TALEN-mediated targeted mutagenesis of bcas2. (A) A schematic diagram showing the genomic organization of bcas2 with the first 4 exons (E1-E4) and connecting lines depicting introns and the location of the TALEN pair used to generate bcas2 mutants. P1 and P2 show the primer sites for amplification. The NcoI restriction site in the spacer region is used for mutation detection. (B) DNA sequencing identified a deletion of 5 bases in the third exon of bcas2, which forms a premature stop code in the coding region of bcas2 in the mutant line. (C) Real-time PCR analysis shows a 50% decrease in bcas2 mRNA expression in heterozygous embryos and almost undetectable bcas2 mRNA in bcas2^−/− mutants at 3 dpf. (D) Western blot analysis shows similar expression of BCAS2 in siblings and heterozygous embryos but greatly decreased expression in bcas2^−/− mutants at 3 dpf.