视紫红质基因启动子的分离及视网膜特异表达载体的构建

Isolation of the rhodopsin gene promoter and construction of a retina-specific expression vector

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 5 2 4bpDNA片段 ,然后插入质粒pEGFP 1GFP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 5 2 4bp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。

ObjectiveTo construct a retina specific expression vector with green fluorecent protein (GFP) as reporter for gene delivering to retina,especially for gene therapy of retinal diseases.MethodsA 524 bp DNA fragment was isolated from mouse liver genomic DNA by mouse rhodopsin gene specific primers and polymerase chain reaction(PCR),and then the fragment was inserted into the MCS (Multiple Cloning Site) of the pEGFP-1 vector.Liposome-coating recombinant pmRho-EGFP plasmid was transferred into cultured human retinal pigment epithelium (RPE) cells and several other types of cell lines were injected in vivo under the retina of RCS rats or into the vitreous of SD rats.ResultsThe expression of GFP was observed in RPE cells with significant difference in numbers and amount in comparison with other cell lines.GFP was also seen in retina cells of rats.ConclusionThe 524 bp DNA fragment derived from 5’ flank sequence of mouse rhodopsin gene functions as a promoter and can drive retina specific expression in vitro and in vivo.