Making New Cell Lines with the NHEJ Repair Reporter System installed.

Summary: The DR150 kit will allow you to install the NHEJ reporter in any cell line that can be stably transfected.  NHEJ is activated after transfecting the newly engineered cell line with the mega-endonuclease I-Sce1.

Details:  This is a customized cell-based reporter system designed to examine NHEJ in any cell line.  It is useful to screen or identify agents (drugs, natural products, small molecules, synthetics, miRNAs, and genes) that affect or impact NHEJ DNA repair in virtually any cell lineage. The kit uses GFP as an in situ readout for the NHEJ pathway.  We include all plasmids necessary to program the in any line that can be transfected (i.e., the NHEJ Reporter DNA plasmid (NHEJ-GFP) may be placed  in the customers cell line model.  Also include is the I-Sce1 expression plasmid to allow the customer to activate NHEJ DNA repair in newly made cell lines.

The Figures show the performance of the system: Fig 1 shows the NHEJ genetic construct and illustrates the formation of WT GFP following I-Sce1 transfection.  Fig 2 shows FACS scatter plots at time zero, and 48h after I-Sce1, in addition to tracking of the same single cell before and after 36 or 48h of NHEJ.  The live imaging data clearly show the clonal nature of NHEJ derived WT-GFP cells and that these cells form daughter cell descendants.  Thus, the GFP+ cells are healthy and viable. 

Included: 

1. NHEJ-GFP plasmid (store at -20^o or 4^oC). DNA concentration is described on the tube label  (a total of 50ug). This plasmid has a G418 marker for selection.  This is the NHEJ reporter plasmid that will be stably transfected in your cells of interest.
2. pSCE1 expression plasmid (DNA concentration is described on the tube label, containing a total of 50ug). ). There are no selectable markers with this plasmid.  This is the plasmid to be used in transfecting newly engineered cells of your choosing to initiate NHEJ in the cells.
3. G418 (Neo) at 50mg/ml (1mL total).
4. Fully detailed instructions on how to create and test new cell lines.