利用TALEN技术构建猪内源性基因<em>Tiki1</em>打靶猪模型

doi:10.3969/j.issn.1674-7968.2019.10.016

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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.

Key words： Transcription activator-like effector nuclease (TALEN) gene editing technology Tiki1 Gene targeting Swine model

Received: 22 March 2019

ZTFLH:

S828.9+9

Corresponding Authors: lai_liangxue@gibh.ac.cn; yan_quanmei@gibh.ac.cn

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	Articles by authors
	WU Cai-Xia
	LIU Zhao-Ming
	YAN Quan-Mei
	OUYANG Zhen
	ZHAO Yu
	ZHANG Quan-Jun
	FAN Na-Na
	LAI Liang-Xue

Cite this article:

WU Cai-Xia,LIU Zhao-Ming,YAN Quan-Mei, et al. Construction of Porcine Endogenous Gene Tiki1 Targeted Pig (Sus scrofa) Model Using TALEN Technology[J]. 农业生物技术学报, 2019, 27(10): 1858-1868.

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http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2019.10.016 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2019/V27/I10/1858

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