An improved Tet-on system in microRNA overexpression and CRISPR/Cas9-mediated gene editing
An improved Tet-on system in microRNA overexpression and CRISPR/Cas9-mediated gene editing
复制标题
microRNA 过表达和 CRISPR/Cas9 介导的基因编辑中改进的 Tet-on 系统
DOI：
10.1186/s40104-019-0354-5
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发表时间：
2019-06-10
影响因子：
7
通讯作者：
Gou, Deming
中科院分区：
文献类型：
--
作者：
Kang, Kang;Huang, Lian;Gou, Deming
背景四环素（泰特）调控表达系统已成为一种广泛应用的基因调控工具。本研究旨在改善Tet-on系统的上级regulatory characteristics. ResultsThrough compared factors of transactivators，Tet-responsive elements（TREs），induced expression cassette，and promotors controlling the transactivator，we developed an optimal Tet-on system with enhanced inductive efficiency and lower leakiness.利用该系统，我们成功地进行了有效的可诱导和可逆的microRNA表达，并提出了一个更精确和更容易重现的微调，以确定一个miRNA的目标。最后，将该系统应用于CRISPR/Cas9介导的活化T细胞核因子-5（NFAT 5）基因敲除，NFAT 5是一种保护性转录因子，在细胞凋亡调控中发挥重要作用。
BackgroundTetracycline (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.ResultsBy 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 microRNA, and presented a more precise and easily reproducible fine-tuning for confirming the target of a miRNA. 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.ConclusionsThis study established an improved Tet-on system for powerful and stringent gene regulation in functional genetic studies.