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Proc Natl Acad Sci U S A. 2017 Nov 21;114(47):12501-12506. doi: 10.1073/pnas.1706855114. Epub 2017 Oct 31.

Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration.

Author information

1
Deutsche Forschungsgemeinschaft (DFG)-Center for Regenerative Therapies Dresden, Technische Universität Dresden, 01307 Dresden, Germany; jifengfei@m.scnu.edu.cn elly.tanaka@imp.ac.at.
2
Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
3
Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou 510631, China.
4
Deutsche Forschungsgemeinschaft (DFG)-Center for Regenerative Therapies Dresden, Technische Universität Dresden, 01307 Dresden, Germany.
5
Protein Expression Facility, Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.

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.

KEYWORDS:

CRISPR/Cas9; knockin; neural stem cells; regeneration; satellite cells

PMID:
29087939
PMCID:
PMC5703281
DOI:
10.1073/pnas.1706855114
[Indexed for MEDLINE]
Free PMC Article

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