TALEN RNA Microinjection Protocol

To construct TALEN-L and TALEN-R plasmids, TAL repeats were assembled by the modified Golden Gate assembly method (Sakuma et al, 2013 (link)). TALEN plasmids and the phiC31 integrase expression plasmid (Ishikawa et al, 2018 (link)) were linearized with NotI, purified by phenol/chloroform extraction, and used as a template to synthesize capped mRNAs using the mMESSAGE mMACHINE SP6 kit (Life Technologies) followed by purification with RNeasy MinElute (QIAGEN). Synthesized RNAs were microinjected as described previously in Ishikawa et al (2011) (link) into one-cell-stage embryos at the concentration of 50 ng/μl for TALEN-L and TALEN-R and 100 ng/μl for the phiC31 integrase expression plasmid.