Wu Ren-Huang, Cheng Tsung-Lin, Lo San-Ren, Hsu Hui-Chun, Hung Chuan-Fu, Teng Chiao-Fang, Wu Ming-Ping, Tsai Wen-Hui, Chang Wen-Tsan
Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College, Tainan 701, Taiwan, ROC.
J Gene Med. 2007 Jul;9(7):620-34. doi: 10.1002/jgm.1048.
RNA interference (RNAi) is a powerful and widely used gene silencing strategy for studying gene function in mammalian cells. Transient or constitutive expression of either small interfering RNA (siRNA) or short hairpin RNA (shRNA) results in temporal or persistent inhibition of gene expression, respectively. A tightly regulated and reversibly inducible RNAi-mediated gene silencing approach could conditionally control gene expression in a temporal or spatial manner that provides an extremely useful tool for studying gene function involved in cell growth, survival and development.
In this study, we have developed a lactose analog isopropyl thiogalactose (IPTG)-responsive lac repressor-operator-controlled RNA polymerase III (Pol III)-dependent human RNase P RNA (H1) promoter-driven inducible siRNA expression system. To demonstrate its tight regulation, efficient induction and reversible inhibition, we have used this system to conditionally control the expression of firefly luciferase and human tumor suppressor protein p53 in both transient transfection cells and established stable clones.
The results showed that this inducible siRNA expression system could efficiently induce conditional inhibition of these two genes in a dose- and time-dependent manner by administration of the inducing agent IPTG as well as being fully reverted after withdrawal of IPTG. In particular, this system could conditionally inhibit the expression of both the genes in not only established stable clones but also transient transfection cells, which should greatly increase its usefulness and convenience.
The results presented in this study clearly indicate that this inducible siRNA expression system could efficiently, conditionally and reversibly inhibit gene expression with only very low or undetectable background silencing effects under non-inducing condition. Thus, this inducible siRNA expression system provides an ideal genetic switcher allowing the inducible and reversible control of specific gene activity in mammalian cells.
RNA干扰（RNAi）是一种强大且广泛应用于研究哺乳动物细胞基因功能的基因沉默策略。小干扰RNA（siRNA）或短发夹RNA（shRNA）的瞬时或组成型表达分别导致基因表达的瞬时或持续抑制。一种严格调控且可逆诱导的RNAi介导的基因沉默方法能够以时间或空间方式有条件地控制基因表达，为研究参与细胞生长、存活和发育的基因功能提供了极为有用的工具。
在本研究中，我们开发了一种乳糖类似物异丙基硫代半乳糖苷（IPTG）响应的乳糖阻遏物 - 操纵子控制的RNA聚合酶III（Pol III）依赖性人核糖核酸酶P RNA（H1）启动子驱动的诱导型siRNA表达系统。为证明其严格调控、高效诱导和可逆抑制特性，我们使用该系统在瞬时转染细胞和已建立的稳定克隆中对萤火虫荧光素酶和人肿瘤抑制蛋白p53的表达进行有条件控制。
结果表明，通过给予诱导剂IPTG，该诱导型siRNA表达系统能够以剂量和时间依赖性方式有效诱导这两个基因的条件性抑制，并且在撤去IPTG后完全恢复。特别地，该系统不仅能够在已建立的稳定克隆中，还能在瞬时转染细胞中有条件地抑制这两个基因的表达，这将大大提高其实用性和便利性。
本研究结果清楚地表明，该诱导型siRNA表达系统能够在非诱导条件下以非常低或不可检测的背景沉默效应高效、有条件且可逆地抑制基因表达。因此，该诱导型siRNA表达系统提供了一种理想的基因开关，可对哺乳动物细胞中的特定基因活性进行诱导和可逆控制。
