Abstract
Repair of double strand break by homologous recombination was examined using U2OS cells or RG37 cells harbouring specific substrate developed by Puget et al. (2005) and Dumay et al. (2006), respectively, to measure the repair of DNA double strand breaks by homologous recombination. The substrate is composed of two inactive copies of the GFP gene. The upstream copy is inactive due to the absence of promoter, the downstream copy present a promoter but is inactivated by the insertion of the sequence coding for the recognition site of the I-SceI enzyme. The substrate is stably expressed in cells after its insertion in the genome and present as a unique copy. The unique DNA double strand break is then induced by the expression of the I-SceI enzyme after cell transfection with a plasmid coding for the I-SceI enzyme.
Keywords: DNA double strand break repair, Homologous recombination, Human cells, DNA repair, Genetic instability
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from previous works by Dumay et al. (2006) and Puget et al. (2005). We acknowledge the use of the Toulouse Rio Imaging facilities for flow cytometry analysis. This work was supported by grants from the Ligue Nationale Contre le Cancer (D. Trouche; équipe labellisée), the Association de Recherche contre le Cancer (ARC) as a Programme ARC, the Agence Nationale pour la Recherche (Projet 2011 blanc SVSE8 PinGs), and by an Electricité de France grant (Y. Canitrot).
References
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