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Zebrafish fgfr4 knockout validated by real time quantitative polymerase chain reaction (RT-qPCR). (A) Schematic of wildtype zebrafish fgfr4 mature mRNA transcript and regions targeted for RT-qPCR. Blue triangle indicates approximate fgfr4 crispant mutation site for all strains relative to primers. Orange box indicates region 5’ of mutation site targeted by RT-qPCR primers, and green box indicates region 3’ of mutation site targeted by RT-qPCR primers. Relative expression of fgfr4 mRNA as measured by RT-qPCR targeting the regions 5’ (B) and 3’ (C) of the sequence mutation. Expression is normalized to gapdh and rpl13a. Each point represents a pool of n = 12, 24 hours post-fertilization (hpf) zebrafish embryos derived from a maternal zygotic. Multiple points represent biological replicates, while three technical replicates were used to generate each biological replicate. Error bar is the mean ± standard deviation. P values were calculated using a one-way Brown-Forsythe and Welch ANOVA, correcting for multiple comparisons with a Dunnett T3 test.
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