Quantitative analysis of conditional gene inactivation using rationally designed, tetracycline-controlled miRNAs.
The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo.
Berger
Stefan M
SM
Central Institute of Mental Health, Department of Molecular Biology, D-68159 Mannheim, Germany. stefan.berger@zi-mannheim.de
Pesold
Brigitte
B
Reber
Simone
S
Schönig
Kai
K
Berger
Annette J
AJ
Weidenfeld
Ina
I
Miao
Jun
J
Berger
Martin R
MR
Gruss
Oliver J
OJ
Bartsch
Dusan
D
D000818:Animals; D002460:Cell Line; D004318:Doxycycline / Q000494:pharmacology; D055785:Gene Knockdown Techniques / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008819:Mice, Nude; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012319:RNA Polymerase II / Q000378:metabolism; D012320:RNA Polymerase III / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D035683:MicroRNAs; D012333:RNA, Messenger; D012319:RNA Polymerase II; D012320:RNA Polymerase III; D004318:Doxycycline