Fig. 1

Fig. 1

Generation of a zebrafish Stat5.1 mutation mimicking those in human STAT5B associated with GHISID1. A Schematic diagram of the STAT5B protein domain architecture consisting of N-terminal (yellow), coiled-coil (blue), DNA-binding (green), linker (orange), SH2 (pink), and transactivation (TA) (dark green) domains. B Alignment of human STAT5B with zebrafish stat5.1 sequences around the site of the R152X mutation (green) seen in human GHISID1, showing identical (*), highly similar (:) and similar (.) amino acids. C Nucleotide sequence of zebrafish stat5.1 genomic DNA targeted using CRISPR/Cas9 with the gRNA (orange) shown and the coding phase indicated. D HRM analysis of a sample of embryos injected with stat5.1 gRNA and Cas9, with wild-type embryos in green and other colours representing potential mutants. E–F Genotyping by PCR and gel electrophoresis of representative F1 wild-type (WT) and heterozygous (HET) mutant carriers (E) and F3 WT, HET and homozygous (HOM) fish (F), with the wild-type allele indicated with the green arrow and the mutant allele with red. G Sequence of homozygous wild-type (WT, wt/wt) and loss-of-function (LOF, mdu022/mdu022) mutant stat5.1 showing the chromatogram along with corresponding nucleotides, including a 47 bp insertion (pink text) in the LOF mutant. The coding phases and protein translations are shown, including the alternate phase and resultant five de novo amino acid residues and stop codon (orange text) following T155 in the LOF mutant. H Expression analysis of stat5.1 using qRT.²-PCR on the indicated tissues from WT and LOF adult female (F) zebrafish. Data was normalised to actb and represented as relative fold-change compared to WT, with mean ± SD shown and statistical significance indicated (n = 6)