Figure 9. Subretinal injection followed by electroporation, and analysis of flatmounts. Four different electroporation programs were employed to compare qualitatively their efficacy. These conditions resembled the voltage, pulse duration, and train of four different published techniques to electroporate the retina and RPE sheet in other species and at differing stages of development. A: The conditions were 30 V, 8 pulses, 50 ms duration per pulse, and an interval of 0.1 s between pulses [61]. B: The conditions were 38 V, 5 pulses, 50 ms duration, and 1 s between pulses [62]. C: The conditions were 50 V, 5 pulses, 50 ms pulse duration, and 0.95 s between pulses [63]. D: The conditions included two voltage steps per cycle. The first voltage was 150 V for 0.25 ms and then 5 V for 5 ms. This combination of voltages was applied 5 times [47]. In all four images, cells were transfected and expressed tdTomato in the flatmount. In most cases, tdTomato accumulated in cells of the cornea, ciliary body, and to a somewhat lesser extent in the RPE. In A, B, and C, there is evidence of red fluorescence in the corneal endothelium. Some of the expression was off center from the position of the anode. In one case there was some evidence of burn damage. The scale bar represents 1 mm.