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Edited by Marianne Bronner, California Institute of Technology, Pasadena, CA, and approved September 28, 2017 (received for review May 3, 2017)
Significance
Salamanders have great potential to regenerate damaged organs upon injury, and thus provide an important model for understanding the mechanisms of tissue regeneration; however, genetic studies have been limited due to a lack of gene knockin strategies. In this study, we have established efficient CRISPR/Cas9 mediated gene knockin approaches in the axolotl (Ambystoma mexicanum), which has allowed us to genetically mark two critical stem cell pools for limb and spinal cord regeneration. Our genetic fate mapping establishes the role of PAX7+ satellite cells for limb muscle regeneration. This method opens up the possibility of marking and perturbing gene function inducibly in any definable cell populations in the axolotl, a key functionality required for the precise, rigorous understanding of processes such as regeneration.
Abstract
Salamanders exhibit extensive regenerative capacities and serve as a unique model in regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying regeneration. Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 (∼5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5–15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration.
Footnotes
- ↵1To whom correspondence may be addressed. Email: jifengfei{at}m.scnu.edu.cn or elly.tanaka{at}imp.ac.at.
Author contributions: J.-F.F. and E.M.T. designed research; J.-F.F., M.S., D.K., Y.T., and D.N.D. performed research; J.-F.F. and E.M.T. analyzed data; and J.-F.F. and E.M.T. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. MF996566, MF996567, and MG017611).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1706855114/-/DCSupplemental.
Published under the PNAS license.
CRISPR-mediated knockin in axolotl
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