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Author Notes:

Correspondence to: John M. Nickerson, Ph.D., Department of Ophthalmology, Emory University, Room B5602, 1365B Clifton Road, NE, Atlanta, GA, 30322; Phone: (404) 778-4411; FAX: (404) 778-2231; email: litjn@emory.edu

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Research Funding:

This work was supported by the National Eye Institute (R01EY016470, R01EY014026, P30EY006360, R24EY017045, T32EY007092), an unrestricted grant to the Department of Ophthalmology at Emory University from Research to Prevent Blindness, Inc., the Foundation Fighting Blindness, Fight for Sight, and the Intramural Research Program of the National Eye Institute.

Technical Brief: Subretinal injection and electroporation into adult mouse eyes

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Journal Title:

Molecular Vision

Volume:

Volume 2008, Number 14

Publisher:

, Pages 2211-2226

Type of Work:

Article | Final Publisher PDF

Abstract:

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. 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. VidisicR 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. 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 VidisicR 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.

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This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported License ( http://creativecommons.org/licenses/by-nc-nd/3.0/), which permits distribution, public display, and publicly performance, making multiple copies, provided the original work is properly cited. This license requires copyright and license notices be kept intact, credit be given to copyright holder and/or author. This license prohibits exercising rights for commercial purposes.

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