Inducible, reversible, and stable RNA interference in mammalian cells

Gupta, S., Schoer, R. A., Egan, J. E., Hannon, G. J., Mittal, V. (February 2004) Inducible, reversible, and stable RNA interference in mammalian cells. Proc Natl Acad Sci U S A, 101 (7). pp. 1927-32. ISSN 0027-8424 (Print)

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URL: https://www.ncbi.nlm.nih.gov/pubmed/14762164
DOI: 10.1073/pnas.0306111101

Abstract

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.

Item Type: Paper
Uncontrolled Keywords: Animals Cell Line Ecdysone pharmacology Genetic Techniques Genetic Vectors genetics Humans Mice MyoD Protein biosynthesis genetics RNA genetics metabolism RNA Interference Reproducibility of Results Retroviridae genetics Trans-Activation (Genetics) drug effects Tumor Suppressor Protein p53 biosynthesis genetics
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNAi
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > shRNA
CSHL Authors:
Communities: CSHL labs > Mittal lab
CSHL labs > Hannon lab
Depositing User: CSHL Librarian
Date: 17 February 2004
Date Deposited: 09 Feb 2012 15:24
Last Modified: 09 Nov 2017 17:13
PMCID: PMC357029
Related URLs:
URI: http://repository.cshl.edu/id/eprint/22378

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