|Human: HeLa NHEJ Reporter Cell Line with Tet-on I-Sce1|
|DR5001-iHNHeLa-Tet||25 Assays||$1,995.00||Order Now|
DNA is continually being exposed to genotoxic agents leading to cell death and/or changes in gene expression. Of the various forms of DNA damage, the most dangerous are DNA double-strand breaks (DSBs), which may create serious problems arising from inappropriate recombination such as chromosomal translocations. To deal with the threats posed by DSBs, cells have developed multiple mechanisms to correct the problem using homologous recombination (HR) and non-homologous end-joining (NHEJ). These pathways are further subdivided into more specific sub-pathway processes. In prokaryotes, HR has been known to be a major pathway for the repair of DSBs, while in eukaryotes, NHEJ was thought to predominate. These pathways are largely distinct from one another yet function in complementary ways. While NHEJ is not cell cycle dependent, HR is strongly S-phase dependent. NHEJ involves the ligation of two DNA ends without homology and tends to be error prone while HR is high fidelity and essentially error free. In NHEJ, the broken DNA ends are modified for compatibility and then simply rejoined to restore DNA integrity. The resealing step is aided when there is some degree of micro-homology, even if only a few base pairs. It is not surprising NHEJ will result in mutations; however regeneration of wild type sequence is also common.
TopoGEN scientists have created a novel, cell based kit to follow NHEJ by simply assaying for the presence of GFP positive cells (Fig 1). The Kit uses a mutated GFP reporter that has two I-Sce1 sites disrupting the gene. When I-Sce1 is expressed by turning on the Tet-on promoter with doxycycline, DNA is severed at specific SCE-1 sites, which are subsequently repaired by NHEJ and the cells become GFP positive (over a 2-5d window). These cells can be tracked by live imaging or assayed using Cytometry (Fig. 1B,C) by simple imaging (enumerate fraction of GFP+ cells) or by live imaging (Fig. 1D).
This kit, which includes a reporter cell line, is shipped on dry ice. The frozen cells should be stored at -80° C for no more than one week before thawing and plating. Other reagents should be stored at -20° C upon receipt.