Generating ΔERE Mutant Medaka Using TALEN

Mutant medaka in which an ERE sequence in the npba promoter was deleted (ΔERE) were generated by TALEN-mediated genome editing. TALENs were designed to target the ERE that has been shown to be functional in vitro (Hiraki et al., 2014 (link)). The position and sequence of the targeted ERE and TALEN binding sites are shown in Figure 1—figure supplement 1. The TALE repeat arrays were assembled by the Golden Gate method (Cermak et al., 2011 (link)) as described elsewhere (Ansai et al., 2013 (link)), using the Golden Gate TALEN and TAL Effector Kit 2.0 (Addgene 1000000024). TALEN mRNAs were synthesized by in vitro transcription using the mMessage mMachine SP6 Kit (Thermo Fisher Scientific) and microinjected into the cytoplasm of embryos at the one-cell stage. Potential founders were screened by outcrossing to wild-type fish and testing progeny for mutations at the target site using a mismatch-sensitive T7 endonuclease I assay (Kim et al., 2009 (link)) followed by direct sequencing. A founder was identified that yielded a high proportion of progeny carrying a 7 bp deletion; the progeny were subsequently intercrossed to generate fish homozygous for the deletion. The genotype of each fish was determined by direct sequencing using the primers listed in Supplementary file 2. In all analyses of ∆ERE mutants, their wild-type siblings were used as controls.