A tightly regulated and reversibly inducible siRNA expression system for conditional RNAi-mediated gene silencing in mammalian cells

Autor: Tsung Lin Cheng, Ming Ping Wu, Wen Tsan Chang, Wen Hui Tsai, Hui Chun Hsu, Chuan Fu Hung, Chiao Fang Teng, San Ren Lo, Ren Huang Wu
Rok vydání: 2007
Předmět:
Zdroj: The Journal of Gene Medicine. 9:620-634
ISSN: 1521-2254
1099-498X
DOI: 10.1002/jgm.1048
Popis: Background 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. Material and methods 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. Results 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. Conclusions 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. Copyright © 2007 John Wiley & Sons, Ltd.
Databáze: OpenAIRE
Externí odkaz: