Abstract
Abstract: :
Purpose: To demonstrate in vivo non–viral gene transfer by electroporation into adult C57Bl6 mouse retina. Methods: Nine week or older C57Bl6 mice were properly anesthetized, and injected with 1.5 ml of plasmid carrying CMV–driven Enhanced Green Fluorescent Protein (EGFP) into the subretinal space. Eyes were then electroporated with micropaddles or tweezer electrodes on a BTX ECM 830 electroporator (San Diego, CA). Electroporation parameters ranged from 5 to 80 volts, 5 to 10 pulses, 50 to 100 millisecond pulse intervals and 1.39 to 10.0 mg/ml DNA concentration. Eyes were dilated on day 3 and/or 5 and fundi examined for fluorescence. Animals were sacrificed; eyes were removed and fixed into 4% paraformaldehyde in phosphate buffered saline. Eyes were then sectioned or retinas dissected for flat mounts and inspected on a fluorescent microscope for the presence of EGFP. Results: EGFP–specific fluorescence was exhibited in retinal bipolar, ganglion, pigmented epithelial cells, as well as some photoreceptor outer segments. Higher voltages and plasmid concentrations appeared to improve efficiency. Conclusions: Successful non–viral in vivo gene transfer into adult C57Bl6 mouse retinas can be accomplished by electroporation. Supported by Foundation Fighting Blindness, Research to Prevent Blindness, NIH grant EY10820
Keywords: gene transfer/gene therapy