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

Mao Yang, Xiao Guang Wang, J. Timothy Stout, Pu Chen, Leonard M Hjelmeland, Binoy Appukuttan, Henry K W Fong

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Purpose: 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. Methods: A human RGR cDNA was cloned into a replication-defective lentiviral vector, and recombinant bRGR-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. Results: 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 -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. Conclusions: 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.

Original languageEnglish (US)
Pages (from-to)237-242
Number of pages6
JournalMolecular Vision
Volume6
Issue number32
StatePublished - 2000

ASJC Scopus subject areas

  • Ophthalmology

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    Yang, M., Wang, X. G., Timothy Stout, J., Chen, P., Hjelmeland, L. M., Appukuttan, B., & Fong, H. K. W. (2000). Expression of a recombinant human RGR opsin in Lentivirus-transduced cultured cells. Molecular Vision, 6(32), 237-242.