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Nucleic Acids Research 2005 33(15):e131; doi:10.1093/nar/gni130
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Published online 19 August 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

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

Amarsanaa Jazag1, Fumihiko Kanai1,2,*, Hideaki Ijichi1, Keisuke Tateishi1, Tsuneo Ikenoue1, Yasuo Tanaka1, Miki Ohta1, Jun Imamura1, Bayasi Guleng1, Yoshinari Asaoka1, Takao Kawabe1, Makoto Miyagishi3,4, Kazunari Taira3,4 and Masao Omata1

1Department of Gastroenterology, Graduate School of Medicine, University of Tokyo Tokyo 113-8655, Japan 2Clinical Research Center, University of Tokyo Hospital Tokyo 113-8655, Japan 3Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo Tokyo 113-8656, Japan 4Gene Function Research Center, National Institute of Advanced Industrial Science and Technology Ibaraki 305-8562, Japan

*To whom correspondence should be addressed. Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, Tel: +81 3 5800 9747; Fax: +81 3 5800 8775; Email: kanaif-int{at}h.u-tokyo.ac.jp

Received April 5, 2005. Revised July 10, 2005. Accepted August 7, 2005.

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 ß (TGF-ß) pathway-related Smads—Smad2, Smad3 and Smad4—at the cellular level. We observed distinct phenotypic changes in TGF-ß-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.


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