Figure 1.

Generation of p53 knockout golden Syrian hamsters using the CRISPR/Cas9 system. (a) Schematic of the golden Syrian hamster p53 genomic locus showing the exons and gene targeting site. The sequence of sgRNA in green letters, with the protospacer adjacent motif in red letters to target a region of exon 5 in the p53 golden Syrian hamster gene. The blue two arrows indicate the locations of polymerase chain reaction primers for the sequence. (b). Genotyping results by the Cleavage Detection assay on three golden Syrian hamster pups produced by pronuclear injections of sgRNA/Cas9-p53. Pups F0#1 and F0#3 are wild type; pup F0#2 is genetically modified in the p53 locus. (c) The frequencies of DNA sub-clones with F0#2 are indicated by flanking regions surrounding the sgRNA targeting site identified F0#2 with frame shift mutations. −21 bp: 21-nucleotide deletions; +1 bp: one-nucleotide insertion. (d) Genotyping results with the Cleavage Detection assay on the eighteen F1 pups produced by breeding founder animal F0#2 with a wild type littermate. (e) Subcloning sequencing shows an individual clone has either wild type or p53 knockout alleles. Comparison between wild type sequence and the sequencing result of F1#15 at the p53 genomic locus. The red box shows the insertion of 1 nucleotide in p53 genomic loci. (f) Western blotting assay to compare the expression of p53 protein in wild type and homozygous one-nucleotide insertion hamsters