Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector.
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.
van de Wetering
Marc
M
Hubrecht Laboratory, Centre for Biomedical Genetics, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Oving
Irma
I
Muncan
Vanesa
V
Pon Fong
Menno Tjon
MT
Brantjes
Helen
H
van Leenen
Dik
D
Holstege
Frank C P
FC
Brummelkamp
Thijn R
TR
Agami
Reuven
R
Clevers
Hans
H
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
D003598:Cytoskeletal Proteins; D034741:RNA, Small Interfering; D015534:Trans-Activators; D051176:beta Catenin; D008156:Luciferases