The Recombination And Functional Identification Of A Human Telomerase Reverse Transcriptase Gene Promoter

The formation of tumor cells highly depends on telomerase activity, so it is very important to obtain a high specificity and transcription activity human telomerase reverse transcriptase (hTERT) promotor for tumor gene therapy and controlling tumor form during stem cells transplant. Respecting that natural hTERT promotor is not enough excellent in transcription activity and specificity, we design our research to obtain a high specificity and transcription activity hTERT promotor that expressed in telomerase positive cells. First the 270bp base sequence, which is in upstream of CD start site(ATG)of the natural hTERT promotor, was used as basic frame to design recombination promoter and we put four E-box(CACGTG)repeated sequences into the downstream of the 270bp base sequence. Later the sequences were artificial synthesized by chemosynthesis and subcloned into PUC57 cloning vector. Second, the lentivirus vector expressing enhanced green fluorescent protein (eGFP) was constructed and we replaced the cytomegalovirus(CMV) promotor in upstream of eGFP with recombination hTERT promotor. Third, to identify the function of the recombination promotor, the recombination lentivirus vector was transfected into TLMA positive human embryo kidney EP tumor cells(293FT), hepatoma carcinoma cells(HepGII), gastric carcinoma cells(SGC7901) and TLMA negtive human osteosarcoma cells(U2OS) by liposome. Fourth, to identify the transcription activity and specificity of the recombination hTERT promotor in normal cells, the lentivirus vector was packaged into virus particle and transfected into primary cultured human fibroblasts to detected the expression of the report gene with confocal microscopy. The results showed that the green fluorescent protein was expressed in 293FT,HepGâ…¡,SGC7901 that are telomerase positive cells, but not in the U2OS that are telomerase negtive cells as well as primary cultured human fibroblasts. We can conclued that the recombination hTERT promotors have high specificity and transcription activity in telomerase positive cells.