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

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 i...

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Bibliographic Details
Published in:Nucleic acids research 2005-01, Vol.33 (15), p.e131-e131
Main Authors: Jazag, Amarsanaa, Kanai, Fumihiko, Ijichi, Hideaki, Tateishi, Keisuke, Ikenoue, Tsuneo, Tanaka, Yasuo, Ohta, Miki, Imamura, Jun, Guleng, Bayasi, Asaoka, Yoshinari, Kawabe, Takao, Miyagishi, Makoto, Taira, Kazunari, Omata, Masao
Format: Article
Language:English
Subjects:
Cell Line
DNA-Binding Proteins - antagonists & inhibitors
DNA-Binding Proteins - genetics
Gene Expression
Genetic Vectors
Humans
Methods Online
Phenotype
Protein-Serine-Threonine Kinases
Receptors, Transforming Growth Factor beta - antagonists & inhibitors
Receptors, Transforming Growth Factor beta - genetics
RNA Interference
RNA, Small Interfering - biosynthesis
RNA, Small Interfering - genetics
Signal Transduction
Smad2 Protein
Smad3 Protein
Smad4 Protein
Trans-Activators - antagonists & inhibitors
Trans-Activators - genetics
Transforming Growth Factor beta - antagonists & inhibitors
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Summary: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 β (TGF-β) pathway-related Smads—Smad2, Smad3 and Smad4—at the cellular level. We observed distinct phenotypic changes in TGF-β-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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gni130