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CRIPSR-generated mutation in acmsd causes nonsense-mediated decay of resulting mRNA. A Zebrafish express a single Acmsd protein, sharing 81% protein identity to human ACMSD. A mutation was introduced into exon 6 using CRISPR/Cas9 resulting in a predicted truncated protein. Red region depicts altered amino acids in the mutated protein. B The resulting 70 bp reduction in DNA length allowed for genotyping by standard PCR. acmsd+/+ zebrafish were identified by a single 425 bp band (right), homozygous acmsd-/- mutants by a 355 bp band (left), and heterozygous acmsd+/- mutants by a double band (centre). C DNA sequence of acmsd exon 6 and intron 6. acmsd-/- zebrafish possessed a 71 bp deletion (red) and 1 bp insertion (blue) in this region, resulting in the loss of a 3’ splice site (arrows). acmsd-/- larvae demonstrated reduced acmsd expression compared to their acmsd+/+ siblings at 5dpf (D, n = 3 biological replicates (15 larvae per replicate), p = 0.0012) and in adult brain tissue (E, n = 6 biological replicates (1 brain per replicate), p = 0.0040). Statistics from two-tailed t tests using ddCt values.
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