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

Ren Huang Wu, Tsung Lin Cheng, San Ren Lo, Hui Chun Hsu, Chuan Fu Hung, Chiao Fang Teng, Ming Ping Wu, Wen Hui Tsai, Wen-Tsan Chang

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)620-634
Number of pages15
JournalJournal of Gene Medicine
Volume9
Issue number7
DOIs
Publication statusPublished - 2007 Jul 1

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Drug Discovery
  • Genetics(clinical)

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