Specific and potent RNA interference in terminally differentiated myotubes.
Double-stranded RNA (dsRNA) interference is a potent mechanism for sequence-specific silencing of gene expression and represents an invaluable approach for investigating gene function in normal and diseased states as well as for drug target validation. Here, we report that skeletal muscle myoblasts and terminally differentiated myotubes are susceptible to RNA interference. We employed an approach in which dsRNA is generated by cellular transcription from plasmids containing long (1 kilobase) inverted DNA repeats of the target gene rather than using dsRNA synthesized in vitro. We show that gene silencing by this method is effective for endogenously expressed genes as well as for exogenous reporter genes. An analysis of the expression of several endogenous genes and exogenous reporters demonstrates that the silencing effect is specific for the target gene containing sequences within the inverted repeat. Our method eliminates the need to chemically synthesize dsRNA and is not accompanied by global repression of gene expression. Furthermore, we show for the first time that sequence-specific dsRNA-mediated gene silencing is possible in differentiated, multinucleated skeletal muscle myotubes. These findings provide an important molecular tool for the examination of protein function in terminally differentiated muscle cells and provide alternative approaches for generating disease models.
Yi
Christopher E
CE
Department of Physiological Science, University of California, Los Angeles, 90025, USA.
Bekker
Janine M
JM
Miller
Gaynor
G
Hill
Kent L
KL
Crosbie
Rachelle H
RH
D000818:Animals; D002460:Cell Line; D020868:Gene Silencing; D051379:Mice; D032448:Myoblasts, Skeletal / Q000378:metabolism*; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D014162:Transfection
D012330:RNA, Double-Stranded