[Construction and identification of a tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor].
OBJECTIVE
To construct the tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor.


METHODS
The fragment of the enhanced green fluorescent protein (EGFP) gene was PCR amplified from the pEGFP-N1 plasmid and cloned into the multiple cloning site of pLNCX vector, and the recombinant was named as pLNCX-EGFP. The fragment of human telomerase reserve transcriptase gene promoter was amplified from the human genome by using the human telomerase reserve transcriptase gene specific primers, and cloned into the pLNCX-EGFP vector, where the cytomegalovirus promoter was previously removed using restriction enzymes, in sense orientation relative to the green fluorescent protein coding sequence. Then the expression vector pLNT-EGFP under the control of the human telomerase reserve transcriptase gene promoter, containing green fluorescent protein reporter gene, was successfully constructed. To detect the transcriptional activity of the human telomerase reserve transcriptase gene promoter, transient transfection of this specific expression vector into HLF cell lines with high telomerase activity and WI38 cell lines without telomerase activity was performed.


RESULTS
The expression vector proven by restriction enzymes digestion and sequencing was in correspondence with the design. The results of transient transfection showed that the pLNT-EGFP vector could highly expressed green fluorescent protein reporter gene in telomerase-positive cells, but not in telomerase-negative cells.


CONCLUSIONS
A tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor has been successfully constructed.
Yin
Jia-Jun
JJ
Department of General Surgery, The 4th Affiliated Hospital of Dalian Medical University, Dalian 116001, China.
Hu
Xiang
X
Fu
Pan-feng
PF
Yuan
Xiao-dong
XD
Zhang
Jin-yu
JY
D003587:Cytomegalovirus / Q000235:genetics; D016373:DNA, Antisense / Q000032:analysis; D018076:DNA, Complementary / Q000032:analysis; D004268:DNA-Binding Proteins; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D019098:Telomerase / Q000235:genetics*
D016373:DNA, Antisense; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D019098:Telomerase