Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events.
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 disor...
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| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2021-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0237413&type=printable |
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| author | Alejandra Bernardi Dino Gobelli Julia Serna Paulina Nawrocka Gabriel March-Rosselló Antonio Orduña Piotr Kozlowski María Simarro Miguel A de la Fuente |
| author_facet | Alejandra Bernardi Dino Gobelli Julia Serna Paulina Nawrocka Gabriel March-Rosselló Antonio Orduña Piotr Kozlowski María Simarro Miguel A de la Fuente |
| author_sort | Alejandra Bernardi |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-388a77dad91c4ac9aab9c5687726972a |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2021-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-388a77dad91c4ac9aab9c5687726972a2025-08-20T02:17:48ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-01164e023741310.1371/journal.pone.0237413Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events.Alejandra BernardiDino GobelliJulia SernaPaulina NawrockaGabriel March-RossellóAntonio OrduñaPiotr KozlowskiMaría SimarroMiguel A de la FuenteHomologous 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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0237413&type=printable |
| spellingShingle | Alejandra Bernardi Dino Gobelli Julia Serna Paulina Nawrocka Gabriel March-Rosselló Antonio Orduña Piotr Kozlowski María Simarro Miguel A de la Fuente Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events. PLoS ONE |
| title | Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events. |
| title_full | Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events. |
| title_fullStr | Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events. |
| title_full_unstemmed | Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events. |
| title_short | Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events. |
| title_sort | novel fluorescent based reporter cell line engineered for monitoring homologous recombination events |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0237413&type=printable |
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