The zebrafish ptpn6 mutant generated using CRISPR/Cas9 is a strong null allele. (A) Schematic overview of Shp1. gRNA was targeted to the C-terminal end of the N-terminal SH2 domain, inducing a 7 bp deletion (DEL7) in exon 4, indicated by the red box in the WT sequence. The mutation was confirmed by Sanger sequencing after sub-cloning of F1 DNA. The 7 bp deletion leads to a frameshift and a stop codon in exon 4, ten amino acids downstream of the mutation site. (B) Immunoblots detecting endogenous Shp1 in WT and heterozygous embryos, but not in homozygous ptpn6 mutant embryos. Lysates of transfected HEK293T cells expressing zebrafish Shp1 were used as a control for the detection of zebrafish Shp1. An Akt-specific antibody was used to monitor equal loading.
