Conditional gene silencing in mammalian cells mediated by a stress-inducible promoter.
MicroRNAs (miRNAs) are endogenous non-coding small RNAs, which negatively regulate gene expression in a sequence-specific manner through the RNA interference (RNAi) pathway. Here we describe a new miRNA-based conditional RNAi expression system that relies on cellular stress-response mechanisms in mammalian cells. In our constructs, expression of miRNA mimics is tightly controlled by a heat shock-inducible promoter. This system is highly effective in silencing permanently or conditionally expressed luciferase. The stress inducible vectors also effectively deplete co-expressed pro-apoptotic protein CHOP with heat shock. Furthermore, we demonstrate cloning of a protein-coding sequence between the stress-inducible promoter and the miRNA expression cassette allows simultaneous silencing of a target gene and activation of synthesis of a protein of choice in response to stress stimulation. This new conditional gene silencing approach could be an invaluable tool for various areas of basic and applied research and for therapeutic intervention.
Yang
Wei
W
Department of Anesthesiology, Duke University Medical Center, Durham, NC, USA.
Paschen
Wulf
W
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D020868:Gene Silencing; D017930:Genes, Reporter; D005822:Genetic Vectors; D006358:Hot Temperature; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000096:biosynthesis*
D034741:RNA, Small Interfering; D008156:Luciferases