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


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 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.


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.
Yang
M
M
Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USA.
Wang
X G
XG
Stout
J T
JT
Chen
P
P
Hjelmeland
L M
LM
Appukuttan
B
B
Fong
H K
HK
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003673:Defective Viruses; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics*; D007124:Immunoenzyme Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008861:Microsomes / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012172:Retinaldehyde / Q000378:metabolism; D017299:Rod Opsins / Q000096:biosynthesis* / Q000235:genetics; D014161:Transduction, Genetic; D014801:Vitamin A / Q000378:metabolism
D005136:Eye Proteins; C089754:G protein-coupled receptor RGR; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D017299:Rod Opsins; D014801:Vitamin A; D012172:Retinaldehyde