Enhanced gene disruption by programmable nucleases delivered by a minicircle vector
- Title
- Enhanced gene disruption by programmable nucleases delivered by a minicircle vector
- Author
- Ramakrishna Suresh
- Keywords
- ZINC-FINGER NUCLEASES; PLURIPOTENT STEM-CELLS; TAL EFFECTOR NUCLEASES; DOUBLE-STRAND BREAKS; EXPRESSION IN-VIVO; HUMAN IPS CELLS; HOMOLOGOUS RECOMBINATION; INDUCED MUTATIONS; EMBRYO MICROINJECTION; SURROGATE REPORTERS
- Issue Date
- 2014-11
- Publisher
- Nature Publishing Group
- Citation
- Gene Therapy, 2014, 21(11), P.921-930
- Abstract
- Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) is of great value in biomedical research, medicine and biotechnology. Minicircle vectors,which lack extraneous bacterial sequences, have several advantages over conventional plasmids for transgene delivery. 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. Surrogate reporter assays and T7 endonuclease analyses revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the target sites than those in the conventional plasmid group. Quantitative PCR and reverse transcription-PCR showed higher vector copy number and programmable nuclease transcript levels, respectively, in 293T cells after minicircle versus conventional plasmid vector transfection. In addition, 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. Taken together, our results show that gene disruption 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://www.nature.com/articles/gt201476http://hdl.handle.net/20.500.11754/53293
- ISSN
- 0969-7128; 1476-5462
- DOI
- 10.1038/gt.2014.76
- Appears in Collections:
- GRADUATE SCHOOL OF BIOMEDICAL SCIENCE AND ENGINEERING[S](의생명공학전문대학원) > ETC
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