We work on developing gene therapies for congenital and early onset muscular dystrophies and neuromuscular disorders. And to accomplish this, we use different gene therapy modalities to address the underlying genetic causes of those disorders. The continuous development of viral and non-viral gene therapy vectors has been very important to our field.
As the new improved viral and non-viral gene therapy vectors allow safer and more efficient gene therapies to be developed, this in turn, enhances the safety and reduces the time to get those therapies ready for the patients. In this protocol, we provide thorough and detailed optimized steps of performing mouse lateral tail-vein injections. And the aim of this protocol is to assist experimenters in consistently performing the injection procedures successfully.
To begin, insert the mouse tail into the restrainer tubes top opening. Slowly pull the mouse into the tube, and place the plug ahead of the mouse to prevent it from escaping. To make a restraining sleeve for a smaller mouse, cut at the nose end of the cone to ensure the mouse's nose is not obstructed and it has enough room to breathe.
Then cut the back of the cone similar to the length of the mouse. Place the mouse in the tube restrainer as previously described. While holding the mouse's tail, let the mouse walk into the cone.
Slide the restrainer cone into the tube. Ensure that the mouse's tail is all the way out of the back of the tube, and the mouse has room to breathe inside the cone. Secure the tube plug immediately in front of the cone's nose opening.
Fill the 15 milliliters conical tube with warm water. Place the tube restrainer with the mouse inside it on the elevated platform. Dip the restrained mouse's tail in warm water for at least one minute until the lateral veins are clearly dilated and visible.
Place the uncapped adeno-associated virus or AAV containing 1.7 milliliters microcentrifuge tube in a tube rack. With the dominant hand, insert the needle vertically into the tube. Once the needle is inside the tube, hold the tube with a non-dominant hand.
Then raise the tube and the syringe simultaneously to eye level while ensuring the needle does not touch the tube wall. Rest both arms on the table and slowly pull the dose into the syringe. Expel air bubbles from the syringe over a disposable absorbent pad.
Hold the injectate for 40 seconds to warm it up. After ensuring tail-veins are clearly visible, remove the tube restrainer from the elevated platform and place it directly on the table. Position the mouse in the restrainer with its feet downwards for easy handling of the tail.
Quickly wipe the tail with gauze to dry it. Then wipe the tail with an alcohol swab, and again, dry it with dry gauze. Rotate the tail approximately 90 degrees to the left or the right so that one of the two lateral veins is facing upwards.
Locate a suitable injection site within the middle third of the tail. Hold the tail with the non-dominant hand using the thumb and index finger immediately distal to the injection site. Fold the tail over the index finger so the injection site lies flat on the index finger.
Then pull back on the tail to stretch it and the injection site is completely horizontal. Hold the syringe using the index and middle fingers of the dominant hand on either side of the syringe's barrel flange while keeping the thumb ready at the plunger. Rest both hands on the table and place the needle directly on and parallel to the tail and the vein with the bevel facing upwards.
Now apply downward pressure on the needle and slide it forward into the vein to inject the AAV solution into the vein. After administering the dose, slowly withdraw the needle and immediately apply pressure with gauze at the injection site for at least 10 seconds until the bleeding stops. Histological imaging of fluorescent reporter protein post-injection showed that tdTomato was expressed diffusely throughout the liver and quadriceps indicating that AAV9 broadly reached and transduced different regions of both tissues.
