小鼠视网膜体外电转化及Transwell体外培养的方法

In vitro electroporation and retinal explant cultures in transwell of mouse retina

目的介绍小鼠视网膜体外电转化及体外培养的方法 ,并对其关键技术步骤进行探讨。方法将新生小鼠处死后,取出眼球分离视网膜,利用电转化仪将携带绿色荧光蛋白(green fluorescent protein,GFP)报告基因的质粒转入视网膜细胞,然后在Transwell小室中铺片培养,利用免疫荧光方法检测Rhodopsin基因的表达。结果视网膜体外培养10d,Rhodopsin和DAPI染色显示视网膜形态完整。通过电转化方法将GFP质粒导入视网膜细胞,进行体外培养5～10 d后可检测GFP报告基因的表达。结论视网膜体外电转化结合体外培养方法是一种快速、高效研究视网膜相关基因的方法 ,可以应用于视网膜疾病、视网膜发育及相关基因功能等研究,具有独特的优势。

Objective This paper described a method for in vitro electroporation and retinal explant cultures of mouse retina.And the key technical steps were discussed.Methods The newborn mice were sacrificed,removed the eye and separated retina.Using the electric conversion instrument,the GFP gene was transfected into retinal cells.Then the retina was placed and cultured in transwell chamber.The gene expression was detected by the frozen sections and immunofluorescence staining.Results Retinal explant in vitro culture for 10 days,Rhodopsin and DAPI staining showed the retinal morphology was complete.The GFP plasmid was transfected into retinal cells by electroporation and cultured in vitro.After 5-10 days of culture,the expression of GFP reporter gene can be detected.Conclusion This technique is a fast and efficient method for the study of retinal disease,retinal development and function of related genes,and this method has unique advantages.