Figure 1.

The rps19 gene in zebrafish is targeted using TALENs. (a) Partial structure and sequence of the zebrafish rps19 gene. The binding sites of the left and right TALENs were underlined. The intron sequence was shown in lowercase and the exon in uppercase. P1 and P2 showed the primer sites for amplification. E2, E3, and E4 represent the second, third, and fourth exon of the rps19 gene, respectively. (b) Identification of mutations by enzyme digestion. PCR products (∼305 bp) were processed with an enzyme that can recognize and cut at mismatch region. The presence of lower bands (∼135 bp and ∼170 bp) suggested a mutation. (c) Genomic sequences of the rps19 mutations. Deletions and insertions were shown in dashes and red letters, respectively. The premature stop codons produced in the two independent mutations were underlined. (d) Real-time PCR result showed rps19 gene expression was reduced by more than 99% in mutant 1. ***P<0.001; Student's t-test). W, WT control; M, mutant; IC, internal control; L, DNA Ladder; bp, base pair