AAV Craniotomy

Weigh mice.

Administer intraperitoneal mixture of ketamine (ButlerAnimal Health Supply) and xylazine (Lloyd Laboratories) at concentrations of 110 and 10 mg/kg of body weight, respectively.

Confirm induction of anesthesia.

Place mouse into stereotactic frame (David Kopf Instruments) and place eye lubricant.

Remove fur from skull area using either electric shaver or depilatory cream.

Sterilize skull area and then cut open with scalpel.

Adjust brain so that it is level. Locate bregma and zero X and Y axes on stereotaxic frame.

Move to injection coordinates and drill small hole into the skull.

Zero the Z axis on the stereotaxic frame.

Prepare AAV solution by diluting AAV in PBS with 500mM Mg2/Cl to a final dilution of total of 2x10¹¹ genomic particles/mL.

Using a 10 μL stereotactic syringe with a 33 G needle attached to a micro-infusion pump (World Precision Instruments), withdraw the AAV solution.

With the loaded syringe, navigate the needle to the desired X, Y, Z coordinates.

Infuse the brain with AAV at a flow rate of 0.1 - 0.4 μL/min.

To prevent reflux, after each infusion, leave the injection needle in place for 5 min, withdraw a short distance (0.3 - 0.5 mm), and then leave in the new position for an additional 2 min before complete removal of the needle.

Close skin with non-absorbable sutures.

Inject a total of 2x10¹¹ genomic particles/mL of vector is injected into the appropriate coordinates.

Give meloxicam 2mg/kg subcutaneously.

Perform health checks every day for the first 72 hrs post-injection.

Remove sutures 15 days post-injection.