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A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

机译：微型设备可以高效地局部电穿孔胚胎并减少细胞损伤

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摘要

The ability to follow and modify cell behaviour with accurate spatiotemporal resolution is a prerequisite to study morphogenesis in developing organisms. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has been used with this aim in different model systems. However, current localised electroporation strategies suffer from insufficient reproducibility and mediocre survival when applied to small and delicate organisms such as early post-implantation mouse embryos. We introduce here a microdevice to achieve localised electroporation with high efficiency and reduced cell damage. In silico simulations using a simpleelectricalmodel ofmouse embryos indicated that a dielectric guide-based design would improve on existing alternatives. Such a device was microfabricated and its capacities tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Transfection was efficiently and reproducibly restricted to fewer than four visceral endoderm cells without compromising cell behaviour and embryo survival. Combining targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos revealed that, like leading DVE cells, non-leading ones send long basal projections and intercalate during their migration. Finally, we show that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation stages to organ explants. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in the mouse embryo. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices.
机译：以准确的时空分辨率跟踪和改变细胞行为的能力是研究发育中的生物形态发生的先决条件。为此目的，已经在不同的模型系统中使用了电穿孔，即通过质膜的电渗透将外源分子递送到靶细胞群中。但是，当前的局部电穿孔策略在应用于小型且脆弱的生物体（如早期植入后的小鼠胚胎）时，其再现性不足且存活率中等。我们在这里介绍一种微型设备，可实现高效的局部电穿孔并减少细胞损伤。在使用鼠标胚胎的简单电模型进行的计算机模拟中，基于电导的设计将改善现有的替代方案。这种设备是微制造的，其功能通过靶向远端内脏内胚层（DVE）进行测试，DVE是建立前后轴必不可少的迁移细胞群。转染被有效且可重复地限制在少于四个内脏内胚层细胞，而不会影响细胞行为和胚胎存活。将荧光标记物的定向镶嵌表达与转基因胚胎中的实时成像相结合，发现与领先的DVE细胞一样，非领先的DVE细胞在迁移过程中也会传递较长的基础突起并插入。最后，我们证明了我们的微系统的使用可以扩展到从植入前阶段到器官外植体的各种胚胎学背景。因此，我们已经通过实验验证了一种为研究小鼠胚胎的形态发生提供量身定制的工具的方法。此外，我们为特设电穿孔设备的开发制定了全面的战略。

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