Tumor-specific expression detected with the use of an expression vector driven by human telomerase reverse transcriptase gene promoter.
To construct the tumor-specific expression vector driven by human telomerase reserve transcriptase gene promoter, we amplified a fragment of the enhanced green fluorescent protein (EGFP) gene from the pEGFP-N1 plasmid and cloned it into the multiple cloning site of the pLNCX vector, then named the recombinant as pLNCX-EGFP. The fragment of human telomerase reverse transcriptase gene promoter (hTERT) was amplified from the human genome with the use of human telomerase reserve transcriptase gene-specific primers and cloned into the pLNCX-EGFP vector, from which the cytomegalovirus promoter had previously been removed through the use of 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, which contains green fluorescent protein reporter gene-was successfully constructed. To detect the transcriptional activity of the human telomerase reserve transcriptase gene promoter, we conducted transient transfection of this specific expression vector into human lung fibroblast (HLF) cell lines with high telomerase activity and normal human fetal lung fibroblast (WI38) cell lines without telomerase activity. The results of transient transfection showed that the pLNT-EGFP vector strongly expressed the green fluorescent protein reporter gene in telomerase-positive cells but not in telomerase-negative cells.
Yin
Jiajun
J
Department of General Surgery, Dalian Railway Hospital and Fourth Affiliated Hospital of Dalian Medical University, China.
Fu
Panfeng
P
Wang
Xiaobo
X
Jin
Gang
G
Xiu
Ruijuan
R
D001483:Base Sequence; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D019098:Telomerase