Reporter constructs for analysis of DSB repair. (A) Reporter plasmid for analysis of NHEJ. The reporter cassette consists of a GFP gene under a CMV promoter with an engineered intron from the rat Pem1 gene, interrupted by an adenoviral exon (Ad). The adenoviral exon is flanked by I-SceI recognition sites in inverted orientation for induction of DSBs. In this construct, the GFP gene is inactive; however, upon induction of a DSB and successful NHEJ, the construct becomes GFP+. SA indicates splice acceptor; SA, splice donor; shaded squares, polyadenylation sites. (B) Reporter plasmid for analysis of HR. The reporter cassette consists of two mutated copies of GFP-Pem1. In the first copy of GFP-Pem1, the first GFP exon contains a deletion of 22 nt and an insertion of two I-SceI recognition sites in inverted orientation. The 22 nt deletion ensures that GFP cannot be reconstituted by a NHEJ event. The second copy of GFP-Pem1 lacks the ATG and the second exon of GFP. Upon induction of DSBs by I-SceI, gene conversion events reconstitute an active GFP gene. (C) Repair products of the reporter plasmid for the analysis of HR shown in (B). GC indicates gene conversion; SSA, single-strand annealing. Only gene conversion leads to reconstitution of the GFP activity.