Figure 3

GluN2 and GFP transgene expression in vitro from optimized AAV vector plasmids. Representative ICC images from transfections with optimized AAV plasmids designed to express Flag-GluN2A/B or GFP transgenes utilizing one of the following promoters: 1.3αCaMKII, 0.4αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV, TRE3G, EFS, or RSV. Plasmids containing transgenes controlled by the neuron specific, 0.4αCaMKII, 1.3αCaMKII, 0.5synapsin and 1.1synapsin promoters were transfected into N2A cells. Plasmids containing transgenes controlled by CMV, TRE3G, EFS and RSV promoters were transfected into 293FT cells. TRE3G promoter containing plasmids were co-transfected with the pTet-Off plasmid. Twenty four hours after transfection, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Plasmids with neuron specific promoters were able to confer GFP expression but were not able to confer GluN2 expression. Plasmids containing transgenes controlled by CMV, TRE3G, EFS and RSV promoters were capable of conferring GluN2 and GFP expression. Untransfected cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. (Scale bar = 20 μm).