Single small-interfering RNA expression vector for silencing multiple transforming growth factor-beta pathway components

Amarsanaa Jazag; Fumihiko Kanai; Hideaki Ijichi; Keisuke Tateishi; Tsuneo Ikenoue; Yasuo Tanaka; Miki Ohta; Jun Imamura; Bayasi Guleng; Yoshinari Asaoka; Takao Kawabe; Makoto Miyagishi; Kazunari Taira; Masao Omata

doi:10.1093/nar/gni130

Single small-interfering RNA expression vector for silencing multiple transforming growth factor-beta pathway components

Nucleic Acids Res. 2005 Aug 19;33(15):e131. doi: 10.1093/nar/gni130.

Authors

Amarsanaa Jazag¹, Fumihiko Kanai, Hideaki Ijichi, Keisuke Tateishi, Tsuneo Ikenoue, Yasuo Tanaka, Miki Ohta, Jun Imamura, Bayasi Guleng, Yoshinari Asaoka, Takao Kawabe, Makoto Miyagishi, Kazunari Taira, Masao Omata

Affiliation

¹ Department of Gastroenterology, Graduate School of Medicine, University of Tokyo Tokyo 113-8655, Japan.

Abstract

Although RNA interference (RNAi) is a popular technique, no method for simultaneous silencing of multiple targets by small-hairpin RNA (shRNA)-expressing RNAi vectors has yet been established. Although gene silencing can be achieved by synthetic small-interfering RNA (siRNA) duplexes, the approach is transient and largely dependent on the transfection efficiency of the host cell. We offer a solution: a simple, restriction enzyme-generated stable RNAi technique that can efficiently silence multiple targets with a single RNAi vector and a single selection marker. In this study, we succeeded in simultaneous stable knockdown of transforming growth factor beta (TGF-beta) pathway-related Smads--Smad2, Smad3 and Smad4--at the cellular level. We observed distinct phenotypic changes in TGF-beta-dependent cellular functions such as invasion, wound healing and apoptosis. This method is best suited for an analysis of complex signal transduction pathways in which silencing of a single gene cannot account for the whole process.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Line
DNA-Binding Proteins / antagonists & inhibitors
DNA-Binding Proteins / genetics
Gene Expression
Genetic Vectors*
Humans
Phenotype
Protein Serine-Threonine Kinases
RNA Interference*
RNA, Small Interfering / biosynthesis
RNA, Small Interfering / genetics*
Receptor, Transforming Growth Factor-beta Type II
Receptors, Transforming Growth Factor beta / antagonists & inhibitors
Receptors, Transforming Growth Factor beta / genetics
Signal Transduction*
Smad2 Protein
Smad3 Protein
Smad4 Protein
Trans-Activators / antagonists & inhibitors
Trans-Activators / genetics
Transforming Growth Factor beta / antagonists & inhibitors*

Substances

DNA-Binding Proteins
RNA, Small Interfering
Receptors, Transforming Growth Factor beta
SMAD2 protein, human
SMAD3 protein, human
SMAD4 protein, human
Smad2 Protein
Smad3 Protein
Smad4 Protein
Trans-Activators
Transforming Growth Factor beta
Protein Serine-Threonine Kinases
Receptor, Transforming Growth Factor-beta Type II