Generation of scamp5a KO zebrafish using the CRISPR/Cas9 system. (A), Schematic diagram shows the genomic structure of scamp5a with 7 exons, indicating the site of genome editing. The target site is located in the fourth exon of the scamp5a gene. Target sequences are highlighted in red with underline. PAM sequences are highlighted in blue. (B), DNA sequence analysis identified the scamp5a mutant zebrafish line with a 14 bp deletion in exon 4 of scamp5a gene. WT, wild type. (C), The 14 bp deletion in scamp5a causes frame-shift and is predicted to cause a premature stop codon, resulting in a truncated mutant Scamp5a proteins with only 83 amino acids left. (D), Kaplan-Meier survival curve of WT, scamp5a+/-, scamp5a-/- larvae (log-rank Mantel-Cox test). (E), Lateral view of WT and scamp5a-/- larvae at 2.5 dpf. Scale bars: 200 μm. (F), The body length of WT and scamp5a-/- larvae at 2.5 dpf. WT (n=37), scamp5a+/- (n=70) and scamp5a-/- (n=35) larvae. The data in panel (D) and (F) are presented as mean ± SEM. n≥ 3 independent experiments. Significance levels are denoted as follows: ns, not significant, **P<0.01, ***P<0.001
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