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 Citations (Scopus)

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

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Opsins
Lentivirus
Cultured Cells
Retinal Pigment Epithelium
Proteins
COS Cells
Vitamin A
Cell Line
Gene Transfer Techniques
Retinoids
Transgenes
Complementary DNA
Western Blotting
Cell Membrane
Viruses

ASJC Scopus subject areas

  • Ophthalmology

Cite this

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.

Expression of a recombinant human RGR opsin in Lentivirus-transduced cultured cells. / Yang, Mao; Wang, Xiao Guang; Timothy Stout, J.; Chen, Pu; Hjelmeland, Leonard M; Appukuttan, Binoy; Fong, Henry K W.

In: Molecular Vision, Vol. 6, No. 32, 2000, p. 237-242.

Research output: Contribution to journalArticle

Yang, M, Wang, XG, Timothy Stout, J, Chen, P, Hjelmeland, LM, Appukuttan, B & Fong, HKW 2000, 'Expression of a recombinant human RGR opsin in Lentivirus-transduced cultured cells', Molecular Vision, vol. 6, no. 32, pp. 237-242.
Yang M, Wang XG, Timothy Stout J, Chen P, Hjelmeland LM, Appukuttan B et al. Expression of a recombinant human RGR opsin in Lentivirus-transduced cultured cells. Molecular Vision. 2000;6(32):237-242.
Yang, Mao ; Wang, Xiao Guang ; Timothy Stout, J. ; Chen, Pu ; Hjelmeland, Leonard M ; Appukuttan, Binoy ; Fong, Henry K W. / Expression of a recombinant human RGR opsin in Lentivirus-transduced cultured cells. In: Molecular Vision. 2000 ; Vol. 6, No. 32. pp. 237-242.
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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.",
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AU - Wang, Xiao Guang

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AU - Hjelmeland, Leonard M

AU - Appukuttan, Binoy

AU - Fong, Henry K W

PY - 2000

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AB - 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.

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