Injecting Recombinant Adeno-Associated Vectors in a Spinal and Bulbar Muscular Atrophy Mouse Model

Begin with a restrained Spinal and bulbar muscular atrophy or SBMA-affected mouse.

Clean its tail and warm it to dilate the vein.

SBMA is caused by a mutated androgen receptor or AR gene that produces mutant AR mRNA.

This mRNA synthesizes toxic proteins in motor neurons that impair signal transmission and result in muscle weakness.

Next, take a syringe containing recombinant adeno-associated virus or AAV vectors with a plasmid encoding therapeutic microRNA.

Insert the needle into the tail vein and inject the solution.

Now, Apply pressure to stop bleeding, then return the mouse to its cage.

Once in circulation, the viral vector reaches motor neurons that control muscle function.

The virus enters the cell via endocytosis, releasing the plasmid.

The plasmid produces mature microRNA with complementary sequences to the mutant AR mRNA.

These microRNAs bind to AR mRNAs, triggering their degradation.

This inhibits toxic protein accumulation and helps to improve neuromuscular function.

Divide the AAV microRNA plasmid stock into 100 to 200-microliter aliquots, containing a viral load of 10 to 100 trillion genomes per milliliter. Next, load one aliquot into a syringe and attach 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.