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Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe

Abstract

This protocol is an extension to: Nat. Protoc. 1, 1552–1558 (2006); doi:10.1038/nprot.2006.276; published online 9 November 2006

This article describes how to reliably electroporate with DNA plasmids rodent neuronal progenitors of the hippocampus; the motor, prefrontal and visual cortices; and the cerebellum in utero. As a Protocol Extension article, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol describes how to electroporate mouse embryos using two standard forceps-type electrodes. In the present protocol, additional electroporation configurations are possible because of the addition of a third electrode alongside the two standard forceps-type electrodes. By adjusting the position and polarity of the three electrodes, the electric field can be directed with great accuracy to different neurogenic areas. Bilateral transfection of brain hemispheres can be achieved after a single electroporation episode. Approximately 75% of electroporated embryos survive to postnatal ages, and depending on the target area, 50–90% express the electroporated vector. The electroporation procedure takes 1 h 35 min. The protocol is suitable for the preparation of animals for various applications, including histochemistry, behavioral studies, electrophysiology and in vivo imaging.

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Figure 1: Crafting of the third electrode.
Figure 2: Configuration of the surgery setup with a pregnant mouse laying on the operating heating platform.
Figure 3: DNA injection in the embryo using a commercial needle or a glass micropipette.
Figure 5: Experimental configuration for electroporation of the hippocampus.
Figure 6: Experimental configuration for electroporation of the motor cortex.
Figure 7: Experimental configuration for electroporation of the prefrontal cortex.
Figure 8: Experimental configuration for electroporation of the visual cortex.
Figure 9: Experimental configuration for electroporation of the Purkinje cells of the cerebellum.
Figure 4: Examples of in utero electroporation of mouse embryos (E15.5) with the use of different sizes (top labels) of forceps-type electrodes and the additional third electrode.
Figure 10: Long-lasting expression of the proteins of interest by tripolar in utero electroporation.
Figure 11: Confocal image showing bilateral expression of the fluorescent protein EGFP in rat hippocampi at P21 obtained by a single electroporation episode with the tripolar-electrode configuration at E17.5. Black cells are neurons expressing EGFP.
Figure 12: Tripolar in utero electroporation allows in vivo chloride imaging in the visual cortex.

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Acknowledgements

We thank S. Sulis Sato and P. Artoni (Scuola Normale Superiore, Pisa, Italy) for performing the in vivo imaging of electroporated neurons, G. Deidda for the video showing visual cortex illumination in a live pup, F. Managò (IIT) for her idea to use a cell scraper as the holder for the third custom-made electrode, G. Pruzzo (IIT) for his technical help in crafting it, C. Garcia (IIT) for samples from long-term in utero electroporation experiments, A. Contestabile (IIT) for the recipe for the cleaning of surgical tools, and S.-I. Lee (Stony Brook University) for his assistance during some surgeries. The work was supported by Telethon nos. GGP10135 and GGP13187, and IIT interdepartmental grant Novel Site.

Author information

Authors and Affiliations

Authors

Contributions

J.S. performed in utero electroporation, wrote the manuscript and made the figures. A.W.C. contributed to in utero electroporation, performed immunohistochemistry, acquired the images and made the figures. J.S. and A.W.C. participated in the design of the experiments. M.d.M. assisted during in utero electroporation and made the Purkinje cell videos. D.G. assisted during in utero electroporation. G.M.R. set up the in vivo chloride imaging and made the video and the figure. L.C. performed in utero electroporation, designed the experiments and wrote the manuscript. G.M.R. and L.C. invented the triple-electrode configuration. All authors revised the manuscript.

Corresponding author

Correspondence to Laura Cancedda.

Ethics declarations

Competing interests

L.C. and G.M.R. are co-inventors on a patent entitled “three-electrode electroporation device” (Patent number WO2012153291).

Integrated supplementary information

Supplementary Figure 1 Equipment for in utero electroporation.

(This figure is also available in Protocol Exchange doi:10.1038/protex.2013.089 (2013) and is reproduced with permission here to aid use of the protocol)

Supplementary Figure 2 Electrode configuration for tripolar in utero electroporation.

(a) The configuration for tripolar in utero electroporation entails two conventional forceps-type electrodes connected to a single polarity by a Y-connector and an additional custom-made third electrode. Scale bar: 5 cm. (b) High magnification of the Y-connector for connection of commercial forceps-like electrodes to a same pole. Scale bar: 1 cm. (This figure is also available in Protocol Exchange doi:10.1038/protex.2013.089 (2013) and is reproduced with permission here to aid use of the protocol)

Supplementary Figure 3 Tools for in utero electroporation.

(a) ring forceps (1), shark-tooth tissue forceps (2), scissors with flat shanks – angular (3), scissors with flat shanks – straight (4), Olsen-Hegar needle holders with scissors (5), scalpel (6), scalpel blade (7). (b) surgical tools, plastic connectors, surgical drape and gauze in a self-sealing autoclavable pouch. Scale bars: 2 cm. (This figure is also available in Protocol Exchange doi:10.1038/protex.2013.089 (2013) and is reproduced with permission here to aid use of the protocol)

Supplementary Figure 4 The fluorescence of the reporter gene can be enhanced by immunostaining with a specific antibody.

(a) Confocal image of EGFP fluorescence in a coronal section of a mouse neocortex at P7 after in utero transfection (at E15.5) in an experiment that resulted in low transfection efficiency. (b) Confocal image of the same slice after immunostaining with anti-GFP antibody (Abcam, cat. no. Ab13970; concentration 1:1000). Images in (a) and (b) were acquired using a confocal microscope with the same parameters. Scale bar: 100 µm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 677 kb)

Identification of EGPF transfection by exposing live pups (P4) electroporated in the visual cortex in utero (E17.5) to a fluorescent-protein flashlight lamp.

This simple and quick method is extremely useful to distinguish upfront successful electroporations from failures. (MOV 1568 kb)

3D cell reconstructions of Purkinje cells of the rat cerebellum at P14.

For 3D reconstructions of the morphology of transfected neurons in brain slices, it is important to achieve a compromise between spatial resolution, time of acquisition and potential bleaching of the chromophores during image acquisition. To achieve this aim, we recommend performing a serial acquisition of tiled z-stacks to cover a complete slice using a 40X oil immersion objective (1.4 NA). The field of view should be 350 × 350 μm−2, with 0.2 × 0.2 × 0.5 voxel size, and should be acquired with 3 line averaging. The power at the sample should be kept as low as possible to prevent bleaching over the relatively long exposures (45 min per slice, typically). Rendering of acquired data can be performed with freely available software packages (e.g., Fiji\3D viewer or pictograph libraries) or with more sophisticated and dedicated consoles (e.g., Imaris or Huygens). (MOV 1014 kb)

3D cell reconstruction of an isolated Purkinje cell of the rat cerebellum at P14.

Image acquisition and cell reconstruction were performed as described for Supplementary video 2. (MOV 4953 kb)

Tripolar in utero electroporation allows in vivo chloride imaging in the visual cortex.

Video for Figure 12. (AVI 19604 kb)

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Szczurkowska, J., Cwetsch, A., dal Maschio, M. et al. Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe. Nat Protoc 11, 399–412 (2016). https://doi.org/10.1038/nprot.2016.014

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