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 the DRG.
   4. Gradually inject 1.0 µL solution of DNA plasmids or RNA oligos into the DRG using the intracellular microinjection dispense systems (30 psi, 8 ms).
      NOTE: The duration of the injection should last no less than 5 minutes.
2. Electroporation
   1. Drop phosphate-buffered saline (PBS) on the tips of the electrodes.
   2. Clean up the bleeding with sterilized square cotton gauze.
   3. Pinch the target DRG with the electrodes gently and apply 5 square electric pulses with the electroporation system.
   4. Close the muscle and skin layers with 5-0 nylon sutures, respectively.
   5. Place the mouse on a heated blanket (35.0 °C) under close attention until it has completely regained sufficient consciousness to maintain sternal recumbency. Return the mouse to the home cage after it has fully recovered from the anesthesia.
      NOTE: It takes approximately 60 min for the mouse to completely recover from anesthesia on the 37 °C blanket. Dissolve one pill (200 mg) ibuprofen into 1,000 ml water (0.2 mg/ml) for daily feeding to relieve the post-surgical pain.
3. Sciatic Nerve Crush
   1. Two or three days (depending on the experimental design) after the DRG electroporation, anesthetize the mouse intraperitoneally with the anesthetic solution (with avertin 400 mg/kg body weight).
   2. Tape the four limbs of the mouse on the corkboard.
   3. Make a 1 cm incision 0.5 cm to the left side along the midline. Cut the muscles, such as the gluteus maximus muscle and the piriformis muscle, longitudinally. Expose the segment of the sciatic nerve between the greater sciatic foramen and the sciatic notch.
   4. Crush the nerve with microsurgery forceps for 12 s and mark the crush site with a 10-0 nylon epineural suture. Make a knot on the dural membrane to mark the crush site.
   5. Close the muscle and skin layers with 5-0 nylon suture.
   6. Place the mouse on a heated blanket (35.0 °C) with close attention until it has completely regained sufficient consciousness to maintain sternal recumbency. Return the mouse to the home cage after it has fully recovered from the anesthesia.
      NOTE: It takes approximately 60 min for the mouse to completely recover from anesthesia on the 37 °C blanket. Dissolve one pill (200 mg) ibuprofen into 1,000 ml water (0.2 mg/ml) for daily feeding to relieve the post-surgical pain.

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