Generation of R26NHEJ knock-in mouse model.

<p>(<b>A</b>) The pROSA26PA-NHEJ vector containing NHEJ reporter construct targeted to the ROSA26 genomic sequence. The construct consists of GFP exons separated by the Pem1 intron, interrupted by the killer exon Ad2. Flanking Ad2 are unique I-SceI recognition sites for inducing DSB. Successful NHEJ repair leads to the reconstitution of GFP. Two <i>loxP</i> sites in direct orientation flanking Neomycin/Kanamycin genes (Neo/Kana) and Bacterial origin of Replication (OriC) are located downstream. This vector was targeted by homologous recombination into the endogenous ROSA26 locus of C57BL/6 mouse ES cells. (<b>B</b>) NHEJ construct integrated into ROSA26 locus in the mouse genome. (<b>C</b>) DNA from G418-resistant ES cell clones was digested with <i>Bam</i>HI and hybridized to a GFP probe (indicated in B). (<b>D</b>) Founder mice were genotyped using PCR primers indicated in (B). The positive control lanes contain PCR reactions with genomic DNA from ES cell clones shown in (C) as a template. Negative control lane contains PCR reactions with DNA from a wild-type C57BL/6 mouse as template and is followed by a No template control.