniature / Q000235:genetics*
chemical_list: D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine