A Kit for Homologous Repair in any Cell Line by Transient Transfection.

• HR-GFP. This reporter gene, when transfected into cells, does not express GFP since the gene is mutated in a twin cassette set up.  The 5’ cassette contains a disrupting I-Sce1 site/stop codon and the 3’ cassette is  WT over at the same region but contains deletions that prevent expression.
• I-Sce1 Expression Plasmid. This is a mega-endonuclease that does not cut anywhere in the human genome, except in the single I-Sce1 site located in the 5’ cassette of HR-GFP

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.

Kit Contents  

1. I-Sce1 gene expression plasmid, pSCE1 (store at -20^o or 4^oC).
2. DR-HRGFP (store at -20^o or 4^oC). This plasmid has a puromycin selection gene.
3. For both plasmids, the DNA concentration is typically 0.5ug/ul (but verify on tube label). Note that the Catalog Number the Kit size which is based on amount of DNA
4. eGFP (Wild type GFP with CMV promoter) transfection control is not included (50ug at 0.5ug/ul) in Kits but is available for purchase (some labs already have eGFP).
5. Optional:  To help establish the system, we offer a HeLa cell line control.