Studying extracellular vesicle transfer by a Cre-loxP method
Studying extracellular vesicle transfer by a Cre-loxP method
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DOI：
10.1038/nprot.2015.138
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发表时间：
2015-12
期刊：
影响因子：
14.8
通讯作者：
A. Zomer;S. Steenbeek;Carrie Maynard;J. Rheenen
中科院分区：
文献类型：
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作者：
A. Zomer;S. Steenbeek;Carrie Maynard;J. Rheenen
细胞外囊泡（Extracellular vesicle， EV）通过在细胞间传递多种生物分子，被越来越多地认为是细胞间通讯的一种重要方式。体外和体内分离的EV的特性对EV转移的生物学功能的理解有很大的贡献。然而，由于无法区分吸收EV的细胞和不吸收EV的细胞，因此在动物体内对EV释放和摄取的研究仍然具有挑战性。最近，一种基于Cre- loxpsystem的技术被开发出来，可以在各种体外和体内环境下荧光标记Cre报告细胞，这些细胞吸收了表达Cre重组酶的细胞释放的ev。在这里，我们描述了Cre+细胞和报告细胞的生成的详细方案，这需要~ 6周，以及随后用这些细胞系研究体外和体内（小鼠）设置的功能性EV转移，这需要~ 2个月。
Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization ofin vitro– orex vivo–isolated EVs has considerably contributed to the understanding of biological functions of EV transfer. However, the study of EV release and uptake in anin vivosetting has remained challenging, because cells that take up EVs could not be discriminated from cells that do not take up EVs. Recently, a technique based on the Cre-loxPsystem was developed to fluorescently mark Cre-reporter cells that take up EVs released by Cre recombinase–expressing cells in variousin vitroandin vivosettings. Here we describe a detailed protocol for the generation of Cre+cells and reporter+cells, which takes ∼6 weeks, and subsequent assays with these lines to study functional EV transfer inin vitroandin vivo(mouse) settings, which take up to ∼2 months.