We selected shha (GenBank ID: 30269), the gene encoding Sonic Hedgehog a, to test the utility of Zwitch. We tested several pairs of TALENs and selected the one that most efficiently induced DNA double-strand breaks (DSBs) in intron 1 of shha (Figure 1B and C). mRNA encoding the TALENs was co-injected with the donor vector (pZwitch-shha-int1) into one-cell-stage embryos. The donor vector includes the RA and LA sequences in the orientation that facilitated the insertion of Zwitch in the non-mutagenic orientation (Figure 1D). The injected fish were examined for LG expression at 7 and 45 days post-fertilization (dpf), and fish that maintained LG expression at 45 dpf were raised to adulthood and outcrossed with WT fish (Figure 1E). We screened 32 LG+ potential founders, finding that 19 of them (59%) transmitted LG expression (Figure 1E). PCR demonstrated that Zwitch was inserted in the correct non-mutagenic orientation in 17 (89%) of the 19 LG+ founders (Figure 1E and F). The precise location of the insertion was further verified using DNA sequencing (Figure 1—figure supplement 2A and B) and Southern blot analysis (Figure 1G). We subsequently established a conditional gene trap line, Tg(shha:Zwitch)vcc8Gt, which is referred to as shhact hereafter. We explained key steps for using Zwitch for other genes in the subsection titled ‘Targeting Zwitch into other genes’ in the Materials and methods.