Yang M, Wang X G, Stout J T, Chen P, Hjelmeland L M, Appukuttan B, Fong H K
Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USA.
Mol Vis. 2000 Nov 18;6:237-42.
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.
我们的目标是制备一种用于生化研究的功能性重组视网膜色素上皮（RPE）视网膜G蛋白偶联受体（RGR）视蛋白，并测试慢病毒载体用于人RGR转基因表达的效率。
将人RGR cDNA克隆到复制缺陷型慢病毒载体中，制备重组hRGR-慢病毒用于转导人视网膜色素上皮（RPE）细胞系ARPE-19和COS-7细胞。通过蛋白质印迹分析检测重组RGR的表达，并通过[3H]全反式视黄醛结合试验测试该蛋白的功能。
用重组病毒转导后，在每种细胞类型中均检测到RGR蛋白，而未处理的细胞中未观察到。转导后，ARPE-19细胞中的RGR表达在长达10天的时间内稳步增加，并至少稳定6个月。转导的ARPE-19细胞产生的RGR蛋白量比转导的COS-7细胞高约100倍。当将ARPE-19细胞的细胞膜与[3H]全反式视黄醛一起孵育时，发色团特异性结合到表达的蛋白上。[3H]全反式视黄醇被ARPE-19细胞摄取后，放射性标记的类视黄醇特异性结合到RGR上。
使用慢病毒衍生的基因递送系统，我们能够在ARPE-19人RPE细胞系中大量表达人RGR蛋白。转导的ARPE-19细胞仍然能够处理全反式视黄醇，并且表达的蛋白能够结合全反式视黄醛发色团。基于慢病毒的功能性RGR表达可用于在培养细胞中研究RGR，并测试静止RPE细胞的体内转导。
