siRNA Produced by Recombinant Dicer Mediates Efficient Gene Silencing in Islet Cells

Abstract

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.

Improved Vascular Engraftment and Graft Function After Inhibition of the Angiostatic Factor Thrombospondin-1 in Mouse Pancreatic Islets

“…Each fraction was placed into a nonadhesive culture dish followed by transfection with 100 nmol/l scramble or an equal mixture of four different TSP-1 siRNA sequences (Table 1) using 10 g/600 l Lipofectamine (Invitrogen, Lidingö , Sweden) in 600 l RPMI 1640. Using this Lipofectamine protocol, we have previously observed uptake of siRNA in Ͼ90% of all islet cells (26). After 3 h, the transfection medium was replaced with culture medium.…”

“…TSP-1 siRNA transfection of islet cells. siRNA transfection was performed as previously described (26). Freshly isolated islets were dispersed at 37°C into single cells by addition of 5 mg/ml trypsin (Sigma-Aldrich) for Ͻ5 min.…”

Silencing Mitogen-activated Protein 4 Kinase 4 (MAP4K4) Protects Beta Cells from Tumor Necrosis Factor-α-induced Decrease of IRS-2 and Inhibition of Glucose-stimulated Insulin Secretion

“…RNA Interference (RNAi)-mediated Knockdown of MAP4K4-MAP4K4 expression was decreased by siRNA transfection as described (28). In brief liposome-siRNA (Lipofectamine 2000 and siRNA (100 nM)) was prepared in 200 l of opti-MEM as described by the suppliers.…”

MAPK Kinase Kinase-1 Is Essential for Cytokine-Induced c-Jun NH2-Terminal Kinase and Nuclear Factor-κB Activation in Human Pancreatic Islet Cells

“…This raises the possibility that NF-B could exert an anti-apoptotic effect in human islet cells, as it does in many other cell types (17), and that the pro-apoptotic effects of JNK activation are in part neutralized by NF-B, thereby explaining the weaker apoptotic response of human ␤-cells compared with rodent ␤-cells. Unfortunately, the effect of siRNA in islet cells is only transient (1-3 days) (26), and the toxic effect of cytokines requires Ͼ7 days of cytokine exposure (27). Thus, a different approach than that presently used would have been necessary to clarify this particular issue.…”

“…MEKK-1 overexpression in primary islet cells is problematic because the liposome-mediated transfection of primary islet cells is less efficient than that of ␤-cell lines (25). However, in a previous study, we achieved successful knockdown of target genes in dispersed primary islet cells by using liposomal reagents and d-siRNA (26). The dsiRNA technique uses the in vitro activity of recombinant dicer to yield a pool of d-siRNA, which seems to be more efficient than synthetic siRNA molecules (22).…”