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Human: HeLa HR Reporter Cell Line with Tet-on I-Sce1
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DR3001-iHRHeLa-Tet 25 Assays $1,495.00 Order Now

DR3001-iHRHeLa-Tet

HeLa Cell System to analyze HR with I-Sce1 activated by Doxycycline in a Tet-on promoter

DNA Repair pathways in animal cells can be divided into two main categories:  HR and NHEJ.  HR (homologous recombination) is a minor pathway but very important in genome stability.  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 reporter based assay for HR can be very beneficial for anti-cancer drug discovery projects, learning more about the process of DNA HR repair and establishing intersecting pathways and druggable pathway targets.  This kit contains an HR GFP Reporter gene.  HR is initiated by the addition of Doxycycline, which activates expression of I-Sce1 (mega-endonuclease).  I-Sce1 introduces a DS DNA break at a single unique site in GFP, which activates the HR process.  The system is hosted in a human cell line (Hela).  The Kit is a powerful mechanistic tool to study subtle details of HR in a cellular context.  Therefore, the results have high physiological relevance.

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 loss of template. To deal with the threats posed by DSBs, cells have developed multiple mechanisms to detect, signal, and repair the regions in chromatin.  Two main pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ), are involved in the repair of DSB.  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 be preferred.  More recently, HR has also been shown to operate in mammals.  These pathways are largely distinct from one another, yet function in complementary ways.  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 the HR process (sometimes referred to as gene conversion), a donor DNA sequence with homology to both sides of the DSB supplies genetic information to repair the DSB. The homologous sequence is copied into the broken locus, making the repaired locus an exact copy of donor sequence, without altering the donor sequence (Fig. 1).

This is a cell based/cell context system to allow researchers to examine and interrogate the HR process in live cells.  The HR Kit uses a twin GFP cassette that converts from GFP negative to GFP positive cells using homologous recombination (HR). DNA repair via HR (as gene conversion) will result since a wild type (homologous) GFP segment is present in close proximity to the DNA break.  To introduce a precise DNA cleavage, a mega-endonuclease (I-Sce1) is activated (Tet-on) and introduces a DS break in the GFP locus of Cassette 1 (Fig. 1). The DS break initiates HR and using the WT sequence as a homology template (located in Cassette 2) the gene converts to WT and GFP positive cells appear (Fig. 2).   HR is triggered by a DS break which is achieved by activating the expression plasmid for I-Sce1 (Fig. 2).  Expression of I-Sce1 is controlled by a Tet-on promoter; therefore, adding Doxycycline will induce a synchronous wave of HR in the entire cell population.  This makes analysis simple and improves resolving power by measuring GFP+ cells in the population.  One major advantage is that the process of HR can be tracked at the level of a single cell through live imaging of GFP+ cells (only GFP+ have completed HR).

Kit Contents

  1. iHR-HeLa Cells frozen in cyroprotection medium.  Delivered on Dry Ice.
  2. Doxycycline stock and Puromycin stock (suitable for 25 Assays).
  3. A detailed protocol describing how to set up and use this kit for assaying HR mechanisms in the cell host provided.
Shipping and Storage Instructions:

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.