Study On Cloning Of Human Telomerase Catalytic Subunit (hTERT) Gene Promoter And Its Activity

ObjectivesWe cloned the hTERT promoter into the pGL3 Vector, resulting in the expression of fluorescent proteins only in telomerase positive cancer cells.MethodsWe uesd RT-PCR and real-time fluorescent quantitative PCR (FQ-PCR, or qPCR) to detect the expression of the hTERT andβ-actin genes in some tumor cell lines and a normal cell line, tested the protein expressing of the hTERT andβ-actin genes by Western-Blotting, amplified and cloned the hTERT core promoter by PCR and inserted into pGL3-Basic vectors, constructed the hTERT promoter recombinant plasmids, detected transcriptional activities of hTERT promoter in cell lines by measuring the luciferase activities.Results1. The RT-PCR showed the expressions of the hTERT mRNA existed in H460, U251, U87, ARO and FRO cells, yet the levels of transcriptional activities were different. But the expression of the hTERT mRNA was do not found in the MRC-5(normal cell). The FQ-PCR showed the expression of the hTERT gene was very high in U251 cells, and the relative concentrations was 0.18% compared withβ-actin (internal reference).2. Western blots demonstrated a positive band at 120 kD, which was the target gene expression of hTERT protein.3. Successfully constructed and identified plasmids pMD-18T and the pGL3 with 3 different length hTERT promoter sequences (260 bp,456 bp and 1453 bp). And successfully constructed and identified plasmid pGL3-hNIS with 260 bp hTERT promoter and hNIS as well.4. Luciferase activity assay showed all three kinds of hTERT promoter fragments could induce the expression of fluorescent proteins, but differences existed in their efficiencies in 5 tumor cells,. The pGL3-204 contained the p260 bp promoter fragment possessed the strongest efficiencies, which could reach 80% of the SV40 promoter, in FRO, H460, U251 and U87cells.ConclusionThe expressions of the hTERT gene was different in tumor cells,and the hTERT promoter could lead the tumor-specific expressions in glioma cells and other tumor cells. The hTERT core promoter had higher promoting efficiency, and could replaced the full length promoter and lead the tumor-specific gene expression.