Abstract: 【Objective】 Keratin 10 (K10) is one of the molecular markers of melanosome transfer from melanocytes to the around keratinocytes, which can be used as the specific promoter for the research on the interactions between melanocytes and keratinocytes. Here, the strongest promoter of K10 will be screened to supply the evidence and basis for the research of the function of K10 and other relative genes. 【Method】 Genomic DNA was extracted from a mouse tail. Six fragments of K10 were amplified and subcloned into pMD18-T vector and sequenced. They were then cloned into pGL0 vector to construct pGL0-F1-F6, was transfected into 293T cells by liposome. The luciferase activity were used to identify the strongest promoter from the 6 fragments of K10 promoter (F1-F6), which was measured by luciferase reporter (GFP) from the cell lysis and the prediction of transcript factor binding sites by bioinformatics of the sequence. After the strongest promoter of K10 was identified, it was used as a specific promoter, which recombined with pGL0 (without CMV) to construct pGL0-F-CDK5. pGL0-F-CDK5 was transfected into keratinocytes of mouse by liposome and then Cyclin-dependent Kinase 5 (CDK5) expression was measured by Immunofluorescence chemistry, luciferase reporter (GFP) and quantitative real-time PCR from the cells, cell lysis and total RNA, respectively. 【Result】 Six fragments (F1-F6) of K10 were amplified, cloned and sequenced with the size of 1 201, 908, 664, 787, 790 and 656 bp, respectively. The luciferase activity of F4 with the size of 787 bp was the strongest as measured by the luciferase reporter gene in 293T cell, which suggested that F4 would be used as a specific promoter. But the luciferase activity of F1-F6 was weaker than that of CMV. In the F4 sequence, the basic conserved region (TATAAAA) and the transcript factor binding sites such as C/EBPβ, GATA, HSF and CAP were found by Promoter Scan software, which promotes the K10 expression in keratinocytes. After transfection, the result of luciferase activity measurement showed that pGL0-F4-CDK5 made GFP expression stronger than pGL0-basic-CDK5 with a significant difference（P＜0.01), and made CDK5 mRNA expression higher than pGL0-basic-CDK5 with a significant difference (P＜0.01), which suggested that F4 was the corn region of K10 promoter with the strongest activity. 【Conclusion】 F4 was identified as the corn region of K10 promoter to have the strongest activity in promoting CDK5 expression in keratinocyte, which suggested that F4 would be used as a specific promoter for the gene function research during the interaction between melanocyte and keratinocyte.

Key words: keratin 10 (K10), promoter, specific promoter, transcription factor, Cyclin-dependent Kinase 5 (CDK5), mouse