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.
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
