To characterize the shhact allele (Figure 2A), we injected one-cell-stage shhact/+ embryos with a Cre expression vector (pUbb-iCRE-GFP; Figure 2—figure supplement 1A and B). Genomic PCR and reverse transcription (RT)-PCR detected the gene trap cassette inversion (Figure 2B) and alternative shha splicing in embryos injected with Cre DNA (Cre+) but not in uninjected embryos (Cre−) (Figure 2C). DNA sequencing verified the precise inversion (Figure 2—figure supplement 2A) and in-frame transcription of shha-P2A-TagRFP mRNA (Figure 2—figure supplement 2B). We injected Flp mRNA into one-cell-stage embryos of the offspring of an incross of shhact/+ fish and detected the excision of the FRT-flanked LG cassette using genomic PCR (Figure 2D and E). EGFP fluorescence was lost or reduced at approximately the expected frequency of the homozygous or heterozygous shhact genotype (Figure 2F and G).10.7554/eLife.24635.005Figure 2.Characterization of the shhact allele.(A) Schematic of Cre-dependent conversion of Zwitch from the non-mutagenic orientation to the mutagenic orientation. Cre activation induces an inversion between loxP or lox5171 sites and the subsequent excision of loxP or lox5171-flanking DNA sequences (Schnütgen and Ghyselinck, 2007), thereby permanently converting Zwitch into the mutagenic form and inducing aberrant shha splicing. (B) PCR analysis of the Zwitch inversion. Genomic DNA from 72 hpf Cre+ and Cre− shhact/+ embryos was analyzed using PCR. (C) RT-PCR analysis of shha expression in 72 hpf Cre+ and Cre− shhact/+ embryos. (D) Schematic of Flp-mediated excision of the FRT-flanked LG tag in the shhact allele. (E) Genomic PCR analysis of Flp-injected (+) or uninjected (−) embryos from a cross of shhact/+ adults. PCR using F3 and R1 primers detected shhact alleles in both samples. Flp mRNA was synthesized from linearized pCS2-FLPo (Materials and methods). (F) Representative image of embryos injected with Flp mRNA. Arrows indicate the lens. (G) Quantification of phenotypes of the embryos analyzed in F. A total of 87 Flp-injected (+) and 99 uninjected embryos (−) were analyzed (****p<1.0 × 10−8 Fisher’s exact test). dpf, days post-fertilization.DOI: http://dx.doi.org/10.7554/eLife.24635.00510.7554/eLife.24635.006Figure 2—figure supplement 1.Cre expression vector.(A) Schematic of pUbb-iCRE-GFP. (B) A representative image of embryos injected with pUbb-iCRE-GFP expressing EGFP (right) on approximately 80% of the body surface (left). Embryos expressing EGFP at similar or greater levels were selected for the analysis presented in Figure 3A–C.DOI: http://dx.doi.org/10.7554/eLife.24635.00610.7554/eLife.24635.007Figure 2—figure supplement 2.DNA sequence of the inverted shhact allele and its transcript.(A) DNA sequence of the inverted shhact allele. (B) DNA sequence of shha-P2A-TagRFP mRNA.DOI: http://dx.doi.org/10.7554/eLife.24635.00710.7554/eLife.24635.008Figure 2—figure supplement 3.TagRFP expression from the inverted shhact allele.Weak expression of TagRFP was detected in cells in the ganglion cell layer (GCL) in the retina of ubb:Cre-GFP; shhact/+ embryos but not in shhact/+ and ubb:Cre-GFP embryos. Confocal projections of z-stacks are shown in three panels from the right, and the bright field image of in situ hybridization of shha mRNA is shown in the far left panel. Dotted lines, approximate border of the GCL. Scale bar, 50 μm.DOI: http://dx.doi.org/10.7554/eLife.24635.008