Figure 4.

Gene transfer to primary motor neurons. EGFP expression in rat and mouse embryonic CNS primary motor neuron cultures infected at MOI 25 and 50 with αp75^NTR-192, αThy1.1, αCAR, and 5pl control pseudotyped HIV-1 lentiviral vectors. Cultures were fixed 4 days after transduction and stained with antibodies to ChAT (Alexa594 secondary antibody staining motor neuron) shown in red, and GFAP (Alexa647 secondary antibody staining astrocytes) shown in blue. Graph shows the efficiency of gene transfer of pseudotyped vectors. Targeted vectors preferentially transduce MNs as compared to 5pl control, which showed no specificity between MNs and astrocytes. The ratio EGFP⁺/ChAT⁺ relative to total ChAT⁺ cell number is plotted in black bars. The ratio of EGFP⁺/GFAP⁺ cell number relative to GFAP⁺ cell number is plotted on white bars. The values are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.0001. Bars = 50 μm. Each type of virus was assessed with the same culture batch in order to compare the differences in transduction levels. 5pl vector was used to control for non-targeted transduction. For each well 10 randomly chosen square fields with sides measuring 125 µm were counted with a computer-assisted imaging program (Rasband, W.S., ImageJ, US National Institutes of Health, USA. http://imagej.nih.gov/ij/). Specific MN transduction was assessed as the percentage of double positive EGFP/ChAT cells on the total of ChAT +ve cells. Specific astrocytic transduction efficiency was assessed as percentage of double positive EGFP/GFAP on the total of GFAP +ve cells. CAR, coxsackievirus and adenovirus receptor; CMV, cytomegalovirus; CNS, central nervous system; EGFP, enhanced GFP; MN, motor neuron.