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PMID: 12776180 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector.

EMBO reports ·Vol. 4 ·No. 6 ·2003-06-00 ·Pages 609-15

van de Wetering M, Oving I, Muncan V, Pon Fong MT, Brantjes H, van Leenen D, Holstege FC, Brummelkamp TR, Agami R, Clevers H

Abstract

We have designed a doxycycline-regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof-of-principle, we have targeted beta-catenin in colorectal cancer (CRC) cells. T-cell factor (TCF) target-gene expression is induced by accumulated beta-catenin, and is the main transforming event in these cells. We have shown previously that the disruption of beta-catenin/TCF4 activity in CRC cells by the overexpression of dominant-negative TCF induces rapid G1 arrest and differentiation. Stable integration of our inducible siRNA vector allowed the rapid production of siRNAs on doxycycline induction, followed by specific downregulation of beta-catenin. In these CRC cells, TCF reporter-gene activity was inhibited, and G1 arrest and differentiation occurred. The inhibition of two other genes using this vector system shows that it should be useful for the inducible knockdown of gene expression.

MeSH Terms
Base Sequence Blotting, Northern Blotting, Western Cell Differentiation Cell Division Colorectal Neoplasms/genetics,therapy Cytoskeletal Proteins/biosynthesis,genetics Down-Regulation Gene Expression Regulation, Neoplastic Genes, Reporter Genetic Techniques Genetic Vectors Luciferases/metabolism Molecular Sequence Data Oligonucleotide Array Sequence Analysis Promoter Regions, Genetic RNA, Small Interfering/genetics Trans-Activators/biosynthesis,genetics beta Catenin
Chemicals
Cytoskeletal Proteins RNA, Small Interfering Trans-Activators beta Catenin Luciferases
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
van de Wetering Marc
Hubrecht Laboratory, Centre for Biomedical Genetics, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Oving Irma
Muncan Vanesa
Pon Fong Menno Tjon
Brantjes Helen
van Leenen Dik
Holstege Frank C P
Brummelkamp Thijn R
Agami Reuven
Clevers Hans
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Article Info
Journal
EMBO reports
Abbr.
EMBO Rep
ISSN
1469-221X
DOI
10.1038/sj.embor.embor865
Published
2003-06-00
Pages
609-15
Language
English
Region
England
NLM ID
100963049
PMCID
PMC1319205
Subset
IM
External Links
PubMed source DOI: 10.1038/sj.embor.embor865 PMC Full Text View journal details
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