Agarose-Embedded Electroporation-Assisted Gene Transfer in Mouse Cortical Interneuron Progenitors

1. Focal DNA injections
   1. Prepare a deoxyribonucleic acid (DNA) mixture of expression vectors: pCAGGs-IRES-GFP (control vector) + pCAGGs-hM3D(Gq)-IRES-RFP, at a concentration of 1 µg/µL for each vector. Add fast green solution (stock 25 mg/mL) in a 1/10 dilution.
   2. Fill a pulled glass micropipette (0.5 mm inner diameter and 1 mm outer diameter) with 10 µL of the DNA mixture and inject small amounts (in the 25-50 nL range) into the selected region (Medial Ganglionic Eminence or MGE/Caudal Ganglionic or CGE) of the slice (Figure 1 and Figure 2).
2. Acute electroporation 
   NOTE: The electroporation should be performed immediately after the focal DNA injection.
   1. Place the small agarose block on the Petri dish electrode and attach the agarose column to the mobile cover electrode using a flat-ended micro-spatula.
   2. Transfer the slice with its supporting membrane onto the agarose block and place the top electrode with the agarose column on top of the selected region (MGE/CGE) of the slice. 
      NOTE: Charging voltages of 125 V (two pulses of 5 ms each, interval 500 ms) will yield a successful electroporation (Figure 3).
3. After the electroporation, place the slice with its supporting membrane in the dish and transfer it to a CO₂ tissue culture incubator at 37 °C.

Figure 1: Representative telencephalic slices used for acute electroporation experiments. (A-C) Telencephalic slices were obtained at three distinct sequential rostrocaudal levels, stained with 4′,6-diamidino-2-phenylindole (DAPI). LGE: lateral ganglionic eminence; MGE: medial ganglionic eminence; CGE: caudal ganglionic eminence. Scale bars = 200 µm. The yellow asterisks indicate the electroporation site in each slice. The white line marks the edge of the ganglionic eminence.

Figure 2: Schematic representation of the experimental workflow. (A) Mouse brain slices are electroporated with appropriate constructs, and (B) after 12 h, modified cortical interneuron (CI) precursors are isolated and (C) transplanted in the pallium of newborn mouse pups (P0−P2). In order to modify the activity of immature CIs, P14 pups that had received cell transplantations were injected with CNO or vehicle for four constitutive days according to the presented protocol. (A') Photograph of the acute mouse brain slice electroporation set-up.

Figure 3: Representative successful acute slice electroporation experiment. (A) Representative coronal section from an E14.5 embryo brain transfected in the CGE with both pCAGGs-IRES-GFP (Green fluorescent protein) and pCAGGs-hM3D(Gq)-IRES-RFP (Red fluorescent protein) plasmids and cultured for 12 h. The section has been immunostained for GFP (A, B, C) and RFP (A, B, D). The boxed area in panel A is magnified to show the expression of both fluorescent reporters (B), GFP (C), and RFP only (D). The white line marks the edge of the ganglionic eminence. B-D: same photo, different channels, or a combination of the two different channels. Scale bars = 200 µm (A), 100 µm (B-D).