Construction of Porcine Endogenous Gene Tiki1 Targeted Pig (Sus scrofa) Model Using TALEN Technology
WU Cai-Xia1,2, LIU Zhao-Ming1,2, YAN Quan-Mei2,*, OUYANG Zhen2, ZHAO Yu2, ZHANG Quan-Jun2, FAN Na-Na2, LAI Liang-Xue2,*
1 College of Veterinary Medicine, Jilin University, Changchun 130062; 2 Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530
Abstract：Functional research experiments on frogs (Xenopus laevis) have confirmed that TraB domain containing 2A (Tiki1) plays a decisive role in the process of head induction, but the function of Tiki1 in the early development of mammals and even humans (Homo sapiens) is not yet clear. Pigs (Sus scrofa) that are very similar to humans in physiology and pathology have become ideal mammalian models for studying Tiki1 functions. In this study, a pair of transcription activator-like effector nuclease (TALEN) plasmids tageting exon 4 of the pig endogenous gene Tiki1 were designed by TALEN technology. TALEN plasmids were validated at the level of porcine parthenogenetic embryos. The targeting efficiency and the affect on embryo development of TALEN plasmids injected at different concentrations were compared. Then plasmids transfection and screening of cells were carried out. In this study, a total of 8 sows were transplanted. 3 sows were pregnant and delivered smoothly, and a total of 12 live pigs and 1 stillborn fetus were obtained. The results showed that 4 of them belonged to the Tiki1 gene targeting positive pig model that expected by enzyme digestion and sequencing. 3 live cloned piglets were obtained from the first sow, and one of the 3 pigs (33.3%) was identified by enzyme digestion and sequencing as the expected Tiki1 gene targeting positive pig model. 6 live pigs and 1 stillbirth were obtained from the second sow. 2 of the 7 piglets (28.6%) were identified by the enzyme digestion and sequencing as the expected Tiki1 gene targeting positive pig model. 3 live cloned piglets were obtained from the third sow. One of the 3 piglets (33.3%) was identified by enzyme digestion and sequencing as the expected Tiki1 gene targeting positive pig model. The Tiki1 gene targeting positive pig model and WT mating were used to obtain 5 live F1 generation piglets. The sequencing analysis showed that the constructed Tiki1 gene targeting positive pig model was inherited by the germline, not chimera. In this experiment, TALEN technology was used to modify the endogenous gene Tiki1 of pig genome for the first time. It provides a powerful tool for efficient gene editing in large animals and a technical basis for the establishment of various biomedical models and agricultural genetically modified pigs in the future.
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