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

Abstract

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 simple electrical model of mouse 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.

Tissue morphodynamics shaping the early mouse embryo

“…At the same time, DVE cells maintain their epithelial characteristics, and exchange neighbors along the migration pathway [35,37]. This behavior not only characterizes the leading DVE cells, but also surrounding cells that will contribute to the AVE [38], establishing this as a coordinated, or collective, cell migration. Genetic studies have identified roles for actin regulators Rac1 and the WAVE complex as well as PTEN in this migration [35,39,40], consistent with the active, directed protrusive behavior.…”

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

“…Temporary in vitro cultivation of embryos at these stages is known to be feasible [72,73]. Therefore, many researchers performed gene delivery (via in vitro EP or injection of viral vectors) into dissected postimplantation embryos to assess gene function, cell movement, and cell lineage [74][75][76][77][78][79][80]. For example, Mellitzer et al [76] microinjected a solution containing dsRNA (100 ng/µL) directed against orthodenticle homeobox 2 (Otx2) or forkhead box protein A2 (Foxa2) and fast green dye into the amniotic cavity of an isolated embryo (Day 7.5 of pregnancy), as shown in Figure 4A.…”

(Invited) Finite Element Model Simulations to Assist the Design of Microdevices Dedicated to the Localized Electroporation of Mouse Embryos

“…These data are similar to the one associated with the best conditions used to run our original SU-8 / Parafilm chip (4). However, thanks to the simulations the present optimal parameters were obtained after a very limited number of tests, ~ 12 per device, whereas in our initial screening work on the first design we performed more than 200 electroporations (4). Finally, it is worth noting that permeation localization also stayed satisfactory since the shape of the dielectric guides was roughly kept constant during the technological evolution process.…”

“…Moreover, microsystem performances could be demonstrated by addressing at E5.5 the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Live imaging of cells discretely labeled with fluorescent proteins allowed us to reveal new facts concerning tissue rearrangement (4). Besides, we showed that the utilization of our device could be extended to a variety of embryological contexts, from preimplantation embryos to organ explants dissected from latter stage organisms (4).…”