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Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination

Nature Protocols volume 9, pages 529–540 (2014)Cite this article

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Abstract

The axolotl (Mexican salamander, Ambystoma mexicanum) has become a very useful model organism for studying limb and spinal cord regeneration because of its high regenerative capacity. Here we present a protocol for successfully mating and breeding axolotls in the laboratory throughout the year, for metamorphosing axolotls by a single i.p. injection and for axolotl transgenesis using I-SceI meganuclease and the mini Tol2 transposon system. Tol2-mediated transgenesis provides different features and advantages compared with I-SceI-mediated transgenesis, and it can result in more than 30% of animals expressing the transgene throughout their bodies so that they can be directly used for experimentation. By using Tol2-mediated transgenesis, experiments can be performed within weeks (e.g., 5–6 weeks for obtaining 2–3-cm-long larvae) without the need to establish germline transgenic lines (which take 12–18 months). In addition, we describe here tamoxifen-induced Cre-mediated recombination in transgenic axolotls.

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Figure 1: Various components of the axolotl transgenics protocol.

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Acknowledgements

This manuscript would not have been possible without the dedicated hard work and commitment from our animal caretakers: H. Andreas, J. Michling, S. Mögel, C. Junghans, M. Armstead and B. Gruhl. We thank S. Borland (former Axolotl colony at Indiana University) for advice on the day/light cycle for mating troubleshooting, and K. Boes and S. Hermann (Center for Regenerative Therapies Dresden) for help in obtaining images and movies for this manuscript. This work was supported by funds from the VolkswagenStiftung, DFG grant nos. TA274/3-1, TA274/3-2, TA274/5-1, a European Research Council Advanced Investigator Grant, central funds of the MPI-CBG and the DFG Center for Regenerative Therapies.

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Authors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany

    Shahryar Khattak, Heino Andreas, Verena Kappert, Maritta Schuez, Tatiana Sandoval-Guzmán & Elly M Tanaka

  2. Technische Universität Dresden, Deutsche Forschungsgemeinschaft (DFG) Center for Regenerative Therapies, Dresden, Germany

    Shahryar Khattak, Prayag Murawala, Maritta Schuez, Tatiana Sandoval-Guzmán, Karen Crawford & Elly M Tanaka

  3. St. Mary's College of Maryland, St. Mary's City, Maryland, USA

    Karen Crawford

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  1. Shahryar Khattak
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Contributions

S.K., P.M., H.A., V.K., M.S., T.S.-G., K.C. and E.M.T. contributed to the various protocols described here. E.M.T. provided financial support and supervised the lab personnel. S.K. and E.M.T. designed the experiments, analyzed the data and wrote the final manuscript.

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Correspondence to Elly M Tanaka.

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Integrated supplementary information

Supplementary Figure 1 Axolotl husbandry.

(a) An axolotl aquarium fitted with a UV filter, biofilter and a continues flow of water. The cages are divided, cleaned manually and juveniles and adults are kept here because of strong water current/flow in the system. (b) Set up of the artemia harvesting tanks. (c) An adult axolotl (adults can reach approx. 30 cm in size). (d) Metamorphosed juveniles (8-10 cm in size). The black axolotl is the wild type and the pinkish one is the naturally occurring white mutant.

Supplementary Figure 2 Axolotl eggs, male and female phenotypes, and intraperitoneal injections

(a) Example of good quality eggs. These are characterized by a smooth surface with dark brown animal pole and a yellowish/beige vegetal pole. The healthy fertilised eggs will orient themselves in such a way that the brown animal pole is always facing up after the dejellying. (b) Example of bad quality eggs. These have a rough surface and often have white lesions that are more visible in the animal pole. Do not inject these eggs or use them as un-injected controls. (c) Female axolotl hind limbs and cloaca in a non-mating/non-egg laying situation. (d) An “open” cloaca after picking up the spermatophores, is ready to lay eggs. (e) A male axolotl hind limbs and cloaca. The cloaca is well shaped and is different from the female axolotl cloaca (c). (f) A glass capillary pulled for preparing an injection needle. This injection needle is filled with the DNA/RNA mix and the tip is broken under the stereomicroscope to get a 5 nl drop size per injection per egg (steps 40-44) (Supplementary Video 5). Scale Bar: 2 mm (g) Intraperitoneal injection of an axolotl larva. Depicted is a Hamilton syringe fitted with a 30 G 1/2 inch 0.3 x 13 mm needle inserted in the lower abdominal cavity of the axolotl larva. The same injection set up is used for intraperitoneal injection of 4-OH tamoxifen and thyroxine solutions. (h) Close up of the intraperitoneal injection showing the needle inserted under the skin into the body cavity. Take care not to touch the internal organs of the animal. The calculated amounts of thyroxine and 4OH-tamoxifen are released into the body cavity.

Supplementary information

Supplementary Figure 1

Axolotl husbandry. (PDF 2539 kb)

Supplementary Figure 2

Axolotl eggs, male and female phenotypes, and intraperitoneal injections. (PDF 6187 kb)

Supplementary Video 1

The video shows the adult axolotl tanks while being fed with fish pellets. One can see both white mutant and dark, wild type axolotls. (WMV 12220 kb)

Supplementary Video 2

The video shows how to harvest artemia. Make sure that the artemia are diluted before feeding them to larvae. (WMV 6639 kb)

Supplementary Video 3

The video shows the habitat of metamorphosed animals. The level of water is very low and rocks/stones are put for the animals to climb up on. (WMV 17613 kb)

Supplementary Video 4

The video starts with a glimpse of spermatophores, followed by showing the female axolotl laying eggs in the mating tanks on artificial plastic leaves. The next step shows harvesting and de-jellying of eggs. (WMV 10598 kb)

Supplementary Video 5

The video starts with setting up the eggs in a pre-cast injection plate, followed by filling up the needle with DNA/RNA mix. Measure drop size for injection and inject 5 nl to every embryo. (WMV 1777 kb)

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Khattak, S., Murawala, P., Andreas, H. et al. Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination. Nat Protoc 9, 529–540 (2014). https://doi.org/10.1038/nprot.2014.040

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