Concurrent enhancement of transcriptional activity and specificity of a retinal pigment epithelial cell-preferential promoter.
OBJECTIVE
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.


METHODS
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).


RESULTS
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.


CONCLUSIONS
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.
Zhang
Dan
D
Department of Molecular Ophthalmology, Lions Eye Institute, Nedlands, Western Australia, Australia.
Sutanto
Erika N
EN
Rakoczy
P Elizabeth
PE
D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D005136:Eye Proteins; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D019076:Transgenes; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D008156:Luciferases; D011506:Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C070633:Gal-VP16