Dissertation > Medicine, health > Basic Medical > Human biochemistry, molecular biology

Construction of Luciferase Transcriptional Targeting Vectors Driven by hTERT Core Promoter Modified by SV40 Enhancer

Author XueLiYing
Tutor ZhangWeiMing;Xu
School Tianjin Medical University
Course Pathology and Pathophysiology
Keywords Telomerase catalytic subunit (hTERT) promoter SV40 enhancer Transcriptional activity Luciferase
CLC R346
Type Master's thesis
Year 2005
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PCR amplification of the purpose of human telomerase catalytic subunit (hTERT) of the core promoter sequences cloned into a luciferase reporter vector containing SV40 enhancer pGL3Basic the pGL3Enhancer in order to compare the hTERT promoter with the hTERT promoter Joint virus SV40 enhancer that hTERT-SV40 chimeric promoter system in telomerase -positive tumor cell lines and telomerase -negative human embryonic lung fibroblast cell line differences in transcriptional activity and specificity , trying to come through the SV40 enhancer enhance the transcriptional activity of the hTERT promoter , while not changing its tumor-specific , targeted cancer in order to provide a preliminary treatment of the theoretical basis and experimental basis . Approach to human genomic DNA as template was amplified by PCR telomerase reverse transcriptase gene 5 'upstream core length 260bp promoter fragment , after its subcloned into pGEM-T Easy vector to construct vector pGEM-hTP, its correct by restriction analysis and DNA sequencing , the use of restriction endonucleases Kpn Ⅰ and Bgl Ⅱ double digested pGEM-hTP carrier, get hTERT core promoter fragment with the same by Kpn Ⅰ and Bgl Ⅱ double digestion luciferase reporter vector pGL3-Basic and pGL3-Ehancer to connect, build pGL3-hTP and pGL3hTP-SV40 recombinant plasmid ; using calcium phosphate transfection system, pGL3-hTP, pGL3-hTP-SV40, pGL3-Control and pGL3 -Basic and pRL-TK plasmids were co-transfected HT-29, SW620, MGC-803 and other telomerase -positive tumor cell lines and normal human fetal lung fibroblast cell line MRC5 , where pGL3-Basic as a negative control , pGL3-Control

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