Dual in Utero Electroporation in Mice to Manipulate Two Specific Neuronal Populations in the Developing Cortex

Abstract

Precise regulation of gene expression during development in cortical neurons is essential for the establishment and maintenance of neuronal connectivity and higher-order cognition. Dual in utero electroporation provides a precise and effective tool to label and manipulate gene expression in multiple neuronal populations within a circuit in a spatially and temporally regulated manner. In addition, this technique allows for morphophysiological investigations into neuronal development and connectivity following cell-specific gene manipulations. Here, we detail the dual in utero electroporation protocol.

Keywords: cortex; development; in utero electroporation; neuron; neuroonal connectivity.

GRAPHICAL ABSTRACT

FIGURE 1

Dual IUE image in the SSC. (A): Image of a postnatal day (P) 14 mouse coronal section containing GFP-expressing L2/3 cortical neurons and tdTomato-expressing L4/5 neurons following IUE at E15.5 and E13.5, respectively. (B): Fluorescent image overlaid with bright field. Bar: 50 µm.

FIGURE 2

Micropipette preparation. (A): Image of an intact glass micropipette that was generated by a vertical puller. The black lines denote the amount of tip that should be removed prior to insertion into the embryo. (B): Image of the large round forceps used to break the tip of micropipette. (C): Image of an adequately broken micropipette with an ∼45-degree-tip. The black lines denote the amount of tip that was removed. (D): An example of a dull pipette. The black lines denote the amount of tip that was removed.

FIGURE 3

Uterine horns exposure during IUE surgery. (A): Image of dissected uterine horns and labeling of desired IUE embryos. Uterine fundus is a useful marker to distinguish the two uterine horns and can be used as a reference to identify the electroporated embryos prior to the second IUE. Label embryos beginning at the uterine knots and continue sequentially to the uterine fundus for each uterine horn. Do not electroporate the embryos at the uterine knot or at the fundus (Left horn: 1, 9; Right horn: 1, 7). Squares surrounding the numbered embryos denote which embryos should receive IUE (Left horn: 2, 4, 6, 8; Right horn: 2, 4, 6). Electroporate embryos in an alternating fashion in the same hemisphere for both IUEs. (B): Image of lateral ventricle injection. Plasmid DNA was injected into E15 embryo, and the right lateral ventricle was filled with fast green.

FIGURE 4

Dual IUE 12-h apart leads to colocalization between the two electroporated cell populations. (A): Images of electroporated neurons in the SSC following a dual IUE 12-h apart (green: pCAG-GFP; red: pCAG-tdTomato). (B): Quantification of the GFP and RFP colocalization and non-colocalization. Bar: 25 μm, 2 slices per mouse, N = 3 mice.