Fig. 1. Generation of Vps13b2−/− mice. (A) A schematic view of the CRISPR/Cas9-mediated generation of Vps13b mutant mice lacking exon 2. The nucleotide and amino acid sequences of wild type (WT) and mutant alleles are shown. The start codon (ATG) is indicated in red and the targeting sites for sgRNAs are underlined in blue. (B) A representative PCR genotyping result for Vps13b WT and mutants. (C) Relative Vps13b mRNA levels in the hippocampus of wild type, heterozygous and homozygous mutant mice measured by quantitative RT-PCR spanning exon 2–3. The mRNA levels were normalized by Gapdh mRNA levels. Vps13b2+/+, n=3 mice; Vps13b2+/−, n=2 mice Vps13b2−/−, n=4 mice. Experiments were triplicated. Data were represented as mean±SEM. (D) Schematics of genomic DNA and mRNA of Vps13b. The target sites of the primers used for RT-PCR are indicated by red arrows. (E) Conventional RT-PCRs were performed by using primers targeting different exons as indicated and cDNAs synthesized from total RNA extracted from the hippocampus. Note that primers targeting exons 2–3 did not produce any PCR product in Vps13b2−/− as shown in (C). Consistently, primers targeting exons 1–4 produce the shorter PCR product, confirming that the exon 2 is deleted in Vps13b2−/−.
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