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siRNA Produced by Recombinant Dicer Mediates Efficient Gene Silencing in Islet Cells
ROBERT HÄGERKVISTa,
DARIUSH MOKHTARIa,
JASON W. MYERSb,
ANDERS TENGHOLMa AND
NILS WELSHa
aDepartment of Medical Cell Biology, Uppsala University, Uppsala, Sweden
bDepartment of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California, USA
Address for correspondence: Nils Welsh, Department of Medical Cell Biology, Uppsala University Biomedicum, P.O. Box 571, S-751 23 Uppsala, Sweden. Voice: +46-18-471-4212; fax: +46-18-471-4059. nils.welsh{at}medcellbiol.uu.se
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.
Key Words: RNAi • siRNA • recombinant Dicer • gene silencing • c-Abl • islet cells • lipofection • d-siRNA • posttranscriptional gene silencing
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