Cloning of human telomerase reverse transcriptase gene promoter and construction of its eukaryotic expression vector

Aim: To construct an eukaryotic expression vector containing human telomerase reverse transcriptase (hTERT) gene promoter and reporter green fluorescent protein(GFP). Methods: PCR amplification was performed using primers based on hTERT gene sequence from GenBank and human genomic DNA as a template. PCR product was directedly ligated into pMD18-T vector for sequencing. The promoter fragment was cloned into a pcDNA3.1(+)-GFP plasmid after it had been identified correctly. A human esophageal cancer cell line EC9706 was transfected with the plasmid containing hTERT promoter and reporter gene GFP, and the transfected cells were observed under fluorescent microscope 36 hours later. Results: A 733bp DNA fragment was amplified. Sequensing and restriction endonuclease digestion demonstrated that the fragment was cloned into the pcDNA3.1(+)-GFP-hTERT vector. Positive transfected cells with green fluorescence were detected under fluorescent microscope. Conclusion: The cloned hTERT promoter fragment in pcDNA3.1(+)-GFP-hTERT plasmid vector is active in human esophageal cancer cell line EC9706.