| Cellular responses to targeted genomic sequence modification using single-stranded oligonucleotides and zinc-finger nucleases. | |
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MedLine Citation:
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PMID: 19071233 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Single-stranded oligonucleotides (ssODNs) and zinc-finger nucleases (ZFNs) are two approaches that are being pursued to achieve sequence specific genome modification. ZFNs induce high rates of homologous recombination (HR) between the target sequence and a given donor by introducing site-specific genomic double-strand breaks (DSBs). The mode of action that is used by ssODNs remains largely unknown, but may involve genomic integration of the ssODNs. In this work, cellular responses following ssODN and ZFN mediated correction of a genomic reporter gene have been investigated in human cells. Comparison of the cell cycle distribution of corrected cells following ssODN or ZFN exposure, established that ssODN corrected cells were arrested in the late S and G2/M cell cycle phases, while ZFN corrected cells displayed normal cell cycle profiles. We demonstrate that after ssODN mediated gene correction, phosphorylation of the damage sensor protein H2AX could be observed in 5.8% and 29% of the corrected cells, using a single copy and a multi copy reporter, respectively. When using the ZFN strategy in a single copy reporter only 1.5% of the corrected cells were positive for gamma-H2AX staining. By direct detection of genomic DSBs we establish that the observed cell cycle arrest following ssODN mediated gene correction could be associated with the presence of unrepaired genomic DSBs. Lastly, we establish that although a mutant cellular mismatch repair (MMR) system as expected enhanced ssODN mediated gene correction, the capacity of the ssODN corrected cells to proliferate was not influenced by the MMR system. In conclusion gene correction by means of the ssODN strategy leads to activation of DNA damage signalling and cell cycle arrest due to formation of unrepaired genomic DSBs in a high proportion of the corrected cells. On the contrary, cells corrected using ZFNs displayed normal cell cycle distribution and lower rates of DNA damage. |
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Authors:
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Petter Angell Olsen; Anita Solhaug; James Alexander Booth; Monika Gelazauskaite; Stefan Krauss |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2008-12-30 |
Journal Detail:
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Title: DNA repair Volume: 8 ISSN: 1568-7864 ISO Abbreviation: DNA Repair (Amst.) Publication Date: 2009 Mar |
Date Detail:
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Created Date: 2009-02-09 Completed Date: 2009-05-27 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 101139138 Medline TA: DNA Repair (Amst) Country: Netherlands |
Other Details:
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Languages: eng Pagination: 298-308 Citation Subset: IM |
Affiliation:
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Section for Cellular and Genetic Therapy, Institute of Microbiology, Rikshospitalet University Hospital HF, Oslo, Norway. petter.angell.olsen@rr-research.no |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Apoptosis
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genetics Cell Line Cell Proliferation / drug effects* DNA Breaks, Double-Stranded / drug effects DNA Mismatch Repair / physiology DNA, Single-Stranded* Deoxyribonucleases / chemistry, pharmacology* Genes, Reporter / genetics Green Fluorescent Proteins Histones / metabolism Humans Mutation Oligodeoxyribonucleotides / genetics, pharmacology* Targeted Gene Repair / methods* Time Factors Zinc Fingers* |
| Chemical | |
Reg. No./Substance:
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0/DNA, Single-Stranded; 0/H2AFX protein, human; 0/Histones; 0/Oligodeoxyribonucleotides; 147336-22-9/Green Fluorescent Proteins; EC 3.1.-/Deoxyribonucleases |
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