Figure 6. Expression of a reporter gene in RPE cells; subretinal injection and electroporation under optimized conditions. Red fluorescence of tdTomato expression was observed following subretinal injection and electroporation under optimized conditions; individual RPE cells were resolved. The cells include neighbors with and without tdTomato fluorescence (red). About 30% of the cells in electroporated area were positive for tdTomato gene expression. This field represents the RPE cells located directly over the anode. TdTomato-expressing cells exhibited polygonal shapes characteristic of normal RPE cells. The electroporation conditions employed were 50 V, 2 mm gap between electrodes, 1 ms pulse duration, and 10 pulses at 1 s intervals. A: In this schematic of the eye, the bleb is in green and the positive electrode in red. The black electrode represents the negative electrode. B: Presented is a wholemount of the eye after bleb formation but without voltage applied to the electrodes. The edges of the flatmounts are outlined in white and form a floret shape. The center of the floret corresponds to the retina while the outer half of the “petals” correspond to the cornea. C: Shown is a wholemount of the eye following subretinal injection and electroporation under the optimized condition. A focused patch of red fluorescent cells is evident near the center of the floret. Each dot represents a separate RPE cell. This region of the retina corresponds to the bleb and the location of the anode. D: High magnification of the fluorescent region shows about 30% of the RPE cells manifesting tdTomato fluorescence. E: Close-up of a cluster of tdTomato fluorescence in cells reveals a cobblestone or polygonal shape. F: Shown is a close-up of a single binucleate RPE cell. In B and C, images are about 9 mm across. The scale bar in D represents 50 μ. The scale bars in E and F represent 25 μm.