Characterization of tirap mutant zebrafish larvae. (A) Mutant DNA and protein sequence of Tirap. A point mutation (T to A) in the TIR domain of zebrafish tirap results in a stop codon. (B) Proportion of genotypes. The proportion of genotypes after the incross of tirap+/- heterozygous. (C) Experimental scheme of body length measurement. (D) Development. The body measurement of tirap+/+ and tirap-/- zebrafish at 5 days post fertilization (5 dpf). The measurements are based on 3 independent experiments with tirap+/+ (n=52) and tirap-/- (n=52). (E, F) qPCR of tirap+/+ and tirap-/- zebrafish embryos. Tirap+/+ and tirap-/- embryos were injected with purified LPS (100 µg/ml) or sterile water as a control at 27 hour post fertilization (hpf). The expression level of il1b and fosl1a were analyzed at 1 hour post injection (hpi) by qRT-PCR. Data (mean ± SD) are combined with 3 biological replicates (n=15 embryos/group). Statistical significance was determined by two-way ANOVA with Tukey's multiple comparison method as a post-hoc test. (G, H, I) Experimental scheme and representative image of macrophages and neutrophils in zebrafish larvae (tirap+/+ and tirap-/-). Scale bar: 50 µm. (J, K) Quantification of neutrophils and macrophages number in tirap mutant and wild-type zebrafish larvae. Number of neutrophils and macrophages in zebrafish larvae under unchallenged conditions. The number of neutrophils for tirap+/+ (n=33) and tirap-/- (n=27) and the number of macrophages for tirap+/+ (n=36) and tirap-/- (n=28) are based on two independent experiments. Statistical significant difference was determined by unpaired t-test, ns, non-significant.
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