Bipartite vectors for co-expression of a growth factor cDNA and short hairpin RNA against an apoptotic gene.
BACKGROUND
Although human islet transplantation is a promising approach for treating type I 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 H1, 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.
Li
Feng
F
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN 38103-3308, USA.
Mahato
Ram I
RI
D017209:Apoptosis / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D042461:Vascular Endothelial Growth Factor A; D012313:RNA; D052247:Nitric Oxide Synthase Type II