Figure 8

GluN2 and GFP transgene expression in vitro from lentiviral vector plasmids. Representative ICC images from transfections with lentiviral 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, or TRE3G. 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 and TRE3G 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. All lentiviral plasmids were capable of conferring GluN2 or GFP expression as intended. Untransfected cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. (Scale bar = 20 μm).