Abstract
目的 分离视紫红质基因启动子,构建以绿色荧光蛋白(GFP)为报告基因的视网膜特异表达载体,为今后视网膜细胞特异的靶向基因转移,特别是为视网膜疾病的基因治疗提供工具.方法根据小鼠视紫红质基因5'端DNA顺序合成引物,通过PCR技术从小鼠基因组DNA中扩增524 bp DNA片段,然后插入质粒pEGFP-1 GFP编码基因上游的多克隆位点处,构建表达载体pmRho-EGFP.采用脂质体包裹pmRho-EGFP,转染体外培养的人视网膜色素上皮(RPE)细胞及其他来源的细胞;注射到SD大鼠视网膜下或玻璃体腔内;或通过电穿孔直接转移到RCS大鼠腓肠肌内.GFP表达采用荧光显微镜观察.结果pmRho-EGFP在体外培养的人RPE细胞中的表达水平明显高于其他组织来源的细胞;体内转染实验证明pmRho-EGFP可在大鼠视网膜神经细胞中表达,但在大鼠腓肠肌中不表达.结论小鼠视紫红质基因5'端524 bp片段具有基本的启动子活性,能够调控基因在视网膜神经细胞及色素上皮细胞中表达,并具有一定的组织和细胞特异性。
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