Purpose: To clone different promoter region of amelogenin gene and analyze their transcriptional activity.
Methods: The upstream regulation sequences of amelogenin gene were retrieved and analyzed. Amplified by PCR from the genomic DNA of mouse C57BL/6J and digested with restriction endonucleases enzyme, different transcriptional regulation sequences of 5'flanking of amelogenin gene including the basal promoter were cloned and ligated with luciferase gene in PGL3-Basic vector. These report vectors were transiently transfected into CHO, Hela and UMR-106 cells and luciferase assay was performed to analyse the transcription activation of these promoters.
Results: 6 promoters different in length were cloned. The activity of luciferase was very strong in Hela cells. On the contrary, the CHO and UMR-106 cells showed weak fluorescence. Luciferase activity fluctuated with the different promoter lengths in Hela cell as well as in CHO and UMR-106 cells. 975 bp and 532 bp of amelogenin 5'flanking DNA had a strong transcriptional activation, but 285 bp of amelogenin 5'flanking DNA had a weaker transcriptional activation.
Conclusion: The sequence of amelogenin promoter can be activated in Hela cell. Hela cell can be used as a good model to study the transcriptional regulation of amelogenin promoter. According to the different activities of different lengths, it is suggested that there were some potential siliencer located between -975 and -532, and some potential activator in the region between -532 and -285.
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