1. Prepare Equipment for Injection

1. Prepare a similar injection setup to the one shown in Figure 1. The central components are: Dissection scope, light source, micromanipulator, Nanoject, metal plate, ice reservoir, dissection tools.

2. To inject the virus, use capillary glass tubes, pulled using a P-97 pipette puller to produce fine tips with high resistance (Figure 2, step 1).

3. Cut the tip of the pipette using fine scissors to reshape the tip such that it would be sufficiently sharp to pierce through the skull, yet sufficiently wide to not break during the process (Figure 2, step 2).

4. Fill the sharp pipettes with mineral oil then attach it to a Nanoject III (Figure 2, step 3; Movie 1).

5. Eject most of the mineral oil using the Nanoject.

6. Load 4μL of vector solution into the pipette (Figure 2, step 4). For accurate movement of the Nanoject-attached-pipette, use an MP-285 micromanipulator.

7. Cool a metal stage by adding ice to the reservoir (Figure 2, step 5).

2. Prepare Pups for Injection

1. Remove P0-P1 pups from their cages and place them on a warm pad.

2. Place a small aluminum plate on ice to cool.

3. Anesthetize each pup on the ice-cold aluminum for 2-3 minutes. Confirm anesthesia by gently squeezing a paw and monitor lack of response.

4. Transfer the pup to the cooled metal plate (Figure 2, step 6). Use a light microscope (Figure 1) to visualize the transverse sinuses (located on the dorsal surface of the mouse head).

5. Use sterilized fine scissors to make a small V-shaped cut (~2mm) in the skin above the right transverse sinus (Movie 2).

3. Transverse Sinus Injection

1. Lay the pup such that the site of injection is perpendicular to the pipette. Place a piece of pudding under the head of the pup to make the head level in the Y-axis (front to back).

2. Gently lower the pipette until the tip touches the skull.

3. Set the micromanipulator coordinates to zero.

4. Advance the pipette through the skull and into the sinus until the tip of the pipette is observed (using the light microscope) to break through the sinus vessel wall (Movie 3, 0:00-10:00s).

5. After waiting for 5 seconds, retract the pipette tip until it is 300-400μm below the surface of the skull, such that the tip resides within the lumen of the sinus (Movie 3, 15:00-21:00s).

6. With no delay, inject 2μL of virus at a rate of 20nL per second.

7. Verify a successful injection by visualizing viral solution flow into the blood stream evidenced by blanching of the sinus (Movie 4). If this is not immediately observed, slowly adjust the depth of the pipette tip (+/- 200μm) while observing the micromanipulator coordinates until the viral plume is observed.

8. Following a 5 second delay, retract the pipette and move the skin back over the cut (Movie 5).

9. Repeat the same cutting and injection procedure targeting the opposite hemisphere. The total volume of virus injected per mouse pup is 4μL.

10. For dual virus injection experiments, one could either mix the two viruses beforehand and co-inject a total volume of 4μL of the two constructs at P1 or inject each virus separately into each sinus (totaling to 4 injections per mouse, 1μL each). The latter approach has proven to be more effective with efficiently expressing both constructs in some cases.

4. Post-injection Care

1. After the injection, fold the skin back, and apply a small amount of VetBond glue to the cut (Movie 6), and dry as much of it as possible using a Kimwipe.

2. Place the pup on a warming pad. After injecting the whole litter, return the pups to their home cage and gently rub them with bedding to prevent rejection by the mother.

Equipment

1. MP-285 micromanipulator (Sutter Instruments, CA, USA)

2. P-97 pipette puller (Sutter Instruments, CA, USA)

3. Capillary glass tubes (3.5’ #3-000-203-G/X. Drummond Scientific Co, PA, USA)

4. Mineral oil (M3516, Sigma-Aldrich, NY, USA)

5. Nanoject III (Drummond Scientific Co)

6. Fine scissors (Fine Science Tools, CA, USA)