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Enhanced gene editing by programmable nucleases delivered by a minicircle vector

Title
Enhanced gene editing by programmable nucleases delivered by a minicircle vector
Other Titles
미니서클 백터를 활용한 유전자 가위의 교정 효율 증가에 관한 연구
Author
아부다드
Alternative Author(s)
Abu-BonsrahKwakuDad
Advisor(s)
김형범 교수
Issue Date
2013-08
Publisher
한양대학교
Degree
Master
Abstract
Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are of great value in biomedical research, medicine, and biotechnology. Transient transfection using conventional plasmids has been the predominant way to deliver programmable nucleases into cells. Minicircle vectors, which lack extraneous bacterial sequences such as an antibiotic resistance gene and a bacterial origin of replication, have been reported to have advantages such as more robust transgene expression, higher efficiency of gene transfer, and greater biosafety as compared to conventional plasmids. Here, for the first time, we delivered programmable nucleases into human cells using transient transfection of a minicircle vector and compared the results with those obtained using a conventional plasmid. A surrogate reporter assay showed that nuclease activity was significantly higher in the minicircle vector group of cells than in the conventional plasmid group (43 ± 1% for ZFN group and 30 ± 1% for TALEN group). A T7 endonuclease assay revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the endogenous target sites of the programmable nucleases than those in the conventional plasmid group (P < 0.001). Tryphan blue staining and flow cytometry after annexin V and propidium iodide staining showed that cell viability was also significantly higher in the minicircle group than in the conventional plasmid group (P < 0.001). Taken together, our results show that gene editing using minicircle vector-mediated delivery of ZFNs and TALENs is a more efficient, safer, and less toxic method than using a conventional plasmid, and indicate that the minicircle vector could serve as an advanced delivery method for programmable nucleases.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/133263http://hanyang.dcollection.net/common/orgView/200000422229
Appears in Collections:
GRADUATE SCHOOL OF BIOMEDICAL SCIENCE AND ENGINEERING[S](의생명공학전문대학원) > BIOMEDICAL SCIENCE(의생명과학과) > Theses (Master)
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