Fig. 1

ZFN and TILLING strategies generate null alleles of zebrafish nf1a and nf1b. (A,B) Scheme of site targeted for ZFN-mediated cleavage in exon 26 of nf1a and exon 17 of nf1b. (C) Alignments of nucleotide sequences from wild-type and mutant nf1a and nf1b alleles. ZFN target sites appear in red. Yellow bases represent insertions that arise from repair of ZFN-induced mutagenic lesions. Black bases correspond to the mutated codon in the nf1a^L1247X allele generated by a TILLING strategy, with the mutated base appearing in blue. (D) ZFN-induced mutagenic lesions in nf1a and nf1b produce frameshift mutations that lead to truncated protein products following short regions of altered translation, which are indicated in green. The nonsense mutation in the nf1a^L1247X allele generated by a TILLING strategy also appears in green. (E) The truncated protein products predicted by the ZFN- and TILLING-induced mutant nf1a/nf1b alleles all harbor complete or partial loss of the neurofibromin GAP-related domain (GRD). (F) Western blot analysis for neurofibromin in protein lysates from 3 dpf wild-type, nf1a^Δ5/Δ5; nf1b^+/+, nf1a^+/+; nf1b^+10/+10 and nf1a^Δ5/Δ5; nf1b^+10/+10 zebrafish larvae (100 μg each) or 293T cells (25 μg) demonstrates absence of Nf1 protein in nf1a^-/-; nf1b^-/- larvae. Equal loading was confirmed by stripping the membrane and reprobing for α-tubulin. (G) Kaplan-Meier survival analysis demonstrates that 100% of nf1a^-/-; nf1b^-/- larvae (n=22) die by 10 dpf as compared with 0% of wild-type larvae (n=27).