The structure of the plasmids used to monitor NHEJ, GCRs, and large deletions.

<p>(A) NHEJ was monitored using the pEJ5-GFP plasmid integrated at interstitial (not shown) or telomeric sites (shown). pEJ5-GFP contains a GFP gene that is initially inactive due to the presence of a puromycin-resistance (puro) gene located between the GFP gene and its promoter. NHEJ occurring between the distal ends of the I-<i>Sce</i>I-induced DSBs at either end of the puro gene results in the activation of the GFP gene. A PCR product generated with oligonucleotide primers spanning one of the I-<i>Sce</i>I sites in the pEJ5-GFP plasmid (red arrows) was digested with I-<i>Sce</i>I endonuclease to determine the frequency of small deletions at a single I-<i>Sce</i>I-induced DSB. Cell clones containing the pGFP-ISceI plasmid integrated at interstitial sites (not shown) or telomeric sites (shown) were used for analysis of large deletions that inactivate the GFP gene. (B) GCRs were monitored using cell clones that contain the pEJ5-GFP plasmid integrated at an interstitial (not shown) or telomeric (shown) site and the pDsRed-ISceI plasmid integrated at an interstitial site at a different location in the genome. The DsRed gene in the pDsRed-ISceI plasmid is initially inactive due to the lack of a promoter, but becomes activated following NHEJ between the I-<i>Sce</i>I-induced DSBs in the pEJ5-GFP and pDsRed-ISceI plasmids. The location of oligonucleotide primers used for PCR to analyze the junctions between the pEJ5-GFP and pDsRed-ISceI plasmids are shown (red arrows). The location of the ampicillin gene and plasmid origin of replication (Amp/ori), chicken β-actin promoter (promoter), puro gene (Puro), GFP coding sequence (GFP), and telomere are shown. Also show is the genomic DNA (solid line), directions of transcription (black arrows), and I-<i>Sce</i>I recognition sites (I-<i>Sce</i>I).