Sequence Analysis of Tyrosine Hydroxylase Promoter in Mice

Objective To analyze the sequence of the tyrosine hydroxylase promoter in mice,and to study the expression regulating ability of different regions of the promoter.Methods Five fragments of TH promoter(3400 bp,2400 bp,2000 bp,1500 bp and 450 bp)amplified by PCR with TaKaRa enzyme,were used to replace the CMV promoter in pcDNA3,and insert into EGFP gene.The recombinant plasmids were transfected instantly into MN-9D cells(TH+)and ECV cells(TH-),respectively.Flow cytometry was used to detect the expression of EGFP gene.Results The expression rates of EGFP in transfected MN-9D cells were 8.01%,7.97%,7.85%,7.72% and 7.74%,with no significant differences among them.In transfected ECV cells,the expression rates of EGFP were 6.51%,6.35%,6.71%,0.89% and 1.02%,showing that the regulating ability of fragments of 3400 bp and 2400 bp in TH-cells have been conspicuously restrained.Conclusion The all promoter fragments have an ability to regulate the expression of genes.It is preliminarily proved that the specialized regulation region of TH promoter is between 2400 bp and 2000 bp.