Targeted mutation of zidh1 using TALENs recapitulated the hematopoietic defects of zidh1^MO. (A) Target site of zidh1 by TALEN. The binding sequence for the TALEN pair were highlighted in cyan (indicated by “left” and “right”) and the BsmI site in yellow. The start codon of zidh1 was bold. E, exon. (B) BsmI digestion of PCR products from pooled genomic DNA of 5 embryos injected with zidh1 TALEN mRNAs in 3 experiments. The cleaved and uncleaved PCR products are indicated. M, marker; WT, wild-type. (C) Representative sequences of uncleaved PCR products. (D) Expression of pu.1 at 18 hpf, l-plastin, mpo, and mpeg1 at 24 hpf; c-myb at 48 hpf; and rag1 at 96 hpf in control and zidh1 TALEN mRNA-injected embryos. (E) The numbers of l-plastin–, mpo-, and mpeg1-positive cells per embryos were counted and are expressed as mean ± standard error of the mean. The numbers in each panel indicates the number of embryos with representative phenotype out of the total number of embryos evaluated. Bars represent 250 μm. (F) Genotyping of 12 zidh1 TALEN and wild-type fish at 3 months old, showing near 100% knockout efficiency based on Bsml digestion of PCR products. The uncleaved (TALENed) and cleaved PCR products are shown. (G, left and middle) May-Grunwald/Giemsa staining of the KM showing hematopoietic cells of different morphologic lineage. Arrowheads, myelomonocytes; asterisks, lymphocytes; black arrows, precursor cells; red arrows, erythrocytes. Original magnification, ×400. (G, right) Mean percentage of nonerythroid cells in the KM, showing statistically significant increase in precursor cell population (5 wild-type and 4 zidh1 TALEN F0 fish were examined). (H, left and middle) Contour plot showing the myelomonocytic (red), precursor (green), lymphocytic (blue), and erythroid lineages (purple). (H, right) The histogram shows the average results of 12 wild-type and TALEN F0 adult fish. n.s., not significant.