An efficient microinjection method for unfertilized eggs of Asian amphioxus Branchiostoma belcheri

Amphioxus is a promising model animal for evolutionary and developmental studies. However, as an emerging model organism, amphioxus lacks most molecular techniques applied in other well-developed model animals. Microinjection is a powerful technique for gene manipulation, and thus it undoubtedly is one of useful approaches in the studies of gene function and embryonic development. Although the method has been exploited in Florida and European amphioxus, it still remains to be optimized and introduced into other amphioxus species. In order to introduce the technique into our lab, we followed and optimized the previous description and successfully performed microinjection on unfertilized eggs of Asian amphioxus Branchiostoma belcheri. We made six solutions for practice: 200 mM KCl, 100 ng/mu l actin-LacZ or 100 ng/mu l actin-RFP vectors, LacZ or RFP vectors without promoter and RFP capped mRNA. More than 99.2 % of eggs injected with KCl were able to be fertilized, 94.3 % of them could hatch normally and 55.9 % survived until 2-day larvae, all of which were nearly equivalent to those obtained from normally fertilized eggs. Embryos injected with two plasmid constructs also showed very high fertilizing and hatching ratios, but normally developing ratios were slightly lower than that of KCl injection. Of those injected embryos, 91.8 % expressed exogenous gene LacZ and 80.5 % exhibited foreign RFP expression, which were driven by a promoter from amphioxus beta-actin gene. The data indicated a successful modified microinjection method for the unfertilized eggs of Asian amphioxus, and those modifications improved the feasibility and efficiency of microinjection on amphioxus.