Protocol

Generation of Transgenic Axolotls (Ambystoma mexicanum)

¹ Center for Regenerative Therapies, Dresden University of Technology, 01307 Dresden, Germany
² Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany

³Corresponding author (elly.tanaka{at}crt-dresden.de)

INTRODUCTION

Injection of RNA, DNA, or morpholinos into amphibian and fish embryos is a standard technique used to test gene function during development or to make transgenic animals. The techniques used to inject axolotl (Ambystoma mexicanum) embryos are very similar to those used for Xenopus embryos, with a few modifications to account for the larger egg size and "softer" cytoplasm. For example, amphibian embryos must be "dejellied" prior to injection. Chemical treatments used to remove the jellycoat for Xenopus eggs are not reliable for axolotl eggs; thus, dejellying is performed manually. In addition, greater injection volumes (typically 50 nL) are used with axolotl embryos, with correspondingly larger total amounts of DNA and RNA, as compared with Xenopus. Injection of plasmid DNA into the axolotl egg results in varying frequencies of integration (depending on the plasmid DNA) within one of the first few cell divisions. The integration efficiency is significantly increased if the plasmid DNA contains SceI meganuclease sites and is coinjected with SceI meganuclease, as described in this protocol.