siRNA produced by recombinant dicer mediates efficient gene silencing in islet cells.
RNA interference (RNAi) is emerging as a powerful and convenient tool for studying gene function and genetic variation. RNAi is mediated by 21- to 23-nucleotide-long, small interfering RNAs (siRNA) produced from larger double-stranded RNAs in vivo by the RNase III family enzyme Dicer. To overcome the problems associated with the use of predesigned synthetic siRNA molecules, a novel method utilizing the in vitro activity of recombinant Dicer has been developed recently. In nonislet cells, it has been demonstrated that a pool of siRNA, generated by Dicer from in vitro transcribed dsRNA (d-siRNA), mediates convenient, efficient, and reproducible gene silencing in various cell types. The aim of this study was to evaluate the ability of d-siRNA to silence endogenous gene expression in pancreatic islet cells. We observed that liposomal transfection mediates efficient transport of siRNA in up to 90% of dispersed islet cells and that d-siRNA mediates almost complete and nontoxic silencing of an endogenous mRNA, the messenger coding for the nonreceptor tyrosine kinase c-Abl. The approach described here using d-siRNA provides an important tool for elucidating gene function in further studies of pancreatic islets and diabetes pathophysiology.
Hägerkvist
Robert
R
Department of Medical Cell Biology, Uppsala University Biomedicum, Sweden.
Mokhtari
Dariush
D
Myers
Jason W
JW
Tengholm
Anders
A
Welsh
Nils
N
D000818:Animals; D002460:Cell Line; D020868:Gene Silencing / Q000502:physiology*; D006801:Humans; D007515:Islets of Langerhans / Q000378:metabolism*; D008297:Male; D051379:Mice; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D043244:Ribonuclease III / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*
D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D043244:Ribonuclease III