title: Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs.
abstract: Pigs are ideal organ donors for xenotransplantation and an excellent model for studying human diseases, such as neurodegenerative disease. Transcription activator-like effector nucleases (TALENs) are used widely for gene targeting in various model animals. Here, we developed a strategy using TALENs to target the GGTA1, Parkin and DJ-1 genes in the porcine genome using Large White porcine fibroblast cells without any foreign gene integration. In total, 5% (2/40), 2.5% (2/80), and 22% (11/50) of the obtained colonies of fibroblast cells were mutated for GGTA1, Parkin, and DJ-1, respectively. Among these mutant colonies, over 1/3 were bi-allelic knockouts (KO), and no off-target cleavage was detected. We also successfully used single-strand oligodeoxynucleotides to introduce a short sequence into the DJ-1 locus. Mixed DJ-1 mutant colonies were used as donor cells for somatic cell nuclear transfer (SCNT), and three female piglets were obtained (two were bi-allelically mutated, and one was mono-allelically mutated). Western blot analysis showed that the expression of the DJ-1 protein was disrupted in KO piglets. These results imply that a combination of TALENs technology with SCNT can efficiently generate bi-allelic KO pigs without the integration of exogenous DNA. These DJ-1 KO pigs will provide valuable information for studying Parkinson's disease.
authors.affiliation: 1] State Key laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101 [2] Beijing Farm Animal Research Center, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101.
authors.forename: Jing
authors.initials: J
authors.lastname: Yao
authors.affiliation: 1] State Key laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101 [2] Beijing Farm Animal Research Center, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101.
authors.forename: Jiaojiao
authors.initials: J
authors.lastname: Huang
authors.affiliation: 1] State Key laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101 [2] Beijing Farm Animal Research Center, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101.
authors.forename: Tang
authors.initials: T
authors.lastname: Hai
authors.affiliation: 1] State Key laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101 [2] Beijing Farm Animal Research Center, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101.
authors.forename: Xianlong
authors.initials: X
authors.lastname: Wang
authors.affiliation: 1] State Key laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101 [2] Beijing Farm Animal Research Center, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101.
authors.forename: Guosong
authors.initials: G
authors.lastname: Qin
authors.affiliation: 1] State Key laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101 [2] Beijing Farm Animal Research Center, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101.
authors.forename: Hongyong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, P. R. China, 100101.
authors.forename: Rong
authors.initials: R
authors.lastname: Wu
authors.affiliation: 1] State Key laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101 [2] Beijing Farm Animal Research Center, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101.
authors.forename: Chunwei
authors.initials: C
authors.lastname: Cao
authors.affiliation: Biomedical Engineering Department, College of Engineering, Peking University, Beijing, P. R. China, 100871.
authors.forename: Jianzhong Jeff
authors.initials: JJ
authors.lastname: Xi
authors.affiliation: State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, P. R. China, 100101.
authors.forename: Zengqiang
authors.initials: Z
authors.lastname: Yuan
authors.affiliation: 1] State Key laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101 [2] Beijing Farm Animal Research Center, Institute of Zoology, Chinese Academy of Sciences, P. R. China, 100101.
authors.forename: Jianguo
authors.initials: J
authors.lastname: Zhao
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D019976:Cloning, Organism; D004262:DNA Restriction Enzymes / Q000235:genetics; D005260:Female; D005700:Galactosyltransferases / Q000235:genetics; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D005818:Genetic Engineering; D008297:Male; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D053652:Nuclear Transfer Techniques; D011993:Recombinant Fusion Proteins / Q000235:genetics; D034421:Sus scrofa / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D005700:Galactosyltransferases; C530267:alpha-1,3-galactosyltransferase 1, porcine; D004262:DNA Restriction Enzymes