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


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.
Wu
Ren-Huang
RH
Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College, Tainan 701, Taiwan, ROC.
Cheng
Tsung-Lin
TL
Lo
San-Ren
SR
Hsu
Hui-Chun
HC
Hung
Chuan-Fu
CF
Teng
Chiao-Fang
CF
Wu
Ming-Ping
MP
Tsai
Wen-Hui
WH
Chang
Wen-Tsan
WT
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D009875:Operator Regions, Genetic / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D034741:RNA, Small Interfering; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; D007544:Isopropyl Thiogalactoside; D008156:Luciferases