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Advanced microinjection protocol for gene manipulation using the model newt Pleurodeles waltl.

The International journal of developmental biology (2019-06-30)
Toshinori Hayashi, Mie Nakajima, Mitsuki Kyakuno, Kanako Doi, Ikumi Manabe, Shouhei Azuma, Takashi Takeuchi
ABSTRACT

Urodele amphibian newts have an outstanding history as experimental animals in various research fields such as developmental biology and regeneration biology. We have reported a model experimental system using the Spanish newt, Pleurodeles waltl, and it enables reverse/molecular genetics through gene manipulation. Microinjection is one of the core techniques in gene manipulation in newts. In the present study, we examined the conditions of the microinjection method, such as egg preparation, de-jelly solution, and formulation of injection medium. We have successfully optimized the injection protocol for P. waltl newts, and our improved protocol is more efficient and lower in cost than previous methods. This protocol can be used for the microinjection of plasmid DNA with I-SceI or mRNA, as well as genome editing using the CRISPR-Cas9 system. This protocol will facilitate research through gene manipulation in newts.