高效的基因敲入在蝾螈中的应用及其在测试卫星细胞在肢体再生中的作用。
Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration.
机构信息
Deutsche Forschungsgemeinschaft (DFG)-Center for Regenerative Therapies Dresden, Technische Universität Dresden, 01307 Dresden, Germany;
Research Institute of Molecular Pathology, Vienna Biocenter, 1030 Vienna, Austria.
出版信息
Proc Natl Acad Sci U S A. 2017 Nov 21;114(47):12501-12506. doi: 10.1073/pnas.1706855114. Epub 2017 Oct 31.
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 () based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the reporter gene and a larger membrane-tagged (∼5-kb) cassette into axolotl and 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.
蝾螈表现出广泛的再生能力，是再生研究中的独特模型。然而，由于缺乏靶向基因敲入方法，标记和操作对于理解再生机制至关重要的一些细胞群体变得困难。在这里，我们基于 CRISPR/Cas9 技术在蝾螈中建立了高效的基因敲入方法。我们使用一种不依赖同源性的方法，成功地将报告基因和一个更大的膜标记的（约 5kb）盒插入蝾螈和基因组位点。根据整合的 DNA 片段的大小，5-15%的 F0 转基因蝾螈的转基因呈阳性。使用这些技术，我们已经标记和追踪了 PAX7 阳性卫星细胞，它们是蝾螈肢体再生中肌肉发生的主要来源。我们的工作为蝾螈再生的分子和细胞研究带来了关键的遗传工具。
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