FIGURE 1.

Generation of zebrafish per2 mutant. A, the TALEN sites targeting the second exon of per2 gene. The left and right TALEN sites are highlighted in cyan, and the BslI site in the spacer in yellow. B, TALEN efficiencies shown by gel analysis. The targeted fragment was PCR-amplified from pooled genomic DNAs of five embryos microinjected with capped TALEN mRNAs at a concentration of 250 ng and then digested with BslI. The uncleaved and cleaved PCR products are indicated. Mutagenesis efficiencies were estimated by the ratios of intensities of uncleaved bands and the sum of cleaved bands and uncleaved bands quantified with NIH ImageJ software. WT, wild type; M, marker. C, types of indel mutations in the per2 TALEN target site shown by representative sequencing results of the uncleaved PCR fragments. D, screening of heritable mutants. F₀ founder fish were out-crossed with wild-type fish to produce F₁, and the DNAs extracted from F₁ embryos were used for identifying mutant fish. Two of 15 fish were found to carry heritable mutations (lanes 4 and 10). E, two mutated fish lines. One has an 11-bp deletion, the other has a 13-bp insertion (upper), and both are frameshift mutations that result in truncated proteins (lower). AA, amino acids.