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Fig. 2 Construction of tdh−/− zebrafish line using CRISPR/Cas9 system. (A) Schematic diagram of the targeting strategy and validation of the result by genomic DNA sequencing. The mutant allele harbors a 5-bp deletion (red box) in the tdh locus that causes a premature stop codon occurred at H213 with three extra AAs. The sequenced region, containing intron and exon parts, is the gRNA-targeting region. Dr, Danio rerio; MTS, mitochondria targeting sequence; Tdh, l-threonine dehydrogenase. (B) RT-qPCR analysis of tdh mRNA expression in wild-type (WT) and tdh−/− embryos at 3 and 5 dpf. Data are presented as the mean ± SD of triplicate reactions; two-tailed t-test; ****P < 0.0001. (C) Whole-mount in situ hybridization (WISH) analysis of tdh mRNA expression in WT and tdh−/− embryos at 48 h post fertilization (hpf). The close-up view of the tail region is shown in the lower panels. Red arrows denote the caudal hematopoietic tissue (CHT) of WT or tdh−/− zebrafish. Scale bar = 500 μm.