2 3 Development of Tyrosinase Promoter - Based Fluorescent Assay for Screening of Anti - 4 melanogenic Agents 5 6

27 For screening of skin-whitening ingredients that modulate inhibition of melanogenesis, 28 tyrosinase promoter-based assay using a 3D spheroid culture technique is a beneficial tool to 29 improve the accuracy of raw material screening in cosmetics through mimicking of the in 30 vivo microenvironment. Although the advantages of high-throughput screening (HTS) are 31 widely known, there has been little focus on specific cell-based promoter assays for HTS in 32 identifying skin-whitening ingredients that inhibit accumulation of melanin. The aim of this 33 study was therefore to develop a large-scale compatible assay through pTyr-EGFP, an 34 enhanced green fluorescent protein (EGFP)-based tyrosinase-specific promoter, to seek 35 potential melanogenesis inhibitors for cosmetic use. Herein, a stably transfected human 36 melanoma cell line expressing EGFP under the control of a 2.2-kb fragment derived from the 37 tyrosinase gene was generated. Spontaneous induction of the tyrosinase promoter by 3D 38 spheroid culture resulted in increased expression of EGFP, providing a significant correlation 39 with the tyrosinase mRNA level, and subsequent inhibition of tyrosinase activity. Importantly, 40 the pTyr-EGFP system provided successful tracking of the changes in the live image and 41 real-time monitoring. Thus tyrosinase promoter-based fluorescent assay using a 3D spheroid 42 culture can be useful as a screening system for exploring the efficiency of anti-melanogenesis 43 ingredients.