Journal
JOURNAL OF GENE MEDICINE
Volume 11, Issue 9, Pages 764-771Publisher
WILEY
DOI: 10.1002/jgm.1357
Keywords
iNOS; islet transplantation; RNAi vectors; shRNA; VEGF
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Funding
- National institute of Health (NIH) [RO1 DK69968]
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Background Although human islet transplantation is a promising approach for treating type 1 diabetes, its success is limited as a result of the poor survival rate of transplanted islets. Expression of a growth factor gene to promote revascularization and silencing of pro-apoptotic genes before transplantation may improve the outcome of islet transplantation. Methods In the present study, we constructed bipartite plasmid vectors to co-express a vascular endothelial growth factor (VEGF) cDNA and short hairpin (sh)RNA targeting inducible NO synthase (iNOS) gene. First, we screened shRNA sequences against human iNOS by transfecting plasmids encoding shRNA targeting different start sites of human iNOS. Then, the effect of different promoters [such as H1, U6 and cytomegalovirus (CMV)] and micro RNA backbones on gene silencing was determined. Results No statistical difference in iNOS gene silencing was observed for the shRNA with HI, U6 and CMV promoters. In addition, a conventional shRNA showed better silencing of the iNOS gene compared to shRNA containing mir375 and mir30 backbones. A bipartite plasmid was also constructed with mir30-shRNA and a VEGF cDNA controlled by a single CMV promoter. This plasmid showed a better silencing effect compared to plasmid without VEGF cDNA. Conclusions In the present study, we have successfully constructed bipartite vectors co-expressing a VEGF cDNA and a shRNA against the iNOS gene. These vectors could be attractive candidates for improving the survival of transplanted islets. Copyright (C) 2009 John Wiley & Sons, Ltd.
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