Figure 5. Generation of conditional knockout mice.

(a) Schematic illustration to generate a conditional allele of the paxillin gene by insertion of two LoxP sites. The sequences of CRISPR RNA (crRNA) to target the Cas9 nuclease to intron 5 (left) and intron 6 (right) are shown in red. The protospacer adjacent motif (PAM) is shown in green. In the oligo donor sequence, the LoxP site is highlighted in yellow. (b) PCR screening using mutant-specific primers identified one (#5) of 12 mice had successful insertion of both LoxP sites in the paxillin allele. (c) Sub-cloning sequencing confirmed the precise integration of LoxP sites in mouse #5. (d) PCR genotyping of paxillin floxed (F) mice crossed with Sox2-Cre mice using primers spanning the two LoxP sites. The presence of an amplified DNA fragment (440 bp) indicates that DNA between the two LoxP sites was deleted by Cre recombinase.