Zinc-finger nuclease-mediated targeting of the zebrafish socs4b gene and evaluation of the socs4bΔ18 mutation. Sequence of zebrafish socs4b WT (A) and socs4bΔ18 (B) alleles, including the corresponding chromatograms, with the sequences targeted by the zinc-finger nuclease (ZFN) pair indicated and the nucleotides deleted in the socs4bΔ18 mutant shown by the grey box. Hyphens represent 258 nucleotides of intervening sequences prior to the first in-frame methionine. The respective protein translations are presented below, with the native start methionine of Socs4b WT indicated by a blue box and the next in-frame methionine in Socs4bΔ18 with a purple box. In vitro transcription/translation analysis of Socs4b WT and Socs4bΔ18 sequences along with a no template control (NTC) (C). The blue arrowhead indicates full-length Socs4b WT protein product and the purple arrowhead indicates the truncated Socs4bΔ18 protein product. Quantitative RT-PCR analysis of the socs4a, socs4b, socs5a, and socs5b genes performed on total RNA extracted from socs4b WT and socs4bΔ18 embryos, expressed as a fold-change socs4bΔ18 relative to socs4b WT embryos, with error bars indicating the standard error of the mean (D). Statistical analysis determined all differences as p > 0.05. Original blot can be found in Supplementary Figure S2.
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