Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration
Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration
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DOI：
10.1073/pnas.1706855114
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发表时间：
2017-11-21
影响因子：
11.1
通讯作者：
Tanaka, Elly M.
中科院分区：
文献类型：
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作者：
Fei, Ji-Feng;Schuez, Maritta;Tanaka, Elly M.
火蜥蜴表现出广泛的再生能力，在再生研究中是一个独特的模式。然而，由于缺乏有针对性的基因敲击方法，对于理解再生机制至关重要的一些细胞群体的标记和操作一直是困难的。在此，我们建立了基于CRISPR/Cas9技术的高效基因敲打方法。利用不依赖同源性的方法，我们成功地将Cherry报告基因和一个较大的膜标记Cherry-ERT2-Cre-ERT2(类似于5-kb)插入到Axolot1 Sox2和Pax7基因组中。根据要整合的DNA片段的大小，F0转基因Axolot1中有5-15%的转基因呈阳性。利用这些技术，我们已经标记和追踪了PAX7阳性的卫星细胞，作为在轴突肢体再生过程中促进肌肉生成的主要来源。我们的工作为紫杉醇再生的分子和细胞研究提供了一个关键的遗传学工具。
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 (similar to 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.