title: Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector.
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.
authors.affiliation: Hubrecht Laboratory, Centre for Biomedical Genetics, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
authors.forename: Marc
authors.initials: M
authors.lastname: van de Wetering
authors.forename: Irma
authors.initials: I
authors.lastname: Oving
authors.forename: Vanesa
authors.initials: V
authors.lastname: Muncan
authors.forename: Menno Tjon
authors.initials: MT
authors.lastname: Pon Fong
authors.forename: Helen
authors.initials: H
authors.lastname: Brantjes
authors.forename: Dik
authors.initials: D
authors.lastname: van Leenen
authors.forename: Frank C P
authors.initials: FC
authors.lastname: Holstege
authors.forename: Thijn R
authors.initials: TR
authors.lastname: Brummelkamp
authors.forename: Reuven
authors.initials: R
authors.lastname: Agami
authors.forename: Hans
authors.initials: H
authors.lastname: Clevers
mesh_terms: D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation; D002455:Cell Division; D015179:Colorectal Neoplasms / Q000235:genetics / Q000628:therapy*; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000235:genetics; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics*; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D051176:beta Catenin
chemical_list: D003598:Cytoskeletal Proteins; D034741:RNA, Small Interfering; D015534:Trans-Activators; D051176:beta Catenin; D008156:Luciferases