Zebrafish Transgenesis and Mutagenesis

Zebrafish embryos were injected at the one-cell stage with a glass microneedle and a microinjector (World Precision Instruments). For transgenesis tol2 mRNA, transcribed from pCS2FA with the mMESSAGE mMACHINE SP6 Transcription Kit, a kind gift from Koichi Kawakami, was injected at a concentration of 25 ng µl⁻¹ together with Tol2 destination vectors^{69 (link)}. For mutagenesis Cas9 mRNA was in vitro transcribed from the MLM3613 plasmid (Addgene plasmid #42251) using the mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Thermo Fisher Scientific); sgRNA target sequences were cloned into DR274 (Addgene plasmid # 42250) and transcribed with the MAXIscript T7 transcription kit (Thermo Fisher Scientific). Subsequently, 1 nl of a mixture of 600 ng ml⁻¹Cas9 mRNA and 50 ng ml⁻¹ sgRNA were co-injected into one-cell stage embryos^{29 (link)}.