ATF4 oxygen dependent degradation domain을 이용한 허혈 특이적 치료 유전자 조절 시스템

Title
ATF4 oxygen dependent degradation domain을 이용한 허혈 특이적 치료 유전자 조절 시스템
Other Titles
Hypoxia-specific gene expression system using the ATF4 oxygen dependent degradation domain for gene therapy
Author
조수희
Alternative Author(s)
Cho, Su Hee
Advisor(s)
이민형
Issue Date
2012-02
Publisher
한양대학교
Degree
Master
Abstract
Hypoxia region is generated in most solid tumor due to the lack of blood supply. To take advantage of this condition, we developed a hypoxia-specific gene expression system using the oxygen dependent degradation (ODD) domain from activating transcription factor 4 (ATF-4). ATF4 is a basic leucine-zipper (bZip) transcription factor, which regulates amino acid metabolism, cellular redox state, and anti-stress responses. To evaluate the ATF4 ODD domain for hypoxia specific gene expression, pSV-Luc-ATF4 ODD was constructed. For the construction of pSV-Luc-ATF4 ODD, the luciferase cDNA without the stop codon was inserted into pSV vector. Then, the chemically synthesized ATF4 ODD inserted at the downstream of the luciferase cDNA. In vitro transfection assay was performed and the cells were incubated under hypoxia or normoxia. The results showed that luciferase expression was induced under hypoxia in the pSV-Luc-ATF4 ODD transfected cells. In RT-PCR, the luciferase mRNA levels were not different between normoxia and hypoxia, suggesting that the induction of luciferase expression was a post-translational event. For cancer therapy, pSV-TK-ATF4 ODD was constructed. To evaluate the cytotoxicity under hypoxia condition, the cells was treated with the ganciclovir (GCV) under hypoxia and normoxia condition and MTT assay was performed. The results showed that the cells transfected with pSV-TK-ATF4 ODD had lower cell viability under hypoxia than normoxia. Similarly, as a result of caspase3/7 assay showed that the level of caspase3/7 as a marker of apoptosis is the high compared to control. In vivo rat brain tumor model was established by the injection of the C6glioblasoma cells into brain using stereotaxic method. Then, pSV-TK-ATF4 ODD was injected through local injection and GCV was injected intraperitoneally. The results showed that the tumor sizes decreased more efficiently than brain tumor model without DNA injection. In conclusion, a post-translational regulation system using the ATF4 ODD domain may be useful for the development of the cancer specific gene therapy system.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/137402http://hanyang.dcollection.net/common/orgView/200000418281
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIOENGINEERING(생명공학과) > Theses (Master)
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