Electroporation-mediated gene transfer in muscle is an easy and efficient technique to investigate changes in muscle physiology. We found that electroporation does not compromise muscle contractility. Electroporation of gene constructs into muscle in vivo is a simple and efficient procedure that does not require more extensive packaging of gene constructs into viral vectors.
It is difficult to visualize, isolate, and inject the EDL due to its location and small size. Practicing on a mouse carcass and injection of biological ink such as India ink may assist in providing evidence of sufficient injection. Demonstrating the procedure will be Brian Hain, a post doc from my laboratory.
Before injecting the mouse, confirm the surgical plane of anesthesia using forceps with the absence of the toe pinch reflex, then transfer the mouse to a nose cone resting on a circulating water plate at 37 degrees Celsius for the rest of the procedure. Once the mouse is placed in a supine position, remove hair from both hind limbs using small hair clippers, then sanitize the injection area with alternating 70%ethanol and betadine. For the injection locate the tibialis anterior or TA tendon visible through the skin on the lateral side of the lower leg and mark the superior end of the TA muscle.
Then insert a 30-gauge needle mounted on a 50-microliter micro syringe into the muscle at a shallow five-degree angle one to two millimeters superior to the myotendinous junction until the needle reaches the superior end of the muscle. Depress the plunger while slowly retracting the needle along the injection path to deliver 50 microliters of the plasmid solution into the muscle. The muscle should swell.
Set the timer for one minute and measure the thickness of the leg at the TA muscle, then set the caliper electrodes to the measured thickness. Then set the electroporator voltage to 12.5 volts per millimeter. After one minute, place the caliper electrodes closely around the lower limb without being overly tight to deliver five square wave pulses with 20-millisecond duration and 200-millisecond intervals.
The muscle should twitch with each pulse. Repeat the procedure with the other limb using the control plasmid vector. When done, remove the mouse from the nose cone and allow the mouse to recover on a heating pad set to 37 degrees Celsius.
Once recovered, return the mouse to its cage. With the mouse in a supine position, locate the tibia bone anterior crest visually and through gentle palpitation. Use a scalpel to make a shallow incision through the skin on the lateral side of the tibia anterior crest five millimeters inferior to the knee to two millimeters superior to the TA myotendinous junction.
With the help of small scissors bluntly dissect the fascia, exposing the TA muscle, and then separate the TA muscle from the tibia gently by pulling the muscle to reveal the extensor digitorum longus or EDL. Keep the TA clear from the EDL during the procedure using small curved forceps. Insert the 30-gauge needle into the EDL longitudinally until the needle reaches the superior end of the muscle and inject 10 microliters of the plasmid solution as demonstrated before.
The EDL muscle should swell. Measure the thickness of the leg at the EDL and set the caliper electrodes to the measured thickness to deliver five square wave pulses as explained earlier. When done, close the incision using disposable 4-0 non-absorbable nylon sutures.
Later remove the mouse from the nose cone and allow it to recover on a heating pad set to 37 degrees Celsius. Administer an appropriate subcutaneous analgesic immediately after the surgery and 12 to 24 hours following the surgery. Once recovered return the mouse to the home cage.
After the injection and electroporation of the pcDNA3-EGFP plasmid in the TA and EDL muscles, the GFP expression was observed indicating DNA uptake in the muscle fibers. When imaged at a lower magnification, the EDL muscle transfection efficiency could be visualized through the appearance of green fibers versus black fibers. The injected and electroporated EDLs had similar tetanic responses at 100 Hertz compared to the control non-injected or electroporated muscles.
The injected and electroporated EDL, the tetanic muscle force, specific tetanic muscle force, time to peak tension, and half relaxation time were not compromised compared to the untouched control. It is important to accurately identify the muscle to be injected and to efficiently deliver the plasmid solution. A number of histological and biochemical measurements can be made using this protocol.
Target protein cellular localization, target gene knockdown, transcriptional reporters, and an array of tissue staining can be conducted. This technique has been used to identify a vast array of changes in molecular signaling in muscle during physiological and pathophysiological conditions. The investigation of the muscle contractility following gene transfer is feasible using this technique.
