文章基本信息

标题：Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration
本地全文：下载
作者：Ji-Feng Fei ; Maritta Schuez ; Dunja Knapp 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2017
卷号：114
期号：47
页码：12501-12506
DOI：10.1073/pnas.1706855114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 ^T2 -Cre-ER ^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.
关键词：CRISPR/Cas9 ; knockin ; neural stem cells ; regeneration ; satellite cells