Inducible, reversible, and stable RNA interference in mammalian cells.
RNA interference is a powerful genetic approach for efficiently silencing target genes. The existing method of gene suppression by the constitutive expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes but limits the analysis of genes essential for cell survival, cell cycle regulation, and cell development. We have developed an inducible U6 promoter for synthesis of shRNAs in both human and murine cells. Cells containing stably integrated shRNA expression constructs demonstrate stringent dosage- and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer ecdysone. Inducible suppression of human p53 in glioblastoma cells shows striking morphological changes and defects in cell cycle arrest caused by DNA damage, as expected. Remarkably, the inducibility is reversible after withdrawal of the inducer, as observed by reappearance of the protein and a restoration of the original cell phenotype. Inducible and reversible regulation of RNA interference has broad applications in the areas of mammalian genetics and molecular therapeutics.
Gupta
Sunita
S
Cancer Genome Research Center, Cold Spring Harbor Laboratory, 500 Sunnyside Boulevard, Woodbury, NY 11797, USA.
Schoer
Rebecca A
RA
Egan
James E
JE
Hannon
Gregory J
GJ
Mittal
Vivek
V
D000818:Animals; D002460:Cell Line; D004440:Ecdysone / Q000494:pharmacology; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D017570:MyoD Protein / Q000096:biosynthesis / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics
D017570:MyoD Protein; D016159:Tumor Suppressor Protein p53; D004440:Ecdysone; D012313:RNA