Expression of a recombinant human RGR opsin in Lentivirus-transduced cultured cells

• Published in: Molecular vision 2000-11, Vol.6, p.237-242
• Main Authors: Yang, M, Wang, X G, Stout, J T, Chen, P, Hjelmeland, L M, Appukuttan, B, Fong, H K
• Format: Article
• Language: English
• Subjects: Animals; Autoradiography; Blotting, Western; Cattle; Cells, Cultured; COS Cells - metabolism; Defective Viruses; Electrophoresis, Polyacrylamide Gel; Eye Proteins - biosynthesis; Eye Proteins - genetics; Genetic Vectors; HIV - genetics; Immunoenzyme Techniques; Mice; Mice, Inbred C57BL; Microsomes - metabolism; Pigment Epithelium of Eye - metabolism; Receptors, Cell Surface - biosynthesis; Receptors, Cell Surface - genetics; Receptors, G-Protein-Coupled; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Retinaldehyde - metabolism; Rod Opsins - biosynthesis; Rod Opsins - genetics; Transduction, Genetic; Vitamin A - metabolism
• ISSN: 1090-0535

Our goals were to produce a functional recombinant RPE retinal G protein-coupled receptor (RGR) opsin for biochemical studies and to test the efficiency of a lentiviral vector for transgene expression of human RGR. A human RGR cDNA was cloned into a replication-defective lentiviral vector, and recombinant hRGR-Lentivirus was prepared for transduction of the ARPE-19, a human retinal pigment epithelium (RPE) cell line, and COS-7 cells. Recombinant RGR expression was detected by Western blot analysis, and functionality of the protein was tested by a [3H]all-trans-retinal binding assay. RGR protein was detected in each cell type after transduction with recombinant virus and was not observed in untreated cells. RGR expression in ARPE-19 cells increased steadily for up to 10 days after transduction and was stable for at least 6 months. The transduced ARPE-19 cells produced approximately 100-fold higher amounts of RGR protein than the transduced COS-7 cells. When cell membranes from the ARPE-19 cells were incubated with [3H]all-trans-retinal, the chromophore bound specifically to the expressed protein. Uptake of [3H]all-trans-retinol into the ARPE-19 cells was followed by specific binding of radiolabeled retinoid to RGR. Using a Lentivirus-derived gene delivery system, we were able to express high amounts of human RGR protein in the ARPE-19 human RPE cell line. The transduced ARPE-19 cells remain able to process all-trans-retinol, and the expressed protein is capable of binding to the all-trans-retinal chromophore. The Lentivirus-based expression of functional RGR can be used to study RGR in cultured cells and to test in vivo transduction of quiescent RPE cells.