Construction of bcas3 KO zebrafish by CRISPR/Cas9 technology. (A), Whole-mount in situ hybridization (WISH) for bcas3 mRNA in WT zebrafish embryos at different development stages. Scale bars: 200 μm. (n = 20 embryos per group). WT: wild type. (B), Temporal pattern of mRNA expression of zebrafish bcas3 gene at different stages. mRNA extracts from whole-embryos were analyzed by RT-PCR for bcas3 mRNA levels at different zebrafish embryo stages. (n = 20 embryos per group). (C), Schematic diagram shows the genomic structure of zebrafish bcas3 with all 24 exons and the site of genome editing marked. The gene editing target site is located in the 12th exon of bcas3 gene. Target sequences are underlined. PAM sequences are highlighted in red. (D), DNA sequence analysis identified the bcas3−/− insertion mutant zebrafish line with a 22 bp insertion in exon 12 of bcas3 gene. (E), The 22 bp insertion in bcas3 causes frame-shift and is predicted to cause a premature stop codon, resulting in a truncated mutant bcas3 proteins with only 302 amino acids left. (F-G), Real-time RT-PCR analysis showed that the bcas3 mRNA level in 10 hpf (hours post fertilization) embryos and brains of 4 mpf (months post fertilization) bcas3−/− zebrafish was reduced compared to WT controls. (n = 3, per group). The rpl13a gene was used as endogenous control. Data are shown as mean ± SEM. Unpaired Student’s t-test was used to analyze RT-qPCR. Significance levels are denoted as follows: **P < 0.01, ***P < 0.001
|
