Figure 3
Identification of CRISPR/Cas9-mediated multiplexed genome-modified mice targeted to Agbl1 and Agbl2 loci by PAGE-based genotyping analysis.
SgRNAs targeting Agbl1 and Agbl2 loci were mixed prior to microinjection into the cytoplasm of oocytes. Pups from F0 generation were genotyped for targeted modification in Agbl1 and Agbl2 loci. Results from agarose gel electrophoresis of PCR products are shown for the Agbl2 (A) and Agbl1 (D) loci. (B) Using the PAGE-based genotyping protocol, genomic indel mutations in the Agbl2 locus were detected in mice # 4, #8, #9 and #11, while all others and wildtype controls exhibited only homoduplex DNA. (C) Sequence analysis confirmed the identification of indel mutations in mice #4, #8, #9 and #11 from Figure 3B. (E) PAGE analysis identified heteroduplex DNA from the Agbl1 locus in mice #2, #4, #9, #10 and #12. (F) Sequencing analysis confirmed the identification of indel mutations in mice #2, #4, #9, #10 and #12 from Figure 3E. Note that mice #4 and #9 (shown in red) possessed indel mutations in both Agbl2 and Agbl1 loci. M, DNA marker; WT, wildtype; Mut, mutant.
