Recoverin Expression in the R28 Retinal Precursor Cell Line

• Published in: In vitro cellular & developmental biology. Animal 1997-07, Vol.33 (7), p.499-502
• Main Authors: Gail M. Seigel, Aimee L. Mutchler, Adamus, Grazyna, Eileen L. Imperato-Kalmar
• Format: Article
• Language: English
• Subjects: Adenovirus E1A Proteins - genetics; Antibodies; Antigens, Neoplasm - genetics; Calcium-Binding Proteins - genetics; Cell culture techniques; Cell Line, Transformed; Cell lines; Cell nucleus; Cellular immunity; Cultured cells; Eye Proteins; Gene Expression; Hippocalcin; Humans; Lipoproteins; Nerve Tissue Proteins; Photoreceptor Cells - metabolism; Photoreceptors; Recoverin; Retina; Retina - metabolism; Scientific Reports; Stem Cells - metabolism; Tumor cell line; Vimentin
• ISSN: 1071-2690; 1543-706X
• DOI: 10.1007/s11626-997-0091-5

In vitro studies of photoreceptor cell development have been limited by the availability of pure retinal cultures with unlimited lifespan. To date, human retinoblastoma tumor-derived cell lines have been most commonly used as models for the study of photoreceptor differentiation in vitro. The most studied of these include Y79 cells. WERI-RB1 cells. Such retinoblastoma cells are highly tumorigenic in vivo and often show anomalous gene regulation that differs from that of the normal retina. Therefore, retinoblastoma-derived cells may not always be the best model system for the study of retinal cell growth and differentiation in vitro and have serious limitations for in vivo experimentation. As an alternative to transformed, tumorigenic retinal cell lines, this laboratory recently developed a 12S E1A-immortalized retinal cell culture. The 12S portion of the E1A gene contains the immortalizing, but not the transforming functions of the gene. The resulting retinal cell line R28 has been propagated in vitro for over 100 passages, exhibits nontransformed growth properties, and has a significant glial cell component. In this study, we show that R28 cells exhibit immunoreactivity to recoverin, a calcium-binding protein expressed in photoreceptors and Y79 retinoblastoma cells, which regulates rhodopsin phosphorylation and is hypothesized to play a role in cancer-associated retinopathy. Because R28 cells represent a virtually unlimited supply of immortalized, nontumorigenic cells expressing recoverin, the cell line may provide a unique and useful way to study the regulation of recoverin gene expression and its integral role in photoreceptor differentiation and function in vitro and in vivo. For all experiments in this study, 12S E1A-immortalized retinal cells derived from PN6 Sprague-Dawley rats were used. Animals were used in accordance with the Association for Research in Vision and Ophthalmology's statement for the Use of Animals in Ophthalmic and Vision Research. Establishment of the cell culture, designated E1A-NR.3, has been described previously. Briefly, a psi 2 12S E1A replication-defective retroviral vector was used to immortalize retinal tissue, which was then selected for 2 wk on the basis of neomycin resistance. Immortalized cells were maintained at 37 degree C in a 5% CO sub(2) incubator under the following culture conditions: Dulbecco's modified Eagle's medium (DMEM +) with 10% calf serum, 1 x minimal essential medium (MEM) nonessential amino acids (GIB