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Nonhomologous end-joining (NHEJ) repair assay

SS Steven T. Sizemore
MZ Manchao Zhang
JC Ju Hwan Cho
GS Gina M. Sizemore
BH Brian Hurwitz
BK Balveen Kaur
NL Norman L. Lehman
MO Michael C. Ostrowski
PR Pierre A. Robe
WM Weili Miao
YW Yinsheng Wang
AC Arnab Chakravarti
FX Fen Xia
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The system used to measure NHEJ repair of I-SceI-induced DSBs has been described previously.17 Briefly, pEJ is an NHEJ-dependent GFP-reporter assay stably integrated into the genome of the U87 cells. GFP translation is prevented in this reporter by the presence of an out-of-frame ATG in the 5′-untranslated region between the CMV promoter and the GFP open reading frame. Two repeated I-SceI sites in inverted orientation flank this out-of-frame ATG. Cleavage by I-SceI removes the out-of-frame ATG and creates non-cohesive double-stranded ends. Repair of this DSB by NHEJ results in GFP translation, which was measured by flow cytometry (FACSort, Becton-Dickinson).

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