Boulanger Ana, Redmond T Michael
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Curr Eye Res. 2002 May;24(5):368-75. doi: 10.1076/ceyr.24.5.368.8523.
To examine the expression and promoter activation of the retinal pigment epithelium (RPE)-preferentially expressed Rpe65 gene in the commonly available RPE cell lines.
Reverse transcription coupled to polymerase chain reaction (RT-PCR) was performed after total RNA extraction from different RPE (ARPE-19, monkey, hTERT-RP1 and D407) and non-RPE (COS-7, HeLa, HepG2 and HS27) cell lines. Promoter activity was assayed by transient transfection of luciferase reporter constructs containing nested deletions of the 5' flanking region of the mouse Rpe65 gene. The involvement of a putative TATA box in the basal promoter expression was studied by site-directed mutagenesis in D407 cells and binding of TATA box-related transcription factors to that region was demonstrated by Electrophoretic Mobility Shift Assays (EMSA).
Expression of the human RPE65 cDNA was observed in all the RPE cell lines tested, and in COS-7 cells (monkey RPE65 cDNA). Transient transfections of the mouse Rpe65 promoter/luciferase transgene containing nested deletions of the Rpe65 5' flanking region showed that fragments containing bases -655 to +48 and -1240 to +48 generated specific promoter activity only in the D407 cell line. A promoter fragment from -49 to +48 directed basal promoter activity in all the cell lines tested. Part of this basal activity was due to a putative TATA box that specifically binds transcription factors contained in a D407 nuclear extract.
Although transcription of the Rpe65 gene occurs in all the tested cell lines, we find that the D407 cell line is the only one capable of directing specific mouse Rpe65 promoter activity. This limits the study of the transcriptional regulation of the mouse Rpe65 gene in vitro to this particular cell line.
检测视网膜色素上皮（RPE）优先表达的Rpe65基因在常用RPE细胞系中的表达及启动子激活情况。
从不同的RPE（ARPE - 19、猴、hTERT - RP1和D407）及非RPE（COS - 7、HeLa、HepG2和HS27）细胞系中提取总RNA后，进行逆转录聚合酶链反应（RT - PCR）。通过瞬时转染含有小鼠Rpe65基因5'侧翼区嵌套缺失的荧光素酶报告构建体来检测启动子活性。通过在D407细胞中进行定点诱变研究假定的TATA框在基础启动子表达中的作用，并通过电泳迁移率变动分析（EMSA）证明TATA框相关转录因子与该区域的结合。
在所检测的所有RPE细胞系以及COS - 7细胞（猴RPE65 cDNA）中均观察到人类RPE65 cDNA的表达。对含有Rpe65 5'侧翼区嵌套缺失的小鼠Rpe65启动子/荧光素酶转基因进行瞬时转染，结果显示，含有碱基-655至+48和-1240至+48的片段仅在D407细胞系中产生特异性启动子活性。从-49至+48的启动子片段在所有检测的细胞系中均指导基础启动子活性。这种基础活性部分归因于一个假定的TATA框，它特异性结合D407核提取物中所含的转录因子。
尽管Rpe65基因在所检测的所有细胞系中均有转录，但我们发现D407细胞系是唯一能够指导小鼠Rpe65启动子特异性活性的细胞系。这限制了在体外对小鼠Rpe65基因转录调控的研究仅能在该特定细胞系中进行。
