Concurrent enhancement of transcriptional activity andspecificity of a retinal pigment epithelial cell-preferential promoter

[摘要] Purpose: To develop a transgene expression system in retinal pigmentepithelial cells with the aim of enhancing the transcriptional activityof a weak RPE-specific/preferential promoter.Methods: The transgene expression system was established byintroducing a chimeric transcriptional activator (GAL4-VP16) and its DNAbinding sequence and using truncated human and mouse RPE65 promoters incombination with a luciferase reporter gene. Two groups of expressionplasmids were constructed for transfection. The group forco-transfection contained two DNA constructs where the reporter andGAL4-VP16 were separately expressed in pLuc and pGV series. The othergroup, pLuc-GV series, was prepared as single DNA constructs expressingboth the reporter and GAL4-VP16. The transcriptional activities of theDNA constructs were assayed by transfection of human RPE cells (RPE51and D407) and other cell lines (HEK293, COS-1, Hela, HepG2, and F2000).Results: We found that the transcriptional activity of the humanRPE65 promoter was dramatically enhanced 10-13 fold in RPE cellsco-transfected with DNA constructs phR65luc and phR65GV when compared tothe human RPE65 promoter alone. A comparatively lower, 4-5 fold,increase was observed following transfection with the single DNAconstruct phR65luc-GV. In RPE cells, when the transcriptional responsesto GAL4-VP16 expression were compared between the RPE65 promoter ofphR65luc and the minimal promoter of pLuc, the increase intranscriptional activity was about 10 fold higher in phR65lucconstructs. Low or non-significant enhancement of promoter activity wasobserved with these constructs following transfection of the non-RPEcell lines.Conclusions: Our results indicate that the current transgeneexpression system dramatically amplifies transcriptional activity ofweak and cell-specific/preferential promoters (e.g., the hRPE65promoter) whilst retaining relative cell specificity.

[学科分类] 生物化学/生物物理