Fluorescence-based quantification of pathway-specific DNA double-strand break repair activities: a powerful method for the analysis of genome destabilizing mechanisms.
This chapter provides instructions for the application of a fluorescence-based assay to examine different DNA double-strand break (DSB) repair pathways in primary mouse embryo fibroblasts (MEFs). The assay relies on targeted DSB formation in one of a series of repair substrates and subsequent repair-mediated reconstitution of the EGFP reporter. We present protocols for efficient introduction of extra-chromosomal repair substrate together with I-SceI endonuclease expression vector and subsequent measurement of DSB repair events down to frequencies of 0.001%. Concomitant transfection of plasmid and siRNA enables assessment of DSB repair under conditions of knockdown of protein expression, allowing to evaluate the contribution of single factors. Since the proteins of interest frequently have dual roles in DSB repair surveillance and checkpoint control, our assay procedure concomitantly corrects for transfection efficiencies, growth-, death-, and expression-related changes and also integrates the examination of the cell cycle status.
Böhringer
Michael
M
Department of Obstetrics and Gynaecology, University of Ulm, D-89075 Ulm, Germany.
Wiesmüller
Lisa
L
D000818:Animals; D002453:Cell Cycle; D004249:DNA Damage; D004260:DNA Repair; D005453:Fluorescence; D016678:Genome; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins