Fig. 2

Mutagenesis. A: A 3.6 kb genomic region of gsdf including exons (e) 1–5 with the TALEN target sequence: TALEN cleavage site (small letters in red), TALEN binding sites in black and BslI restriction site (bracket), along with genotyping primer sites (F, R1 and R2 in green). B: PCR analysis of seven G0 injected embryos at 1 dpf using genotyping primers F and R1 shows a 251 base pair (bp) fragment in wild-types (WT) that digests with BslI to produce fragments of 150 bp and 101 bp. Seven injected embryos (1–7) contained a substantial load of mutations that destroyed the BslI site, leaving the 251 bp fragment undigested, in addition to the WT bands. C: Raising injected embryos and subsequent breeding produced stable lines carrying -8 bp, -1 bp, and -14 bp deletions. Talen cleavage site shown in red. A translated portion of gsdf (in green letters) appears below the -14 bp sequence. D: To verify transcription of the -8 bp deletion, we amplified a 275 bp fragment using primer F in exon-1 and R2 in exon-2 shown in A; results verified the 8 bp deletion, which confirmed the genomic sequence in C. E: TALEN-induced mutations are predicted to result in premature stop codons (*). The translated portion of Gsdf (in black and green letters) indicates the wild-type sequence, while red letters indicate the predicted frame-shifted sequences in gsdf mutants. Protein coding domains: purple, signal peptide; salmon, precursor; blue, TGFB-family domain; gray, C-terminus; e, exons; arrow, cleavage site.