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

Abstract

Source: Denaxa, M., el al., Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains. J. Vis. Exp. (2019)

This video demonstrates the procedure for injecting a plasmid vector carrying the gene of interest into interneuron progenitors in an embryonic brain slice, followed by electroporation for plasmid entry and gene expression. These modified progenitors can later be used for treating brain disorders.

Protocol

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.

Results

Figure 1: Representative telencephalic slices used for acute electroporation experiments. (A-C) Telencephalic slices were...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Medium/Supplements
Neurobasal medium	GIBCO (ThermoFisher Scientific)	21103-049	Neuron basic medium
Equipment
Electroporator	BTX	ECM 830 generator
Injector for acute slice electroporation	Eppendorf	FemtoJet Microinjector
Kite Manual Micromanipulator	WPI	KITE-M3-R
Platinum Elecrode (I)	Protech International Inc.	CUY-700-1
Platinum Elecrode (II)	Protech International Inc.	CUY-700-2
Steel Base Plate	WPI	5479
Other Material
Glass capillaries for electroporation	VWR	1B100-4
Organ tissue culture dishes	BD Biosciences (Falcon)	353037