Mmessage sp6 polymerase kit

A translation-blocking antisense morpholino, targeting X. tropicalis katnal2 (Table S1), was injected at a range between 1 pmol to 6 pmol, with X. tropicalis blastomeres receiving 2 nl and X. laevis 10 nl. Mouse KATNAL2-GFP (Ververis et al., 2016 (link)) was subcloned into pCS108 and in vitro transcribed using NotI restriction enzyme and the mMessage SP6 polymerase kit (Thermo Fisher Scientific). One blastomere at the twocell stage was injected with 300 pg of in vitro transcribed mRNA. Centrin4-CFP/Centrin4-RFP (Antoniades et al., 2014 (link); Park et al., 2008 (link)) was in vitro transcribed using AscI restriction enzyme and the mMessage SP6 polymerase kit (Thermo Fisher Scientific); 300 pg of mRNA was co-injected with 300 pg KATNAL2-GFP mRNA. The negative control for morpholino injection was an injection of 300 pg of Centrin4-CFP mRNA alone.