The overall goal of this method is to select and deliver a microRNA using a recombinant Adeno-associated virus vector. Then deliver it to skeletal muscle and motor neurons of mice, and to assess their efficacy in ameliorating disease phenotypes. This method can help answer the important question of whether a microRNA based silencing strategy can be employed to treat neuromuscular conditions.
The main advantage of this technique is the use of Adeno-associated serotype 9 virus to deliver a microRNA through systemic circulation to the spinal chord and skeletal muscle of mice, to directly silence a target disease gene. Divide the AAV-microRNA plasmid stock into 100 to 200 microliter aliquots containing a viral load of ten to one hundred trillion genomes per milliliter. Next, load one aliquot into a syringe, and attached a 27 to 30 gauge needle.
Now, using a standard mouse restrainer, secure a mouse with its tail exposed. Then, clean the tail with 70%alcohol and place a warm pad under the tail for about 30 seconds to increase vasodilation. Now, at the distal portion of the tail, insert the needle into the tail vein with the bevel up and at a shallow angle, about 15 degrees.
Then, inject the virus and wait a few seconds before removing the needle. After removing the needle, apply moderate pressure using two fingers to stop any bleeding. Then, return the animal to its home cage.
During the tail vein injection, it is not uncommon to see a flash of blood in the hub of the needle. While injecting, there should be no resistance. If the tail starts to blanch and swell, remove the needle and try again proximal to the previous entry location.
For these tests the mice should be older than four weeks, and be sure to collect a baseline measure before treatment. The testing room should be quiet and dark without disturbances. Bring the mice into the room at least 30 minutes before conducting any tests.
Do not disturb the animals during this period of acclimatization. Start with the hanging wire test. Pick up the mouse by its tail and place it on the center of the wire grid.
Then, raise the grid 15 to 20 inches and slowly invert the screen to give the mouse time to adjust its grip. Once the grid is completely inverted, start the timer and record the time to fall. Allow the mouse 60 seconds to hang on, if it falls sooner, then return it to the grid and repeat the procedure.
If the mouse hangs a full 60 seconds, register 60 seconds for the score and return the mouse to its cage. Allow each mouse three tries to hand 60 seconds. Otherwise, score the longest hang time.
After euthanizing the mouse harvest the spinal cord and then harvest the quadriceps skeletal muscle. To snap freeze the harvested tissues, half fill a stainless steel bowl with 2-methylbutane and submerge the bottom quarter of the bowl in liquid nitrogen. Wait two minutes before proceeding.
After a few minutes, transfer the sample into the 2-methylbutane. When the sample turns white, immediately transfer it into a container embedded in dry ice. Later, store the sample at 80 degrees Celsius.
For biochemical analysis and microRNA quantification, place the harvested tissue in a cryovial and freeze it in liquid nitrogen. Then, store it a 80 degrees Celsius. SBMA mice were injected with one hundred trillion genomes of AAV microR-298 at five weeks of age.
SBMA mice carry the human androgen receptor transgene with an abnormally expanded polyglutamine tract. By ten weeks, they normally develop signs of neuromuscular disease. The lumbar spinal cord and quadriceps muscle were harvested at several time points after treatment for analysis.
Quantitative RT-PCR showed that the micro-R298 expression peaked in the skeletal muscle after eight weeks, and that the expression peaked in the spinal cord at 12 weeks. Ten weeks after the injection, GFP was detected in both tissues. And GFP expression co-localized with the motor neuron marker choline acetyltransferase.
In a follow-up experiment, mice were randomly injected with the virus, or vehicle, at seven weeks of age and were then weighed and assay behaviorally weekly, up to 40 weeks of age. Ten weeks after the injection, the treated mice started gaining weight and started showing improved motor performance. Quantitative realtime PCR revealed that the treatment reduce AR transcript levels in the affected tissues.
After watching this video, you should have good understanding of how to deliver a microRNA using a recombinant Adeno-associated virus vector through tail vain injection. And how to perform behavioral assays that can help to assess the efficiency of the delivered microRNA in ameliorating lower limb atrophy and weakness in mice. The implication of this technique extends toward gene therapy approaches, because a safe and effective gene therapy requires targeted tissues specific transfer of the therapeutic transgene.
We first had the idea for this method when we realized that disease caused by toxic gain of function mechanisms, such as SBMA, would benefit by over expression of naturally occurring microRNAs specifically targeting the three-prime repair of the disease transcript.
