An improved Tet-on system in micro RNA overexpression and CRISPR/Cas9-mediated gene editing  

作　　者：Kang Kang Lian Huang Qing Li Xiaoyun Liao Quanjin Dang Yi Yang Jun Luo Yan Zeng Li Li Deming Gou 

机构地区：[1]Department of Biochemistry and Molecular Biology,Carson International Cancer Center,Shenzhen University Health Sciences Center,Shenzhen,Guangdong 518060,People’s Republic of China [2]Shaanxi Key Laboratory of Molecular Biology for Agriculture,College of Animal Science and Technology,Northwest A&F University,Yangling,Shaanxi 712100,People’s Republic of China [3]Shenzhen Key Laboratory of Microbial Genetic Engineering,College of Life Sciences and Oceanography,Shenzhen University,Xueyuan Ave 1066,Shenzhen,Guangdong 518060,People’s Republic of China

出　　处：《Journal of Animal Science and Biotechnology》2019年第4期1089-1100,共12页畜牧与生物技术杂志（英文版）

基　　金：partly supported by National Natural Science Foundation of China(31571199,81570046,91739109,81870045,and 81700054);the Shenzhen Municipal Basic Research Program JCYJ20150729104027220 and JCYJ20170818144127727;Interdisciplinary Innovation Team Project of Shenzhen University

摘　　要：Background: Tetracycline(Tet)-regulated expression system has become a widely applied tool to control gene activity. This study aimed to improve the Tet-on system with superior regulatory characteristics.Results: By comprehensively comparing factors of transactivators, Tet-responsive elements(TREs), orientations of induced expression cassette, and promoters controlling the transactivator, we developed an optimal Tet-on system with enhanced inducible efficiency and lower leakiness. With the system, we successfully performed effective inducible and reversible expression of micro RNA, and presented a more precise and easily reproducible fine-tuning for confirming the target of a mi RNA. Finally, the system was applied in CRISPR/Cas9-mediated knockout of nuclear factor of activated T cells-5(NFAT5), a protective transcription factor in cellular osmoregulation.Conclusions: This study established an improved Tet-on system for powerful and stringent gene regulation in functional genetic studies.Background: Tetracycline(Tet)-regulated expression system has become a widely applied tool to control gene activity. This study aimed to improve the Tet-on system with superior regulatory characteristics.Results: By comprehensively comparing factors of transactivators, Tet-responsive elements(TREs), orientations of induced expression cassette, and promoters controlling the transactivator, we developed an optimal Tet-on system with enhanced inducible efficiency and lower leakiness. With the system, we successfully performed effective inducible and reversible expression of micro RNA, and presented a more precise and easily reproducible fine-tuning for confirming the target of a mi RNA. Finally, the system was applied in CRISPR/Cas9-mediated knockout of nuclear factor of activated T cells-5(NFAT5), a protective transcription factor in cellular osmoregulation.Conclusions: This study established an improved Tet-on system for powerful and stringent gene regulation in functional genetic studies.

关 键 词：CRISPR/Cas9 DOXYCYCLINE microRNA NFAT5 TETRACYCLINE 

分 类 号：Q78[生物学—分子生物学]