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Construction of Transcriptional Targeting Replication-defective Recombinant Adenoviruses Containing FCY1 Driven by Modified hTERT Core Promoter

Author: HuaZuo
Tutor: XinXiaoYan
School: Fourth Military Medical University
Course: Surgery
Keywords: hTERT promoter Estrogen response element Hypoxia response element Targeting transcription Ovarian Cancer
CLC: R737.31
Type: Master's thesis
Year: 2003
Downloads: 40
Quote: 0
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Abstract


Experimental study of gene therapy in ovarian cancer, enzymatic prodrug strategy has been widely used. However, the policy itself and can not be achieved selective ovarian cancer will continue to produce heavier side effects. In recent years, targeting transcription has become a research hotspot of cancer gene therapy, a number of studies have been targeting transcription and enzymatic prodrug strategy to combine, and reached to a certain extent, tumor-specific killing effect. However, the promoter for transcriptional targeting in many other organizations have low levels of expression activity, thus limiting the clinical application of this method. The present study attempts to build efficient in ovarian cancer-specific activation of the promoter sequence, body before the regulation of the promoter sequence for the transcription of the drug-converting enzyme genes. Ovarian estrogen environment, the characteristics of the tumor microenvironment as well as the relationship of telomerase and tumor, we build the estrogen response element (ERE) and hypoxia response element (HRE) tandem repeat sequences as cis-acting elements of hTERT core the promoter double modification, in order to achieve tissue-specific and tumor-specific. To further evaluate the promoter activity modified positioning and ovarian cancer cells in vitro to determine its clinical value, we constructed guided by these promoters yeast-derived cytosine off the ammonia enzyme FCY 1 The gene expression of recombinant replication-defective adenovirus. One modified hTERT core promoter: estrogen response element (ERE) were constructed and hypoxia response element (HRE) tandem repeat sequence-specific promoter sequence, and insert them into the hTERT core promoter sequence upstream of ovarian cancer. Shuttle plasmid: the promoter sequence of yeast-derived cytosine deaminase (FCY 1 ) gene was inserted into the shuttle plasmid. Fourth no huge master's degree thesis thirteen recombinant adenovirus: HEK 293 cells were co-transfected with the shuttle plasmid and helper plasmid, the typical cytopathic 7 * days after transfection reaction the CPE), that is fine Wu Wang neon refractive enhancements and fall off the end of the culture plate. Fourth, the identification of recombinant adenovirus: DNA was extracted from the virus in the culture supernatant, using a self-designed for FCYI primers for PCR, were identified virus obtained results can be amplified 122hP objective fragment. Five recombinant adenovirus amplification and titer determination: the identification of the virus were amplified, and the end-point dilution test viral titer was determined 6 two XIO'Pfu / mlo modified by ERE and HRE tandem repeat sequences of hTERT core The recombinant adenovirus promoter guide FCY transcription successfully constructed, the value targeted the transcriptional gene therapy in ovarian cancer for the evaluation of the ERE, HRE and hTERT core promoter laid the foundation.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Ovarian tumors
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