Knockout of Zebrafish arl4aa Perturbed Definitive HSC Development in F0 Somatic Mutants (A) Knockout of arl4aa by a TALEN pair (E2-2) targeting the translation start site. Upper panel: the TALEN pair; middle panel: genotyping of single embryos (numbers 1–5) injected with TALEN E2-2. Mean knockout efficiency was 51% ± 5.5% (n = 5); lower panel: DNA sequence of three mutant clones from F0 embryos. (B) Knockout of arl4aa by two TALEN pairs targeting exons 1 and 2 (E1 + E2). Upper panel: the TALEN pairs, primer positions indicated by red arrows. Middle panel (top): genotyping of single embryos (numbers 1–4) injected with E1 + E2, showing an amplicon of 319 bp. Middle panel (bottom): knockout efficiency was based on qPCR amplifying arl4aa intron 1 existing fold, which was deleted by E1 + E2. Mean knockout efficiency was 26.3% ± 2% (n = 10). Knockout efficiency was correlated with hematopoietic phenotype, being 31% ± 2.6% (n = 5) and 21.6% ± 0.7% (n = 5) for embryos whose c-myb expression was completely absent and reduced. Lower panel: DNA sequencing of three mutant clones from F0 embryos. (C) c-myb expression in control (left arm TALEN E2-2L) (upper), E2-2 injected (middle), and E2-2 and arl4aa mRNA co-injected (lower) embryos at 48 hpf. (D) c-myb expression in control (top), E1 + E2 injected embryos showing absent (second) and reduced (third) c-myb expression, and E1 + E2 and arl4aa mRNA co-injected (bottom) embryos. Data are shown as means ± SEM. Scale bars, 250 μm (in WISH). The numbers in each WISH showed the numbers of embryos with typical gene expression as shown over the total number of embryos injected in each group.
|
