Conclusions
A standardized surgical method and a fast distinct indicator of successful surgery were essential to establishing a gene delivery system based on subsequent electroporation. With the surgery better controlled, electroporation was adequate to transfect a substantial number of RPE cells in a defined position in the globe.
Methods
Mouse eyes were injected subretinally with reporter plasmid DNA, nanobeads, or buffer. A blunt needle was introduced across the cornea, through the pupil, into the vitreous, until it made contact with the neural retina. Gentle pressure was applied to the needle, forcing it to puncture the retina and enter the subretinal space. Fluid was injected subretinally, raising large blebs evident on the mouse fundus. Subretinal injection surgery and outcomes were monitored and evaluated by video recording. Vidisic aided in fundus examination of the blebs. Pores in RPE cells, across which the plasmid in the fluid could diffuse, were created by several short electrical bursts. Expression of the delivered gene, tdTomato, in the plasmid was examined under fluorescence microscopy to identify targeted cells. Electroporation conditions were varied from 0 to 200 V, 5 to 10 pulses, 0.1 ms to 100 ms duration of each pulse, and a space of 1.5 to 2 mm between electrodes on the cornea and sclera.
Purpose
Our goal was to improve and standardize the procedure for subretinal injection of mouse eyes. Also, we wished to optimize conditions for electroporation of retinal pigment epithelium (RPE) cells in the mouse eye with naked plasmids.
Results
A critical sign of surgical success was the appearance and persistence of three large blebs in the mouse eye. This was illustrated by video recordings from two different systems. Application of Vidisic to the cornea made immediate examination of the fundus possible at the end of the surgery. An 80% success rate was readily achieved by following this method. Once a successful subretinal injection technique was established, electroporation conditions were evaluated. Parameters of 50 V, 1 ms pulse duration, 5-10 pulses, 1 s apart and electrodes spaced 1.5-2 mm apart with the anode placed on the sclera in the vicinity of the blebs produced a tight pattern of RPE transfection at approximately 30% efficiency. Conclusions: A standardized surgical method and a fast distinct indicator of successful surgery were essential to establishing a gene delivery system based on subsequent electroporation. With the surgery better controlled, electroporation was adequate to transfect a substantial number of RPE cells in a defined position in the globe.