This video demonstrates intraspinal microinjection of recombinant adeno-associated virus into the dorsal horn of the mouse spinal cord to achieve localized neuronal transduction, enabling targeted gene expression for studying sensory circuit function and behavior.
מאמר שיטה
Intraspinal Delivery of Recombinant Adeno-Associated Virus for Targeted Neuronal Transduction
30 צפיות
⸱
29 באוקטובר 2026
במאמר זה
תקציר
פרוטוקול
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
- Intraspinal Injections
- Move the tip of the micropipette above one of the holes in the dura and then down until a slight dent is noted in the dura. To target the injection to the spinal dorsal horn, move down 500 µm in 100 µm consecutive increments (speed 1 mm/s) and then 200 µm up to allow for mechanical stabilization of the tissue.
NOTE: The final depth of the tip is 300 µm in this case, but can be adapted depending on the target area.- If the tissue is resistant to penetration, the micropipette might be catching on the dura. In this case, retract and move slightly to overlie the hole, or use the needle again to perforate the dura properly before repeating the penetration attempt.
- Program the pump to a target injection volume of 300 nL at an injection speed of 50 nL/min and press the start button to begin the infusion.
- After the injection is completed, check the scale to see whether the virus level has dropped and leave the micropipette in place for an additional 3 min to allow the pressure to equilibrate before slowly retracting the syringe.
- Repeat step 1.1-1.3. for the other two injection sites.
- Move the tip of the micropipette above one of the holes in the dura and then down until a slight dent is noted in the dura. To target the injection to the spinal dorsal horn, move down 500 µm in 100 µm consecutive increments (speed 1 mm/s) and then 200 µm up to allow for mechanical stabilization of the tissue.
- Suturing and Recovery
- Remove the spinal clamps and the cushion below the mouse.
- Close the wound in layers with interrupted stitches, suturing the superficial tissue layers with absorbable sutures and the skin with non-absorbable sutures. Apply iodine disinfectant to the sutured wound.
- Terminate the anesthesia and leave the animal on the heat mat until it recovers before returning it to its home cage.
- Postoperative Care
- Monitor the health of the animal on the day of surgery, the next day, and then every 2-3 days. Ensure that the animal has a normal gait, a healthy appearance (weight, eyes, fur, and behavior), and that the wound is healing.
- Continue analgesic treatment (e.g., 0.1-0.2 mg/kg buprenorphine subcutaneously) as necessary, three times per day.
חומרים
| שם | חברה | מספר קטלוג | הערות |
|---|---|---|---|
| Equipment | |||
| Anesthesia unit: Oxymat3 oxygen concentrator | Weinmann | NA | |
| Anesthesia unit: VIP 3000 Veterinary Vaporizer | Midmark | NA | |
| Heat mat: Mio Star Thermocare 100 | Migros | 717614700000 | |
| Surgical microscope (OPMI pico) | Zeiss | NA | |
| Small animal stereotaxic apparatus | Kopf | NA | |
| Hamilton 701 RN 10 μl glass microliter syringe | Hamilton | 7635-01 | |
| Name | Company | Catalog Number | Comments |
| Consumables and Chemicals | |||
| Thin-wall glass capillary, 1mm outside diameter | World Precision Instruments | TW 100-3 | |
| Syringes (1, 5 and 20 ml) | B. Braun | (9166917V, 4606051V, 4606205V) | |
| Surgical sutures Safil Quick+ 4/0, absorbable | B. Braun | C1046220 | |
| Surgical sutures Premilene 5/0, non-absorbable | B. Braun | C0932191 | |
| Sterile PBS or saline (0.9%) | NA | ||
| Ethanol, 70% (disinfectant) | NA | ||
| Iodine solution (e.g. Braunol) | B. Braun | 18380 | |
| Anaesthetics (e.g. Attane isoflurane) | Provet | 2222 | |
| Analgesics (e.g. buprenorphine: temgesic) | Indivior | GTIN: 7680419310018 | |
| Ophthalmic ointment (e.g. vita-pos) | Pharma medica | GTIN: 4031626710635 | |
| Cotton swabs (e.g. from) | IVF Hartmann | 1628100 | |
| Facial tissues (e.g. from) | Uehlinger AG | 2015.10018 | |
| Superfrost plus microscope slides | ThermoScientific | J1800AMNZ | |
| Name | Company | Catalog Number | Comments |
| Mice | |||
| C57BL/6J mice (wildtype) | The Jackson Laboratory | RRID:IMSR_JAX:000664 | |
| Rorbtm1.1(cre)Hze/J mice (RORβCre) | The Jackson Laboratory | RRID:IMSR_JAX:023526 | |
| Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mice (R26Tom) | The Jackson Laboratory | RRID: IMSR_JAX:007914 | |
| Name | Company | Catalog Number | Comments |
| Viral vectors | |||
| AAV1.CB7.CI.eGFP.WPRE.rBG (AAV1.CAG.eGFP) | Penn Vector Core | AV-1-PV1963 | |
| AAV1.CAG.flex.eGFP.WPRE.bGH (AAV1.CAG.flex.eGFP) | Penn Vector Core | AV-1-ALL854 | |
| AAV1.CAG.flex.tdTomato.WPRE.bGH (AAV1.CAG.flex.tdTomato) | Penn Vector Core | AV-1-ALL864 | |
| AAV1.EF1a.flex.DTA.hGH (AAV1.EF1a.flex.DTA) | Penn Vector Core | Custom production | |
| AAV1.hSyn.DIO.hM3D(Gq)-mCherry.hGH (AAV.flex.hM3D(Gi)) | Penn Vector Core | Custom production |
