Fig. 2.

Generation of base-substituted mice at the Spp1 and Tyr loci. (A) Schematic illustration to generate a three-base-substituted allele at the Spp1 locus. A serine residue in exon 5 was replaced with an aspartic acid residue (TCA to GAC; red letters). A gRNA was designed to cut in close vicinity of the serine residue (underlined in black and red). An ssODN was designed to carry the three-base substitution. (B) Schematic illustration to generate a one-base-substituted allele at the Tyr locus. A guanine in exon 1 was changed to thymine (red letter). An ssODN was designed to carry the one-base substitution. Black boxes indicate the protospacer adjacent motif (PAM) sequences. Arrows indicate the primer sets for PCR. Blue underlined sequences indicate the recognition sites of restriction enzymes for the RFLP analyses.