The use of an EIA-immortalized retinal culture to study IRBP gene regulation

Purpose. To determine whether a non-tumorigenic/non-transformed, immortalized rat retinal cell culture can be used as a model for the study of photoreceptor gene expression. Methods. Immuno-staining with anti-interphotoreceptor retinoid-binding protein (IRBP) was performed on E1A-NR.3 cultures and primary, uninfected rat retina cells. Gel shift and footprint analyses of the IRBP gene were performed on this culture and on the bovine retina. Results. Significant immunoreactivity to IRBP was observed in the primary retinal cells. About 15% to 20% IRBP-positive cells were observed in the immortalized culture. In negative controls, no immunoreactivity was observed in either the primary or immortalized culture. In footprint analysis using a probe that confers retina-specific IRBP expression, two DNase I protected regions appeared in the retina and the culture: one that was associated with the ubiquitous SP1 factor was observed in all tissues, and the other included a photoreceptor conserved element (PCE). In gel shift analysis using the PCE-containing sequence as a probe, specific band shifts in both the retina and the culture were identified. Conclusions. These results of DNA/protein binding assays demonstrate that the immortalized cells retain the ability to regulate IRBP gene expression in a manner similar to that in the retina. These cells and future isolated clones will be tested to determine their eligibility for the study of other photoreceptor gene regulation.