Figure 1

Generation and validation of vma21 mutant zebrafish.

(A) Schematic of the zebrafish (Danio rerio) vma21 gene. Mutations in exon 2 of vma21 were created using CRISPR-Cas9 gene editing. (B) Two mutant lines were generated: (1) a 1 base pair (bp) deletion and (2) a 14 bp deletion with a 21 bp insertion. A compound heterozygous line was also generated by crossing the two mutant lines together. (C) Representative western blot of Vma21 and b-actin (housekeeping protein) protein levels in both the vma21 1 bp deletion (vma21^Δ1/Δ1) and the 14 bp deletion with 21 bp insertion (vma21^{Δ14ins21/Δ14ins21}) homozygous mutants compared to their respective wild-type/heterozygous sibling controls at 5 days post fertilization. Vma21 is ~11 kDa in size and b-actin is ~42 kDa. (D) Quantitative analysis of Vma21 western blot normalized to b-actin loading controls shows that Vma21 protein levels are significantly reduced in both vma21 homozygous mutants (fivefold less) compared to their respective sibling controls. Each western blot had a total of three biological replicates. For each sample, n = 30 zebrafish were utilized. All values are shown as mean ± SEM. Significance at **P < 0.01; ***P < 0.001 (unpaired two-tailed t test). Source data are available online for this figure.