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Fig. 2.Generation of a Xenopus tropicalis CRISP/Cas9 sf3b4 mutant line. (A) Schematic of the genomic sf3b4 locus, and Sf3b4 protein. The relative position of the sgRNA target sequence is indicated (red flags). CDS1-CDS6 represent exons 1-6. (B) Alignment of wild-type (WT) and 5bp or 31bp mutated nucleotide sequences. The PAM sequence is indicated (magenta). A frameshift in the mutant nucleotide sequence results in an incorrect protein sequence with an early stop (asterisk) after amino acids 5 and 28, respectively (cyan). Shown underlined is the targeted nucleotide sequence. (C) Western blot analysis of protein extracts from WT (+/+), heterozygote (+/) and homozygote (/) F2 tailbud-stage embryos (NF stage 25), using an anti-Sf3b4 antibody. -Tubulin is shown as a loading control. n=10. (D) ImageJ quantification of the western blot. (E) sf3b4 mutant embryos at stage 25 have reduced head length and width compared to WT. The distances measured to quantify head length (L) and depth (D) are indicated on the embryo at the top for each genotype. Lateral views, anterior to right, dorsal to top. (F) Graph plotting the length and width for all three genotypes. ns, not significant. *P<0.05, **P<0.01 (Welch's two-tailed unpaired t-test). n=9 (WT), 14 (Het), 8 (Null). Scale bars: 150m.
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