Johnson Christiana J, Berglin Lennart, Chrenek Micah A, Redmond T M, Boatright Jeffrey H, Nickerson John M
Department of Ophthalmology, Emory University, Atlanta, GA 30322, USA.
Mol Vis. 2008;14:2211-26. Epub 2008 Dec 5.
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.
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.
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.
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.
我们的目标是改进并标准化小鼠眼视网膜下注射的操作程序。此外，我们希望优化用裸质粒对小鼠眼视网膜色素上皮（RPE）细胞进行电穿孔的条件。
将报告质粒DNA、纳米珠或缓冲液经视网膜下注射到小鼠眼中。使用钝针穿过角膜、瞳孔，进入玻璃体，直至其接触到神经视网膜。对针施加轻柔压力，使其刺穿视网膜并进入视网膜下间隙。经视网膜下注射液体，在小鼠眼底形成明显的大泡。通过视频记录监测和评估视网膜下注射手术及结果。Vidisic辅助检查大泡的眼底情况。通过几次短暂电脉冲在RPE细胞上形成孔隙，使液体中的质粒能够扩散通过。在荧光显微镜下检查质粒中所传递基因tdTomato的表达，以识别靶向细胞。电穿孔条件的变化范围为0至200V、5至10个脉冲、每个脉冲持续时间为0.1ms至100ms，角膜和巩膜上电极之间的间距为1.5至2mm。
手术成功的一个关键标志是小鼠眼中出现并持续存在三个大泡。来自两个不同系统的视频记录对此进行了说明。在手术结束时，将Vidisic应用于角膜可立即检查眼底。按照此方法很容易实现80%的成功率。一旦建立了成功的视网膜下注射技术，便对电穿孔条件进行评估。参数为50V、1ms脉冲持续时间、5 - 10个脉冲、间隔1s，电极间距1.5 - 2mm且阳极置于靠近大泡的巩膜上，可产生约30%效率的紧密RPE转染模式。
标准化的手术方法和手术成功的快速明确指标对于建立基于后续电穿孔的基因递送系统至关重要。随着手术得到更好的控制，电穿孔足以在眼球中特定位置转染大量RPE细胞。
