Oct 07, 2022
Viral-mediated short-hairpin RNA knockdown
- 1Northwestern University
Protocol Citation: Chuyu Chen, Loukia Parisiadou 2022. Viral-mediated short-hairpin RNA knockdown. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7y819gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 07, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71031
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research (MJFF)
Grant ID: ASAP-020600
Abstract
Stereotaxic injection of viral vectors for gene knockdown experiments : The AAV vectors for knocking down the gene of interest were custom-made with Vector Biolabs using AAV-GFP-U6 vector for AAV1 packaging (Cat no, 7040, Vector Biolabs). They were then unilaterally injected into the striatum of mice as follows.
Just before the procedure
Just before the procedure
The following preparatory steps are followed:
Weigh the animals
Calibrate the David Kopf frame based on the manufacturer’s instructions.
Clean the Hamilton Syringe with 70% ethanol and distilled water.
Shave the fur off the top of the animal’s skull.
Viral injections
Viral injections
Anesthetize the animal with 3-4% Isoflurane. Monitor closely the animal until it reaches anesthesia (typically 5–10 min) and does not respond to a light pinch to the hind paw or tail.
Place the animal in the stereotaxic instrument by carefully placing one ear bar in the ear canal, securing the bar, and holding the animal in place as the other ear bar is placed and secured.
Secure the mouth in the incisor adapter of the stereotaxic instrument. It is critical to ensure that the airway is not blocked.
Cover the anesthetized animal’s eyes with sterile ocular lubricant to keep them moist during the surgery.
Clean the skin’s surface with alcohol and Iodine prep wipes.
Make a midline incision to the skin’s surface’s skull with small surgical scissors or a scalpel.
Clean the skull with cotton swabs.
Identify the coordinates relative to bregma:
- four sites in the striatum
- anterior-posterior 0.8 mm,
- mediolateral 2.4 mm
- dorsoventral –2.8 and −3.6 mm
- anterior-posterior 0.2 mm
- mediolateral 1.8 mm,
- dorsoventral –2.8 and −3.6 mm relative to bregma
Load the viral solution with Hamilton
Find bregma with the syringe needle and zero everything in the stereotactic frame
Determine your coordinates. Deliver virus by 100 nl/min speed (500-700 nl of AAV1-GFP-U6-shPKARIIβ or AAV1-GFP-U6-sh control viruses (5x10^12 genome copies/ml)
After injections, wait 10 minutes to allow the virus to diffuse
Remove the syringe very carefully and close the surgical sutures
Make a subcutaneous injection of Meloxicam right after the surgery and up to 48 hours.
Move the animal to the cage on the heat pad and monitor carefully its behavior.
Perform behavioral and biochemical experiments 4 weeks post operation.