Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs.
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.
Strat
Aurel
A
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center Shreveport, LA, USA.
Gao
Lu
L
Utsuki
Tada
T
Cheng
Bing
B
Nuthalapaty
Sam
S
Mathis
J Mike
JM
Odaka
Yoshinobu
Y
Giordano
Tony
T
D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement; D017930:Genes, Reporter; D006801:Humans; D016899:Interferon-beta / Q000096:biosynthesis; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D010957:Plasmids; D019859:Proto-Oncogene Proteins c-met / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*
D012330:RNA, Double-Stranded; D016899:Interferon-beta; D008156:Luciferases; D019859:Proto-Oncogene Proteins c-met