Zhang Dan, Sutanto Erika N, Rakoczy P Elizabeth
Department of Molecular Ophthalmology, Lions Eye Institute, Nedlands, Western Australia, Australia.
Mol Vis. 2004 Mar 26;10:208-14.
To develop a transgene expression system in retinal pigment epithelial cells with the aim of enhancing the transcriptional activity of a weak RPE-specific/preferential promoter.
The transgene expression system was established by introducing a chimeric transcriptional activator (GAL4-VP16) and its DNA binding sequence and using truncated human and mouse RPE65 promoters in combination with a luciferase reporter gene. Two groups of expression plasmids were constructed for transfection. The group for co-transfection contained two DNA constructs where the reporter and GAL4-VP16 were separately expressed in pLuc and pGV series. The other group, pLuc-GV series, was prepared as single DNA constructs expressing both the reporter and GAL4-VP16. The transcriptional activities of the DNA constructs were assayed by transfection of human RPE cells (RPE51 and D407) and other cell lines (HEK293, COS-1, Hela, HepG2, and F2000).
We found that the transcriptional activity of the human RPE65 promoter was dramatically enhanced 10-13 fold in RPE cells co-transfected with DNA constructs phR65luc and phR65GV when compared to the human RPE65 promoter alone. A comparatively lower, 4-5 fold, increase was observed following transfection with the single DNA construct phR65luc-GV. In RPE cells, when the transcriptional responses to GAL4-VP16 expression were compared between the RPE65 promoter of phR65luc and the minimal promoter of pLuc, the increase in transcriptional activity was about 10 fold higher in phR65luc constructs. Low or non-significant enhancement of promoter activity was observed with these constructs following transfection of the non-RPE cell lines.
Our results indicate that the current transgene expression system dramatically amplifies transcriptional activity of weak and cell-specific/preferential promoters (e.g., the hRPE65 promoter) whilst retaining relative cell specificity.
开发一种视网膜色素上皮细胞中的转基因表达系统，旨在增强弱RPE特异性/优先启动子的转录活性。
通过引入嵌合转录激活因子（GAL4-VP16）及其DNA结合序列，并将截短的人源和小鼠RPE65启动子与荧光素酶报告基因结合，建立转基因表达系统。构建两组表达质粒用于转染。共转染组包含两个DNA构建体，其中报告基因和GAL4-VP16分别在pLuc和pGV系列中表达。另一组pLuc-GV系列则作为表达报告基因和GAL4-VP16的单一DNA构建体制备。通过转染人RPE细胞（RPE51和D407）及其他细胞系（HEK293、COS-1、Hela、HepG2和F2000）来检测DNA构建体的转录活性。
我们发现，与单独的人RPE65启动子相比，用DNA构建体phR65luc和phR65GV共转染的RPE细胞中，人RPE65启动子的转录活性显著增强了10至13倍。用单一DNA构建体phR65luc-GV转染后，观察到相对较低的4至5倍的增加。在RPE细胞中，当比较phR65luc的RPE65启动子和pLuc的最小启动子对GAL4-VP16表达的转录反应时，phR65luc构建体中转录活性的增加约高10倍。在非RPE细胞系转染后，用这些构建体观察到启动子活性的增强较低或无显著增强。
我们的结果表明，当前的转基因表达系统显著放大了弱的细胞特异性/优先启动子（如hRPE65启动子）的转录活性，同时保留了相对的细胞特异性。
