Optimization of in vitro transfection of plasmid delivered short hairpin RNA directed against VEGF

Abstract

Abstract: : Purpose:Previously, we have shown that small interfering RNA (siRNA) directed against vascular endothelial growth factor (VEGF) decreased both VEGF production and neovascularization in a murine model of laser–induced choroidal neovascularization. The purpose of this project is to assess optimal condition for delivering vector–based short hairpin (shRNA) against VEGF in vitro with the goal of testing its effectiveness in suppressing VEGF. Methods:Anti–VEGF shRNA, containing sequence demonstrated to be effective in VEGF suppression, was cloned into pU6 (Ambion) vector containing human U6 promoter. A reporter plasmid containing coral GFP sequence (Genscript) was used to assess transfection efficiency. Human embryonic kidney 293 cells and retinal pigment epithelial ARPE–19 cells were cotransfected with pU6.anti–VEGF shRNA and GFP reporter plasmid using either Lipofectamine (Invitrogen), Fugene6 (Roche), or siPORT–XP1 (Ambion). Transfection efficiency was assessed by fluorescent miscroscopy 24 hours post transfection. Optimal transfection condition was then used to cotransfect GFP reporter plasmid with either pU6 empty vector or pU6.anti–VEGF shRNA. VEGF mRNA and proteins levels were assessed using RT–PCR and ELISA at 24 hours, 48 hours, and 72 hours post transfection. Results:In both cell lines, highest transfection efficiency was achieved using Lipofectamine – 293 cells 50%, RPE cells 30%. In 293 cells, Fugene6 and siPORT–XP1 were comparable in transfection efficiency (both 30–35%). In RPE cells, higher transfection efficiency was achieved with Fugene6 than with siPORT–XP1, 15% and 10%, respectively. At 24 hours post transfection, modest decrease in VEGF protein level was observed in cells transfected with pU6.anti–VEGF shRNA, but not with cells transfected with pU6 empty vector. Conclusions:Lipofectamine achieves higher transfection efficiency than Fugene6 or siPORT–XP1 in 293 and RPE cells. Both Fugene6 and siPORT–XP1 can effectively deliver the plasmids used here into 293 cells. Transfection efficiency of RPE cells is low with both Fugene6 and siPORT–XP1. Anti–VEGF shRNA reduced VEGF protein level at 24 hours post transfection, demonstrating that plasmid mediated delivery of shRNA is a viable way of effecting RNA interference in vitro.

Keywords: cytokines/chemokines • gene/expression • growth factors/growth factor receptors