Fig. 3.

Generation of knock-in mice at the Spp1 locus. (A) Schematic illustration to generate a PITChed allele at the Spp1 locus, mediated by the CRIS-PITCh (v2) system. Four glutamine residues in exons 3 and 4 (three in exon 3 and one in exon 4) were replaced with alanine residues (red stars). Two gene-specific gRNAs were designed within exon 3 and downstream of exon 4. A PITCh donor plasmid was designed to carry the substituted sequences encoding four alanine residues, silent mutations for RFLP analysis (from T to G in exon 3 and from G to A in exon 4) and a point mutation (from T to C) in intron region. Bold black arrows indicate primers for PCR. Yellow and blue arrows indicate the recognition sites of restriction enzymes for the RFLP analyses. (B) Sequence analysis of subcloned PCR products from pups harboring the knock-in allele. The sequences around exon 3 and exon 4 are displayed in the upper and lower panels, respectively. The modified codons encoding four alanines are enclosed in red boxes. The silent mutations for RFLP analyses are enclosed in green boxes. The gRNA-blocking mutation is enclosed in a purple box. The wild-type allele is shown at the top (Spp1 Wild) with the gRNA target sequences (underlined in yellow and blue). The PAM sequences are enclosed in yellow and blue boxes. Uppercase letters indicate exon sequences. Dots indicate the same bases as the wild-type sequence. Dashes indicate deletions. Unintended mutations are enclosed in black boxes. Black underlined sequences indicate NarI and DraI sites in exons 3 and exon 4, respectively.