Int. J. Dev. Biol. 63: 281 - 286 (2019)
doi: 10.1387/ijdb.180297th
© UPV/EHU Press

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

Toshinori Hayashi*,1,2,3, Mie Nakajima1, Mitsuki Kyakuno1,2,3, Kanako Doi1, Ikumi Manabe1, Shouhei Azuma1 and Takashi Takeuchi1

1Department of Biomedical Sciences, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago, Tottori, 2Graduate school of Integrated Science for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima and 3Amphibian Research Center, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan

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.

Keywords:

newt, microinjection, regeneration, transgenesis, genome editing

*Corresponding author e-mail: toshih2@hiroshima-u.ac.jp