In order to investigate the ability of mouse tyrosine hydroxylase (TH) promoter regulating target genes in vitro, high-fidelity PCR was employed to amplify 450 bp, 1500 bp, 2000 bp, 2400 bp and full-length fragments of mouse TH promoter, which were then independently inserted into pcDNA3.0 plasmid as a promoter to regulate EGFP in two different cell lines [MN-9D (TH+) and ECV (TH-) cells]. The results showed that all the five cloned fragments of TH promoter contained the basic promoter elements of eukaryotic cells and the EGFP expression could be regulated by these 5 fragments in MN-9D cells to a similar extent. But, in ECV (TH-) cells, the capacity of 3650 and 2400 bp fragments of TH promoter regulating EGFP was markedly compromised when compared with that of the remaining three fragments. These findings suggested that, in the sequence between -2000 bp ~ -2400 bp of TH promoter, some dominant elements determined the ability of TH promoter regulating target genes in specific cells.
Key words: Tyrosine hydroxylase, promoter, regulation, cloning.
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