Stereotaxic Viral Injection for Targeted Neuronal Gene Editing in the Mouse Brain

Secure an anesthetized mouse in a stereotaxic frame.

Disinfect the shaved scalp, then make a midline incision to expose the skull.

Dry the skull and apply hydrogen peroxide to enhance bregma visibility.

Locate bregma and lambda, then identify the target sites.

Carefully drill through the skull at the target sites.

Attach the virus-filled syringe to the stereotaxic instrument.

The virus encodes Cas9 and a single-guide RNA for genome editing in dentate gyrus neurons, along with a fluorescent reporter to label infected neurons.

Lower the syringe to reach the dentate gyrus, then inject the virus slowly to ensure uniform distribution.

Gradually raise the syringe and repeat the injection at additional coordinates.

Pause briefly to prevent backflow, then withdraw the needle slowly.

Repeat the procedure on the opposite hemisphere.

Finally, suture the incision.

The model is now ready for further studies.

Shave the head of an anesthetized mouse in order to prepare for the injection site. Direct 4% isoflurane into the nose cone. Next, place the mouse in the stereotaxic instrument. Insert the bite bar into its mouth until its teeth drop into the slot before securing the ear bars.

Ensure that the animal's body is on the heating pad and the nose is situated in the nose cone. Next, confirm the anesthesia by giving a foot pinch. Then, apply artificial tears to lubricate the eyes. Subsequently, swab the shaved head with povidone iodine and lidocaine.

Now, make a small incision along the scalp midline. Dry the skull with a swab and apply hydrogen peroxide to help visualize the bregma. Using a dissecting scope, locate the bregma on the skull and place the drill bit above it. Set the digital stereotactic coordinates of the x, y, and z planes to 0.

In order to ensure that the head is leveled on the rostral caudal y-axis, place the drill bit on lambda and level the head so that the z-coordinate is roughly equal to 0 at bregma and lambda. To ensure that the head is leveled along the x-axis, place the drill bit on the midpoint between bregma and lambda and measure the z-coordinates at 1 millimeter from the midpoint on both sides to ensure that they are equal.

Next, lower the isoflurane to 2% before placing the drill over the desired coordinates, and drill through the skull carefully. After drilling all the holes, affix the virus filled syringe onto the stereotaxic instrument. Center the syringe over the hole and set the z-coordinate at the skull to 0. Slowly lower the syringe to the deepest z-depth and begin injection at a rate of 0.25 microliters per minute using a stereotaxic injector.

After the injection is complete at the deepest z-depth, wait one minute before raising it to the next coordinate and begin injection again. Continue this pattern until all the z-injection coordinates are injected. After the last injection, wait two minutes before removing the syringe. Then, remove the mouse from the stereotaxic instrument and suture the scalp. Apply lidocaine and antibacterial cream to the surgical site and administer pain medication before transferring the animal to a heated recovery chamber.