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Expressing exogenous genes in newts by transgenesis

Abstract

The great regenerative abilities of newts provide the impetus for studies at the molecular level. However, efficient methods for gene regulation have historically been quite limited. Here we describe a protocol for transgenically expressing exogenous genes in the newt Cynops pyrrhogaster. This method is simple: a reaction mixture of I-SceI meganuclease and a plasmid DNA carrying a transgene cassette flanked by the enzyme recognition sites is directly injected into fertilized eggs. The protocol achieves a high efficiency of transgenesis, comparable to protocols used in other animal systems, and it provides a practical number of transgenic newts (20% of injected embryos) that survive beyond metamorphosis and that can be applied to regenerative studies. The entire protocol for obtaining transgenic adult newts takes 4–5 months.

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Figure 1: Microinjection into a C. pyrrhogaster egg.
Figure 2: Examples of transgenic C. pyrrhogaster newts generated by the I-SceI method.

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Acknowledgements

We are thankful to E M. Tanaka for her kind and generous gift of pCAGGs-EGFP (Sce). This work was supported by a Grant-in-Aid for Challenging Exploratory Research (20650060) and a Grant-in-Aid for Scientific Research (B) (21300150) from the Japan Society for the Promotion of Science (JSPS) to C.C. and by NIH grant EY10540 to P.A.T.

AUTHOR CONTRIBUTIONS

C.C. designed, directed and analyzed data. M.M.C.-R., S.Y., T.M., K.N., T.H., N.M. and K.D.R.-T. performed experiments and wrote part of the protocols. P.A.T. and C.C. co-wrote the final version of the paper.

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Correspondence to Chikafumi Chiba.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1. Transgenic Cynops pyrrhogaster newts generated by this protocol with pCAGGs-EGFP(Sce) construct.

The movie shows 'Uniform' EGFP-expressing swimming larvae at stage 40 and 50, and a head region, forelimbs and beating heart of another larva at stage 50, and finally a juvenile after metamorphosis. Permission by the University of Tsukuba AUCC was obtained for these experiments. This video was reproduced, with permission, from the data shown in ref. 7 ©Wiley. (WMV 20052 kb)

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Casco-Robles, M., Yamada, S., Miura, T. et al. Expressing exogenous genes in newts by transgenesis. Nat Protoc 6, 600–608 (2011). https://doi.org/10.1038/nprot.2011.334

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