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Customized: You Can Interrogate Homologous Recombination In A Cell Line Of Your Choosing
DNA Repair pathways in animal cells can be divided into two main categories: HR and NHEJ. HR or homologous recombination is a minor pathway but very important in protecting cells from genotoxicity. The process has two key requirements as well: a homologous sequence, usually available after DNA replication when the genome is 4N, and S-phase. A specific HR DNA Reporter system can be very beneficial for anti-cancer drug discovery projects, learning more about the process of DNA homologous recombination repair and establishing intersecting pathways and druggable pathway targets. A cellular context for HR improves the physiological relevance of the results.
HR DNA Reporter Kit contains reagents necessary to measure homologous recombination repair (HR, also known as gene conversion) in a cell line. It includes an HR-GFP reporter with a selectable marker and a DS DNA cleavage nuclease to initiate HR. Because GFP is a neutral gene reporter and not subject to selection, the HR process can easily be examined and assayed by assessing the %GFP or GFP intensity in a population of cells. The system is mobile in that it can be incorporated in any cell lineage or species to adapt to the investigator’s system of study.
This mobile transient assay DNA repair system has been designed to allow researchers to interrogate the HR process in live cells in vivo. The kit is based on a twin GFP cassette that converts from GFP negative to GFP positive cells using homologous recombination (HR). To start the HR process, we introduce a precise DNA cleavage using mega-endonuclease (I-Sce1) in the GFP locus of 5’ Cassette 1. The genome contains no I-Sce1 sites; therefore, an I-Sce1 site in Cassette 1 means that the DS break occurs only at this precise location and not elsewhere. The DS break initiates HR and using the WT sequence as a homology template (located in the 3’ Cassette 2) the gene converts to WT and GFP positive cells appear. In summary, HR is triggered by a DS break which is achieved by activating an expression plasmid for I-Sce1 (Fig. 2).
This technology lends itself to live cell imaging (by tracking single GFP+ cells). Live imaging gives essentially single cell resolution to HR analysis in these cells. In addition, the descendants of DNA repair can be tracked, since these cells are also GFP positive. See this video for further details on live imaging.