We modified FT1 (Ni et al., 2012), a FLEx construct used for random gene trap mutagenesis in zebrafish, and generated a donor vector for the generation of zebrafish with an invertible gene trap cassette via HR (Zwitch) (Figure 1A and Figure 1—figure supplement 1A). Zwitch consists of a removable, lens-specific enhanced green fluorescence protein (EGFP) tag (LG) (Lee et al., 2005) and an invertible splice acceptor site conjugated to a gene expressing a red fluorescent protein (TagRFP) via a 2A self-cleaving peptide sequence (Figure 1A and Figure 1—figure supplement 1B). The region encompassing LG and the gene trap cassette is flanked by unique restriction enzyme sites in which the right arm (RA) and left arm (LA) of homologous sequences are inserted (Figure 1A and Figure 1—figure supplement 1A).10.7554/eLife.24635.002Figure 1.Generation of the shha conditional allele using Zwitch.(A) Schematic of Zwitch. (B) Schematic of the zebrafish shha locus and TALEN used to induce DNA DSBs in intron 1. Exons are indicated by filled boxes with numbers. The binding sites for the TALEN pair are highlighted in blue, and the XbaI site in the spacer region is highlighted in green. (C) The efficiency of the TALENs in introducing DSBs. XbaI digestion of PCR products amplified from the genomic DNA of embryos injected with TALEN mRNAs. The efficiency of the TALEN pair in inducing DSBs (67%) was quantified from the gel image using ImageJ software. (D) Schematic of the strategy used to target shha via TALEN-mediated homologous recombination with pZwitch-shha-int1. (E) The screening process for founders. (F) Genomic PCR analysis of the Zwitch insertion with the correct orientation. (G) Southern blot analysis of the Zwitch-modified shha allele. BGHpA, bovine growth hormone polyadenylation signal; cryaa, α A-crystallin; LA, left arm; RA, right arm.DOI: http://dx.doi.org/10.7554/eLife.24635.00210.7554/eLife.24635.003Figure 1—figure supplement 1.pZwitch vector.(A) Schematic of pZwitch. (B) Sequence of the splice acceptor and P2A components of pZwitch (filled bar in A). The splice acceptor sequence was derived from pFT1 (Ni et al., 2012). pZwitch for the +2 and+3 reading frames was generated by inserting T and TCGAT, respectively, at the indicated site. BP, branch point; ESE, exonic splice enhancer; ISE, intronic splice enhancer; SAC, splice acceptor consensus.DOI: http://dx.doi.org/10.7554/eLife.24635.00310.7554/eLife.24635.004Figure 1—figure supplement 2.DNA sequence of the shhact allele.(A) DNA sequence at the 5′ end of the LA homology sequence of pZwitch-shha-int1. (B) DNA sequence at the 3′ end of the RA homology sequence of pZwitch-shha-int1.DOI: http://dx.doi.org/10.7554/eLife.24635.004