Construction of a R728Q KIF4 transgenic mouse. (A–D) Red (functional mutation) and dark-blue (XhoI site) letters indicate the substitution target site. (A) Schematic illustration showing the locations of the gRNAs and ssODNs. Black bar indicates the position of the gRNA target with the orange bar showing the PAM sequence. ssODNs are shown in green letters (intron area) and blue letters (exon area). (B) The amino acid sequences from wild type (WT) and substituted sequences are shown, with red letters indicating the target and the substitution. (C) WT and donor DNA sequences are displayed in the top panel. The lower panel shows the electropherogram of the direct sanger-sequencing result, in which one of the first-generation male mice received the specific substitution. (D) Schematic illustration showing the locations of the PCR primers, along with the mouse Kif4 locus. Black bars on both sides indicate the position of the PCR primer sequences. (E) Representative image of the genotyping results. The products from genomic PCR of mouse tail DNAs were the same length in the WT and the substituted. Subsequently, XhoI digestion was performed to distinguish the individual genotypes. The blue arrow indicates WT Kif4 and the pink arrow indicates Mut Kif4.