Medaka-microinjection with an Upright Microscope

Background

Medaka is a small freshwater teleost species and has desirable features for use as a vertebrate model, including daily spawning, complete genome sequence, and availability of a number of useful strains. Moreover, the transparency of its eggs enables precise introduction of DNA/RNA/Protein solutions into the cytoplasm of the eggs by microinjection. Microinjection is important for the functional genomic analysis; for example, DNA microinjection is an indispensable technique for generating transgenic fish, and helps us to understand the functions of introduced genes in vivo (Ozato et al., 1986). Furthermore, microinjection of CRISPR/Cas system is able to achieve targeted genome editings such as knockout and knock-in approaches (Ansai and Kinoshita, 2014; Murakami et al., 2017a).

Both of a stereoscopic microscope and an upright microscope can be used for microinjection into fish embryos. Although microinjection with an upright microscope requires attachment of some specific instruments including a micromanipulator and an injection needle holder to the microscope, this method may achieve more precise introduction of solutions into the cytoplasm than that with a stereoscopic microscope due to its high resolution property. In this paper, we described the outline of a microinjection method with an upright microscope. This method can be used together with other protocols, such as those for the generation of gene knockout or gene knock-in lines (Ansai and Kinoshita, 2014; Murakami et al., 2017b).