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Fig. 2
Neutrophil-specific knockout of polg reduced neutrophil motility. (A). Schematic of the design of gateway vectors to clone constructs for neutrophil-specific knockout. (B) Schematic of the gene structure and protein domains of zebrafish polg gene. The sgRNA targets exon 15 in the forward strand. (C) Sequences of sgRNAs (control or polg) produced using our vectors. Note that the first G (blue) is included in the backbone. (D,E) Neutrophils were sorted from 3 dpf embryos of Tg(lyzC:nls-cas9-2A-mCherry/U6a/c:control sgRNA)pu15 (TS-ctrl) and Tg(lyzC:nls-cas9-2A-mCherry/U6a:polg sgRNA)pu16 (TS-polg) lines. T7E1 assay was performed to show the in vivo editing of the polg locus (D), and the ratio of mitochondrial DNA/nuclear DNA content was measured by qPCR in two separate sorts (E). ***P<0.001, **P<0.01 by one-way ANOVA. (F,G) Representative images (F) and quantification (G) of neutrophil motility in the head mesenchyme of 3 dpf larvae. One representative result from three biological repeats is shown. n=64 for TS-ctrl (ctrl) and n=76 for TS-polg (polg) from four different larvae. ****P<0.0001 by Mann–Whitney test. Scale bar: 50 µm.