Altered dopamine release and monoamine transporters in Vps35 p.D620N knock-in mice

Generation of Vps35 p.D620N knock-in mice (VKI). a Schematic of the targeting design showing the murine Vps35 genomic sequence (Ensembl reference ENSMUSG00000031696), 5′ and 3′ homology arms (arrowed), exons 14–17 (blue), engineered loxP site in intron 14, the g.85,263,520G>A (p.D620N) mutation in exon 15 (encoding p.D620N) and endogenous stop codon in exon 17 (TAA). FlpE-recombinase deletion between FRT sites removed the PKG-neo-pA cassette to yield the cVKI allele (not shown). Subsequent Cre-recombinase deletion between loxP sites created the VKI allele. b cDNA sequencing in wild-type mice (top), heterozygous VKI (middle), and homozygous VKI (bottom), between g.8:85263511–85263528 (GRCm38) highlights the g.85,263,520G>A nucleotide mutation in exon 15 that encodes the p.D620N amino acid substitution. c Relative Vps35 mRNA (fold change) expression analysis in cerebellar tissue using quantitative RT-PCR. Data normalized to WT (1-way ANOVA F2,24？=？0.05, p？=？0.95). d Total Vps35 protein levels were quantified by western blotting, showing no difference in Het and Homo mice compared to their WT littermates (1-way ANOVA F2,33？=？0.05, p？=？0.94