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

Abstract

Source: Fricano-Kugler, C. J., et al. Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection. J. Vis. Exp. (2016)

This video demonstrates stereotaxic viral injection for precise neuronal gene editing in the mouse brain. The protocol ensures accurate virus delivery to the dentate gyrus, enabling fluorescent labeling and CRISPR-mediated gene knockout to study neural circuits, gene function, and disease mechanisms in vivo.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Stereotaxic Injection

1. Confirm that the ventilation to the surgery suite is open to ensure proper airflow. Prepare the mouse for surgery by anesthetizing it with 4% isoflurane in an induction chamber. Following anesthesia, shave the head to prepare the injection site.
   1. Place the mouse in the stereotaxic instrument by using tweezers to move the tongue down and to the side. Insert the bite bar into the mouth until the teeth drop into the slot, then secure the ear bars. Ensure that the body of the mouse is on the heating pad and the nose is situated in the nose cone. Direct 4% isoflurane into the nose cone. Confirm anesthesia by pinching the foot with tweezers and ensuring that there is no startle reflex.
   2. Apply artificial tears to lubricate the eyes. Finish preparing the injection site by swabbing the shaved head with alternating rounds of povidone-iodine and lidocaine.
2. Using a scalpel, cut a small incision along the center of the scalp. Dry the skull with a swab and hydrogen peroxide if necessary to help visualize bregma.
   1. Using a dissecting scope, locate the bregma on the skull and place the drill tip on the bregma. Zero (or record) the digital stereotactic coordinates on the x, y, and z planes.
   2. In order to ensure that the head is level on the rostral to caudal y-axis, place the drill bit on lambda and level the head so that the z-coordinate is roughly equal at both bregma and lambda.
   3. In order to ensure that the head is level on the x-axis, place the drill bit at y = 1/2 lambda. Ensure that the z-coordinate is equal to 1 mm on each side (x = +/-1 mm) of the sagittal suture. Adjust the skull if there is a difference in the z-coordinate at 1mm to the left and right of lambda.
3. Place the drill over the desired coordinates. For the dentate gyrus, use the coordinates y = -1.9 from bregma and x = +/- 1.1. Before drilling, lower the isoflurane to 2%, then slowly and carefully drill through the skull.
   1. After drilling all the holes, affix the filled-syringe onto the stereotaxic instrument. Visually center the syringe over the hole and zero the z coordinate at the skull.
   2. Slowly lower the syringe to the deepest z-depth. For the dentate gyrus, the z depths are -2.5, -2.4, and -2.3. Begin injection at a rate of 0.25 µl/min using a stereotaxic injector.
      1. After the injection is complete at the lowest Z-depth, wait 1 min, then raise to the next coordinate and begin injection again. Continue this pattern until all z-injection coordinates are injected. Wait 2 min before removing the syringe after the last injection.
         NOTE: No adverse effects have been noted on the tissue by injecting up to 2 µl of virus per hemisphere in the mouse brain.
   3. Repeat injection for other boreholes.
4. After injection, remove the mouse from the stereotaxic instrument and suture the scalp with 6-0 silk sutures. Apply Lidocaine and an anti-bacterial cream to the wound.
   1. Inject 0.8-1.0 ml saline + Ketoprofen (3-5 mg/kg) via the intraperitoneal (I.P.) route to manage pain. Then, place the mouse into the heated recovery chamber. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return the animal to the company of other animals until it has fully recovered.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Stereotaxic Surgery Reagents
Vet-Syringe vet use T.B. Syringe only 1cc luer slip T.B. 100/bx	Med-Vet International	1CCVLS
Isoflurane
Stainless Steel Scalpel Blades, #10, 100-pk	Med-Vet International	JOR580S
Artificial tear ointment	Med-Vet International	RXPARALUBE-O
PVP PrepSolution	Med-Vet International	HPIV108208H
Normal saline	Med-Vet International	DYND500MLSH
Cotton tipped applicators	Med-Vet International	CTA6
Triple antibiotic ointment	Med-Vet International	RXTRIP-OI15
MONOJECT® Needles Soft Pack 25g x 5/8"	Med-Vet International	25058
6-0 silk sutures	Med-Vet International	MV-711