Fig. 1

Generation of capn3b null mutant allele. a Generation of the capn3b^Δ19Δ14 mutant allele. Upper panel: diagram showing the genomic structure of the zebrafish capn3b gene and the two mutated sites in capn3b^Δ19Δ14 generated by TALEN and CRISPR-Cas9 approaches, respectively. Vertical bar: exon; line connecting vertical bar: intron. Lower panel: highlighting the 19 bp deletion (red letters) in 1st exon generated by the TALEN approach (on the left) and 14 bps deletion (red letters) in 3rd exon by the CRISPR-Cas9 approach (on the right), respectively. The position of nucleotide in the capn3b open reading frame (ORF) from the translation start codon ATG is provided. C120, Capn3b activity center. b Predicted peptide encoded by the capn3b^Δ19 (∆19) and capn3b^Δ19Δ14 (Δ19Δ14) mutant mRNA, respectively. The Δ19 mutation leads to an early stop codon in the capn3b ORF and resulted in a 30 amino acids (aa) long polypeptide, however, the Δ19 mutation potentially allows the ATG codon encoding M⁹⁴ residue of WT Capn3b to be used as an alternative translation start codon to translate an N-terminus truncated peptide which harbors the activity center C120. The Δ19Δ14 mutation creates a new early stop codon in the presumed variant initiated from M⁹⁴ after C120, therefore, capn3b^Δ19Δ14 is likely a null allele. c Western blotting analysis of Capn3b in WT, capn3b capn3b^Δ19Δ14, capn3b^Δ19 at 5dpf. β-Actin: loading control. d Immunostaining of Capn3b in WT and capn3b^Δ19Δ14 mutant embryos at 5dpf. Scale bar: 50 μm