Electroporation-Mediated Transfection of Mouse Dorsal Root Ganglia to Promote Sensory Axon Regeneration

Abstract

Source: Li, Q. et al. Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion. J. Vis. Exp. (2018)

This video demonstrates electroporation-mediated transfection of mouse DRG neurons to study axon regeneration. It outlines the steps for delivering nucleic acid constructs to DRG neurons, inducing sciatic nerve injury, and assessing axonal regeneration.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Dorsal root ganglion (DRG) Injection
   1. Inject an anesthetic solution (dilute avertin in sterile saline at a final concentration of 20 mg/mL, with avertin 200 mg/kg body weight) intraperitoneally to maintain the anesthesia before DRG injection.
   2. Load the deoxyribonucleic acid (DNA) plasmids or ribonucleic acid (RNA) oligos (1 µL per DRG) into the glass capillary pipette.
   3. Insert the tip of the capillary glass pipette carefully into....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ECM 830 Square wave electroporation system	BTX Harvard Apparatus	45-0052	For in vivo electroporation
Tweezertrodes electrodes	BTX Harvard Apparatus	45-0524	For in vivo electroporation, 1 mm flat
Picospritzer III	Parker Instrumentation	1096	Intracellular Microinjection Dispense Systems
Borosilicate Glass Capillaries	World Precision Instruments, Inc.	1902328
Stereo Dissection Microscope	Leica	M80
Microsurgery Forceps	FST by DUMONT, Switzerland	11255-20	Only for sciatic nerve crush
Glass Capillary	World Precision Instruments, Inc.	TW100-4	10 cm, standard wall
Tape	Fisherbrand	15-901-30	For fixing the mouse on the corkboard
2, 2, 2-Tribromoethanol (Avertin)	Sigma-Aldrich	T48402	Avertin stock solution
2-methyl-2-butanol	Sigma-Aldrich	152463	Avertin stock solution
siRNA Fluorescent Universal Negative Control #1	Sigma-Aldrich	SIC003	Non-target siRNA with fluorescence
microRNA Mimic Transfection Control with Dy547	Dharmacon	CP-004500-01-05	Non-target microRNA with fluorescence
Plasmids preparation kit	Invitrogen Purelink	K210016	GFP-coding plasmid preparation
Fast Green Dye	Millipore-Sigma	F7252	For better visualization of the DRG outline during injection
Ketamine	Putney, Inc	NDC 26637-731-51	Anesthesia induction
Xylazine	AnaSed	NDC 59399-110-20	Anesthesia induction
Acetaminophen	McNeil Consumer Healthcare	NDC 50580-449-36	Post-surgical pain relief