Introduction of a frame shift mutation. A The 11097 base pairs of genomic Rag1 are separated in two exons. Of these only Exon 2 contains the complete coding sequence (red bar). Rag1 specific zinc-finger nucleases recognizing 18 bp upstream and downstream of the 6 bp cutting site between position 5233 and 5267 introduced DSBs through the FokI endonuclease domain and initiated NHEJ. BCel-I endonuclease digest of the 310 bp heteroduplex DNA derived from the hybridization of the DNA from LEW wild type rats and founder 58 into 174 bp and 136 bp fragments proofed the mutagenesis of one Rag1 allele in founder 58. G/C: heteroduplex control DNA; LEW: LEW/Ztm wild type DNA.
Zschemisch et al. BMC Immunology 2012 13:60 doi:10.1186/1471-2172-13-60