Published online 11 August 2006
Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs
1 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center Shreveport, LA, USA 2 Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center Shreveport, LA, USA 3 Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center Shreveport, LA, USA
*To whom correspondence should be addressed. Tel: +1 318 675 7791; Fax: +1 318 675 5180; Email: agiord{at}lsuhsc.edu
Received March 8, 2006. Revised May 27, 2006. Accepted July 10, 2006.
A number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand fundamental cellular mechanisms, they require information and knowledge of existing sequences since the RNAi sequences are generated synthetically. An alternative strategy would be to create an RNAi library from cDNA. Unfortunately, the complexity of such a library of siRNAs would make screening difficult. To reduce the complexity, longer dsRNAs could be used; however, concerns of induction of the interferon response and off-target effects of long dsRNAs have prevented their use. As a first step in creating such libraries, long dsRNA was expressed in mammalian cells. The 250 nt dsRNAs were capable of efficiently silencing a luciferase reporter gene that was stably transfected in MDA-MB-231 cells without inducing the interferon response or off-target effects any more than reported for siRNAs. In addition, a long dsRNA expressed in the same cell line was capable of silencing endogenous c-met expression and inhibited cell migration, whereas the dsRNA against luciferase had no effect on c-met or cell migration. The studies suggest that large dsRNA libraries are feasible and that functional selection of genes will be possible.
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