Figure 1.

TALEN-induced mutation in the auts2a gene. A, Zebrafish auts2a gene locus, TALEN target sites and isolated alleles. The TALENs target a pair of binding sites (in blue) flanking a spacer with a restriction enzyme site (in green). Exonic and intronic sequences are shown in upper and lower cases, respectively. In contrast to genomic sequence annotated in the Ensembl (WT), our “in-house” zebrafish AB strain (WT*) has a polymorphism in intronic sequence adjacent to exon 8 (in red). Alleles ncb101, ncb103, ncb104, and ncb105 have the nucleotide deletions that disrupt the donor splice site (in bold) leading to a frameshift after S498 and premature stop codons. Deletion in allele ncb102 does not affect correct splicing and the Auts2a protein sequence. Three single point mutations were introduced in the intron of auts2a^ncb104 allele (in orange) leading to a stop codon creation (underlined). The alternative donor splice sites used to splice mutant auts2a^ncb104 pre-mRNA are highlighted in gray. B, top, Auts2a and Auts2a^ncb104 proteins. The ncb104 mutation causes the loss of the C-terminal portion of Auts2a, comprising PY motif, PR region PR2 and the Auts2 family domain. B, bottom left, RT-PCR analysis of auts2a mRNA, isolated from wild-type (WT), ncb104 heterozygote (HET), and homozygote (HOM) embryos. M, 100-bp DNA ladder (NEB). B, bottom right, Partial protein sequences of mutant alleles. See also Extended Data Figure 1-1.