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DC Field | Value | Language |
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dc.contributor.author | Gravells, P | - |
dc.contributor.author | Ahrabi, S | - |
dc.contributor.author | Vangala, RK | - |
dc.contributor.author | Tomita, K | - |
dc.contributor.author | Brash, JT | - |
dc.contributor.author | Brustle, LA | - |
dc.contributor.author | Chung, C | - |
dc.contributor.author | Hong, JM | - |
dc.contributor.author | Kaloudi, A | - |
dc.contributor.author | Humphrey, TC | - |
dc.contributor.author | Porter, ACG | - |
dc.date.accessioned | 2020-08-10T10:50:56Z | - |
dc.date.available | 2015-01-01 | - |
dc.date.available | 2020-08-10T10:50:56Z | - |
dc.date.issued | 2015-09-30 | - |
dc.identifier.citation | Human Molecular Genetics, 2015, 24 (24), pp. 7097 - 7110 | en_US |
dc.identifier.issn | 0964-6906 | - |
dc.identifier.issn | 1460-2083 | - |
dc.identifier.uri | https://bura.brunel.ac.uk/handle/2438/21375 | - |
dc.description.abstract | © The Authors 2015. Published by Oxford University Press. Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic nonhomologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene. | en_US |
dc.description.sponsorship | This work was supported in part by the Biotechnology and Biological Research Council (BB/H003371/1 to A.C.G.P.), the Medical Research Council (MC_PC_12003 to T.C.H.), Cancer Research UK (C5255/A15935 to S.A.) and University of Oxford (Clarendon Scholarship to S.A.). Funding to pay the Open Access publication charges for this article was provided by the Research Councils UK open access fund. | en_US |
dc.format.extent | 7097 - 7110 | - |
dc.language.iso | en | en_US |
dc.publisher | Oxford University Press | en_US |
dc.subject | dna | en_US |
dc.subject | genes | en_US |
dc.subject | hypoxanthine phosphoribosyltransferase | en_US |
dc.subject | double strand | en_US |
dc.subject | break repair | en_US |
dc.title | Use of the HPRT gene to study nuclease-induced DNA double-strand break repair | en_US |
dc.type | Article | en_US |
dc.identifier.doi | https://doi.org/10.1093/hmg/ddv409 | - |
dc.relation.isPartOf | Human Molecular Genetics | - |
pubs.issue | 24 | - |
pubs.publication-status | Published | - |
pubs.volume | 24 | - |
dc.identifier.eissn | 1460-2083 | - |
Appears in Collections: | Dept of Life Sciences Research Papers |
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FullText.pdf | 899.62 kB | Adobe PDF | View/Open |
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