Probing tumor phenotypes using stable and regulated synthetic microRNA precursors.
RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA-based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA-based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.
Dickins
Ross A
RA
Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Hemann
Michael T
MT
Zilfou
Jack T
JT
Simpson
David R
DR
Ibarra
Ingrid
I
Hannon
Gregory J
GJ
Lowe
Scott W
SW
D000818:Animals; D049109:Cell Proliferation; D016922:Cellular Senescence; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D009369:Neoplasms / Q000378:metabolism*; D009857:Oncogenes; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D012319:RNA Polymerase II / Q000235:genetics*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D013752:Tetracycline / Q000494:pharmacology; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D035683:MicroRNAs; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D012319:RNA Polymerase II; D013752:Tetracycline