학술논문
Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration
Document Type
Report
Author
Source
Proceedings of the National Academy of Sciences of the United States. November 21, 2017, Vol. 114 Issue 47, p12501, 6 p.
Subject
Language
English
ISSN
0027-8424
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-[ER.sup.T2]-Cre-[ER.sup.T2] (~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. CRISPR/Cas9 | knockin | neural stem cells | regeneration | satellite cells