The relationship between melanin production and lipofuscin formation in Tyrosinase gene knockout melanocytes using CRISPR/Cas9 system

Abstract

Age spots are a significant phenotypic marker of aging formed by lipofuscin. Melanin is another skin pigment molecule responsible for skin aging. The present study aims to investigate the relationship between melanin production and lipofuscin synthesis in normal mouse melanoma cell line B16F1 cells and Tyrosinase (TYR) gene knockout cells. TYR gene KO cells were successfully developed using CRISPR/Cas9 system and confirmed by Sanger DNA sequencing analysis. Furthermore, the melanin production and lipofuscin formation were validated through RT-PCR and Western blot analysis. The expression levels of gene microphthalmia-associated transcription factor (MITF), Tyrosinase, tyrosine-related protein-1 (TRP-1), tyrosine-related protein-2 (TRP-2), and antioxidant proteins such as methionine sulfoxide reductase A (MSRA), Catalase and Glutathione reductase (GR) related to melanogenesis was found to be decreased in TYR gene KO cells compared with normal cells. Moreover, lipofuscin formation was increased in TYR gene KO cells compared to normal cells. Therefore, the above findings suggest that melanin production and lipofuscin formation could be linked by the TYR gene in melanocytes.