Development of a high - throughput screening system

Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fi broblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identifi cation of putative materials regulating DNA repair in skin cells. First, we established a modifi ed lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fi broblast WS-1 cells were infected with the modifi ed lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The fi rst step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity aft er pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain eff ective reagents identifi ed in the fi rst step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fi broblasts.