Inducible, reversible, and stable RNA interference in mammalian cells.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 101 ·No. 7 ·2004-02-17 ·Pages 1927-32

Gupta S, Schoer RA, Egan JE, Hannon GJ, Mittal V

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.

MeSH Terms

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 Transcriptional Activation/drug effects Tumor Suppressor Protein p53/biosynthesis,genetics

Chemicals

MyoD Protein Tumor Suppressor Protein p53 Ecdysone RNA

Authors & Affiliations

5 authors, click to expand affiliations / ORCID

Gupta Sunita

Cancer Genome Research Center, Cold Spring Harbor Laboratory, 500 Sunnyside Boulevard, Woodbury, NY 11797, USA.

Schoer Rebecca A

Egan James E

Hannon Gregory J

Mittal Vivek

References (36)

36 references, click to expand

An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus.
Cell. 2000 May 26;101(5):543-53 PMID: 10850496
A system for stable expression of short interfering RNAs in mammalian cells.
Science. 2002 Apr 19;296(5567):550-3 PMID: 11910072
Role for a bidentate ribonuclease in the initiation step of RNA interference.
Nature. 2001 Jan 18;409(6818):363-6 PMID: 11201747
Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action.
Apoptosis. 2000 Apr;5(2):107-14 PMID: 11232238
p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells.
Cancer Res. 2001 Mar 1;61(5):1957-63 PMID: 11280752
Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein.
Nucleic Acids Res. 2001 Apr 15;29(8):1672-82 PMID: 11292839
Regulation of the G2/M transition by p53.
Oncogene. 2001 Apr 5;20(15):1803-15 PMID: 11313928
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
Nature. 2001 May 24;411(6836):494-8 PMID: 11373684
Combined sequential treatment with interferon and dsRNA abrogates virus resistance to interferon action.
J Interferon Cytokine Res. 2001 Jun;21(6):423-9 PMID: 11440640
Argonaute2, a link between genetic and biochemical analyses of RNAi.
Science. 2001 Aug 10;293(5532):1146-50 PMID: 11498593
Effective expression of small interfering RNA in human cells.
Nat Biotechnol. 2002 May;20(5):505-8 PMID: 11981566
Analysis of gene function in somatic mammalian cells using small interfering RNAs.
Methods. 2002 Feb;26(2):199-213 PMID: 12054897
RNA interference: the new somatic cell genetics?
Cancer Cell. 2002 Jul;2(1):17-23 PMID: 12150821
Spatially discrete, light-driven protein expression.
Chem Biol. 2002 Dec;9(12):1347-53 PMID: 12498888
Germline transmission of RNAi in mice.
Nat Struct Biol. 2003 Feb;10(2):91-2 PMID: 12536207
An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo.
Nat Genet. 2003 Mar;33(3):396-400 PMID: 12567186
A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.
Nat Genet. 2003 Mar;33(3):401-6 PMID: 12590264
Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector.
EMBO Rep. 2003 Jun;4(6):609-15 PMID: 12776180
Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference.
J Virol. 2003 Aug;77(16):8957-61 PMID: 12885912
High-throughput selection of effective RNAi probes for gene silencing.
Genome Res. 2003 Oct;13(10):2333-40 PMID: 14525931
Analysis of the p53 gene and its expression in human glioblastoma cells.
Cancer Res. 1994 Feb 1;54(3):649-52 PMID: 8306326
Basal promoter elements as a selective determinant of transcriptional activator function.
Nature. 1995 Apr 13;374(6523):657-60 PMID: 7715708
Transcriptional activation by tetracyclines in mammalian cells.
Science. 1995 Jun 23;268(5218):1766-9 PMID: 7792603
Antisense p53 provokes changes in HeLa cell growth and morphology.
Eur J Cell Biol. 1995 Oct;68(2):122-32 PMID: 8575459
Ecdysone-inducible gene expression in mammalian cells and transgenic mice.
Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3346-51 PMID: 8622939
Staf, a promiscuous activator for enhanced transcription by RNA polymerases II and III.
EMBO J. 1997 Jan 2;16(1):173-81 PMID: 9009278
Requirement for p53 and p21 to sustain G2 arrest after DNA damage.
Science. 1998 Nov 20;282(5393):1497-501 PMID: 9822382
RNAi and double-strand RNA.
Genes Dev. 1999 Jan 15;13(2):139-41 PMID: 9925636
Combined radiation and p53 gene therapy of malignant glioma cells.
Cancer Gene Ther. 1999 Mar-Apr;6(2):155-62 PMID: 10195882
Regulation of PTEN transcription by p53.
Mol Cell. 2001 Aug;8(2):317-25 PMID: 11545734
Evaluation of the tetracycline- and ecdysone-inducible systems for expression of neurotransmitter receptors in mammalian cells.
Eur J Neurosci. 2001 Sep;14(6):968-76 PMID: 11595035
Design of a retroviral-mediated ecdysone-inducible system and its application to the expression profiling of the PTEN tumor suppressor.
Proc Natl Acad Sci U S A. 2001 Nov 6;98(23):13043-8 PMID: 11687610
PTEN protects p53 from Mdm2 and sensitizes cancer cells to chemotherapy.
J Biol Chem. 2002 Feb 15;277(7):5484-9 PMID: 11729185
Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.
Genes Dev. 2002 Apr 15;16(8):948-58 PMID: 11959843
A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.
Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5515-20 PMID: 11960009
Novel alpha 4-integrin ligands on an endothelial cell line.
Biochem Cell Biol. 2000;78(2):99-113 PMID: 10874471

Article Info

Journal

Proceedings of the National Academy of Sciences of the United States of America

Abbr.

Proc Natl Acad Sci U S A

ISSN

0027-8424

DOI

10.1073/pnas.0306111101

Published

2004-02-17

Epub

2004-00-04

Pages

1927-32

Language

English

Region

United States

NLM ID

7505876

PMCID

PMC357029

Subset

External Links

PubMed source DOI: 10.1073/pnas.0306111101 PMC Full Text View journal details

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Inducible, reversible, and stable RNA interference in mammalian cells.

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