Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs

• Published in: Scientific reports Vol. 4; no. 1; p. 6926
• Main Authors: Yao, Jing, Huang, Jiaojiao, Hai, Tang, Wang, Xianlong, Qin, Guosong, Zhang, Hongyong, Wu, Rong, Cao, Chunwei, Xi, Jianzhong Jeff, Yuan, Zengqiang, Zhao, Jianguo
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 05.11.2014; Nature Publishing Group
• Subjects: 13; 13/106; 13/109; 45/41; 49/31; 631/136/1425; 631/61/17/1511; Alleles; Animals; Base Sequence; Cells, Cultured; Cloning, Organism; Colonies; Deoxyribonucleic acid; DNA; DNA Restriction Enzymes - genetics; Female; Fibroblasts; Galactosyltransferases - genetics; Gene Knock-In Techniques; Gene Knockout Techniques; Gene targeting; Genetic Engineering; Genomes; Hogs; Humanities and Social Sciences; Male; Molecular Sequence Data; Movement disorders; multidisciplinary; Mutagenesis, Insertional; Neurodegenerative diseases; Nuclear transfer; Nuclear Transfer Techniques; Nuclease; Oligonucleotides; Organ donors; PARK7 protein; Parkin protein; Parkinson's disease; Recombinant Fusion Proteins - genetics; Science; Somatic cell nuclear transfer; Sus scrofa - genetics; Transcription; Transcription activator-like effector nucleases; Ubiquitin-Protein Ligases - genetics; Xenografts
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep06926

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.