Nickerson John M, Goodman Penny, Chrenek Micah A, Bernal Christiana J, Berglin Lennart, Redmond T Michael, Boatright Jeffrey H
Department of Ophthalmology, Emory University, Atlanta, GA, USA.
Methods Mol Biol. 2012;884:53-69. doi: 10.1007/978-1-61779-848-1_4.
Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, Müller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumulating to high concentrations because there is no dilution due to transport processes or diffusion. Dilution in the interphotoreceptor space (IPS) is minimal because the IPS volume is only 10-20 μl in the human eye and less than 1 μl in the mouse eye. For gene delivery purposes, we wished to transfect the cells adjacent to the IPS in adult mouse eyes. Others transfect these cells in neonatal rats to study the development of the retina. In both neonates and adults, electroporation is found to be effective. Here we describe the optimization of electroporation conditions for RPE cells in the adult mouse eye with naked plasmids. However, both techniques, subretinal injection and electroporation, present some technical challenges that require skill on the part of the surgeon to prevent untoward damage to the eye. Here we describe methods that we have used for the past 10 years (Johnson et al. Mol Vis 14: 2211-2226, 2008).
视网膜下注射是将多种药物、试剂、细胞及治疗手段递送至视网膜光感受器、穆勒细胞和视网膜色素上皮（RPE）细胞的最佳方法之一。注入该区域的药物会积聚在目标细胞的微米范围内，由于不存在运输过程或扩散导致的稀释作用，因此能达到高浓度。光感受器间腔（IPS）内的稀释作用极小，因为人眼的IPS体积仅为10 - 20微升，而小鼠眼的IPS体积小于1微升。为了实现基因递送，我们希望转染成年小鼠眼中IPS附近的细胞。其他人则在新生大鼠中对这些细胞进行转染以研究视网膜的发育。在新生儿和成年人中，电穿孔都被证明是有效的。在此，我们描述了使用裸质粒对成年小鼠眼中RPE细胞进行电穿孔条件的优化。然而，视网膜下注射和电穿孔这两种技术都存在一些技术挑战，需要外科医生具备一定技巧以防止对眼睛造成不良损伤。在此，我们描述了我们在过去10年中所使用的方法（Johnson等人，《分子视觉》14: 2211 - 2226，2008）。
