This article describes a method for gene transfer into Schwann cells using electroporation in an anesthetized neonatal rat model. The technique involves the injection of plasmid DNA and the application of electric pulses to facilitate plasmid uptake.
Begin with an anesthetized neonatal rat placed on a heating pad with an exposed sciatic nerve.
Lift the distal site of the nerve and inject a plasmid DNA solution mixed with a dye. The dye helps to track plasmid delivery.
Place a tweezer-type electrode around the nerve, maintaining a small gap between the electrode arms.
Introduce saline into this gap for current conduction.
Apply an electric pulse surrounding the nerve fibers that creates transient membrane pores in the Schwann cells. Then, invert the electrode positions and apply a second electric pulse. This enhances plasmid DNA entry through the pores.
After electroporation, the cell membrane reseals and traps the plasmid.
Clean the site, seal the skin incision, disinfect it, and allow the rat to recover.
Later, the plasmid DNA translocates to the nucleus and integrates into the Schwann cell DNA, enabling successful gene transfer.
Gently pull the distal side of the nerve using a needle to lift the exposed nerve. Insert the glass micro-pipette into the distal site on the nerve, and inject the DNA solution by applying slight positive pressure until the nerve appears green. Next, place a tweezer-type platinum electrode at about one to two millimeters from the nerve.
Fill the gap between the electrode and the nerve with 0.9% sodium chloride solution. Apply electric pulses to the injection site using an electroporator. After the first pulse set, invert the electrode and apply another pulse set.
Afterward, clean the electroporation site with 0.9% sodium chloride solution. After electroporation, close the incision with cyanoacrylate glue. Then, clean the wound with povidone iodine.
Remove the face mask from the animal, and keep it on the electric warmer for at least an hour to allow full recovery from anesthesia
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