期刊
PLOS ONE
卷 16, 期 4, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0237413
关键词
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资金
- Consejeria de Sanidad CyL [GRS 1971/A/19]
- Consejeria de Educacion CyL [VA114P17]
- Polish National Science Centre [2016/22/A/NZ2/00184]
The study presents a novel assay to measure HR in living cells by integrating HR substrate at a specific locus to quantify HR events, which can further investigate the mechanisms controlling HR.
Homologous recombination (HR) faithfully restores DNA double-strand breaks. Defects in this HR repair pathway are associated with cancer predisposition. In genetic engineering, HR has been used extensively to study gene function and it represents an ideal method of gene therapy for single gene disorders. Here, we present a novel assay to measure HR in living cells. The HR substrate consisted of a non-fluorescent 3' truncated form of the eGFP gene and was integrated into the AAVS1 locus, known as a safe harbor. The donor DNA template comprised a 5' truncated eGFP copy and was delivered via AAV particles. HR mediated repair restored full-length eGFP coding sequence, resulting in eGFP+ cells. The utility of our assay in quantifying HR events was validated by exploring the impact of the overexpression of HR promoters and the siRNA-mediated silencing of genes known to play a role in DNA repair on the frequency of HR. We conclude that this novel assay represents a useful tool to further investigate the mechanisms that control HR and test continually emerging tools for HR-mediated genome editing.
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