(A) Schematic of the non-mutagenic and mutagenic allele, primer sites, and BglI recognition site. (B) A gel image showing the PCR products of the non-mutagenic alleles without (left) and with BglI digestion (middle) and the PCR products of the mutagenic alleles with BglI digestion (right). Genomic DNA was prepared from 96 hpf shhact/+ embryos injected with (+) or without (−) Cre mRNA at single-cell stage. Cre mRNA was prepared from linearized pCS2-iCRE-BFP, in which iCRE cDNA is linked to TagBFP via P2A peptide sequence. (C) Analysis of inversion rate and shha expression levels in single embryos. Embryos were prepared from an incross of shhact/+ fish and injected with Cre mRNA at single-cell stage. Embryos without Cre mRNA injection were used as control (Sample 1). The injected embryos were separated based on the expression of TagBFP at moderate (Sample 2) and high levels (Sample 3 and 4). Individual embryos were genotyped at 96 hpf and genomic DNA and total RNA of each embryo using TRIzol following the manufactured protocol. The upper panels are the gel images used for inversion rate measurement. The numbers on the bottom are the obtained inversion rate in each sample. The lower panels are the results of semi-qRT-PCR analysis of shha expression levels of each sample. (D) Analysis of inversion rate in epicardial cells in zebrafish embryos. Embryos were prepared from crosses of tcf21:DsRed2; shhact/+ fish with shhact/+ fish (Sample 1) and tcf21:CreER; shhact/+ fish with tcf21:DsRed2; shhact/+ fish (Sample 2). Embryos were treated with 4-HT and examined for DsRed2 expression to screen tcf21:DsRed2 transgene and lens EGFP expression to screen tcf21:CreER transgene (Kikuchi et al., 2011). Lens EGFP from tcf21:CreER transgene is considerably stronger than that from shhact alleles in embryos, which enables us to sort tcf21:CreER+ embryos with shhact background. PCR genotyping on shhact alleles were not performed; this does not affect the measurement as the inversion rate is quantified based on the PCR products of the shhact alleles. tcf21:DsRed2+ epicardial cells were isolated by FACS and 1,000 DsRed2+ cells were used for analysis. The estimated inversion rate is 100%; note that the bands from the non-mutagenic allele (N1 and N2) are undetectable in Sample 2 after BglI digestion. (E) Analysis of inversion rate in epicardial cells in the adult zebrafish ventricle. Ventricles were prepared from 4-HT–treated adult tcf21:DsRed2; shhact/+ fish (Sample 1) and tcf21:CreER; tcf21:DsRed2; shhact/+ fish (Sample 2). tcf21:DsRed2+ epicardial cells were isolated by FACS and 5000 DsRed2+ cells were used for analysis. The estimated inversion rate is 100%; note that the bands from the non-mutagenic allele (N1 and N2) are undetectable in Sample 2 after BglI digestion.