Establishment of Stably Transfected Human RPE Cell Lines Over-expressing Oxysterol Binding Proteins

Abstract

Abstract: : Purpose:To generate stably transfected cultured human RPE cell lines over-expressing different oxysterol binding proteins (OSBPs) to use as a stable source of biologically active OSBPs. These cells will also be used to determine whether over expression of OSBPs provide protection or sensitivity to oxysterol and/or oxidized LDL-cholesterol induced cytotoxicity. Methods:A stable host cell line containing an integrated FRT site was generated using Invitrogen's Flp-In technology. The parental or host cell line was then transfected with pcDNA5-FRT-based constructs containing the gene(s) of interest fused to the V5 epitope.. In this study, hTERT, ARPE19 and pre-made Flp-In-293 cell lines were used to establish stably transfected cell lines that over express OSBP4. Results:Several cell lines derived from hTERT cells by transfection with pcDNA5-FRT-OSBP4 showed significantly higher expression of OSBP4 protein than the control hTERT cells by Western blot using antibodies to OSBP4. Antibodies to the V5 epitope also detected the recombinant OSBP4 protein.. Treatment of these cell lines with lethal concentrations of 7-ketocholesterol, oxidized 7-dehydrocholestrol, 20a-hydroxycholesterol and 25-hydroxycholesterol showed a significant reduction in cytotoxicity versus control hTERT cells. Conclusions:OSBP4 was successfully over-expressed in stably transfected hTERT RPE cell line. OSBP4 over-expression significantly protects these cells from oxysterol cytotoxicity. These cell lines will also serve as a source of biologically active OSBP4. Analyses of newly generated cell lines expressing other OSBPs are in progress in both hTERT and ARPE-19 cells.

Keywords: retinal pigment epithelium • cell death/apoptosis • gene/expression