Generation of capn3b null mutant allele. a Generation of the capn3bΔ19Δ14 mutant allele. Upper panel: diagram showing the genomic structure of the zebrafish capn3b gene and the two mutated sites in capn3bΔ19Δ14 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 capn3bΔ19 (∆19) and capn3bΔ19Δ14 (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, capn3bΔ19Δ14 is likely a null allele. c Western blotting analysis of Capn3b in WT, capn3b capn3bΔ19Δ14, capn3bΔ19 at 5dpf. β-Actin: loading control. d Immunostaining of Capn3b in WT and capn3bΔ19Δ14 mutant embryos at 5dpf. Scale bar: 50 μm
This image is the copyrighted work of the attributed author or publisher, and
ZFIN has permission only to display this image to its users.
Additional permissions should be obtained from the applicable author or publisher of the image.
Full text @ Cell Regen (Lond)