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HR DNA Repair Reporter Kit by Plasmid Transfection Assay
Background
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 highly 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.
This 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 Transient Assay-Based Kit 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.
Kit Details
This is a plasmid transfection based reporter system designed to allow the customer to screen or examine any agent that affects the process of HR DNA repair. It will work in most of all cell lines that can be transfected. The kit uses GFP as an in vivo readout for the HR pathway. Kit includes two Plasmids that are validated as high quality DNA (see Figures 1 & 2):
When these two plasmids transfect the same cell, there will be a %GFP readout that reflects the efficiency of HR repair. Since the kit is only based on two DNAs, reasonable transfection efficiencies are possible and the process of HR repair can be examined in virtually any ‘transfectable’ cell lineage. Following transfection, a pooled population of GFP+ cells arise that can be sorted, or followed by live imaging. The efficiency of HR repair is correlated with the % of cells that convert to GFP+ phenotype. It is important to include transfection controls with eGFP in parallel cultures. The eGFP control gene is available for purchase separately from the Kit.
Who Should Buy This Kit?
R&D Labs that are interested in screening for agents, drugs, genes or gene products that specifically target the entire HR DNA repair pathway will find this kit very useful. It is well established that the DNA repair pathway is replete with druggable targets. Agents that target DNA repair may be good anti-cancer or anti-viral drugs. Also, identifying other genes and gene products that interact with DNA repair is important in order to find new drug targets. One major advantage of this kit is that it is operating in the context of a live/living cell. This means that the results will be highly physiological because events are transpiring within the cell in real time. In addition, because it is florescence-based, it is possible to use single cell imaging to track individual cells that undergo repair and also to track the descendants of those cells using time lapse imaging. This kit is based solely on plasmid reporter genes and allows researchers to place the system in any cell line of their choosing. This gives excellent flexibility when the primary interest is a cell line or lineage model. There is no need to make a stable cell line with integrated reporter genes (which takes time) and offers the investigator a quick inroad to drug testing or trans-acting gene effects on HR.
In summary, this kit will allow you to study and analyze HR in a specific cell lineage or cell line using a neutral GFP gene reporter.
