Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events
| Autor: | Piotr Kozlowski, Dino Gobelli, Julia Serna, Gabriel March-Rosselló, María Simarro, Miguel Angel de la Fuente, Alejandra Bernardi, Paulina Maria Nawrocka, Antonio Orduña |
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| Přispěvatelé: | Junta de Castilla y León, National Science Centre (Poland) |
| Rok vydání: | 2021 |
| Předmět: |
Genome engineering
DNA End-Joining Repair Artificial Gene Amplification and Extension Engineering and technology Synthetic genome editing Biochemistry Polymerase Chain Reaction Green fluorescent protein chemistry.chemical_compound Genome editing Neoplasms DNA Breaks Double-Stranded Homologous Recombination Promoter Regions Genetic Transcription activator-like effector nuclease Multidisciplinary Transfection Small interfering RNA Cell biology Nucleic acids TALENs Medicine Hyperexpression Techniques Genetic Engineering Research Article DNA recombination DNA repair Science Green Fluorescent Proteins Bioengineering DNA construction Biology Research and Analysis Methods Fluorescence Cell Line Cell Line Tumor Genetics Gene Expression and Vector Techniques Humans Non-coding RNA Molecular Biology Techniques Molecular Biology Gene Synthetic biology Molecular Biology Assays and Analysis Techniques Biology and life sciences Synthetic genomics Recombinational DNA Repair DNA HCT116 Cells Gene regulation HEK293 Cells chemistry Plasmid Construction RNA Gene expression Homologous recombination |
| Zdroj: | PLoS ONE PLoS ONE, Vol 16, Iss 4, p e0237413 (2021) Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 1932-6203 |
| Popis: | © 2021 Bernardi et al. 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. This work was supported by Consejería de Sanidad CyL (Grant GRS 1971/A/19), Consejería de Educación CyL (Grant VA114P17) and the Polish National Science Centre (Grant 2016/22/A/NZ2/00184). |
| Databáze: | OpenAIRE |
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