Aug 28, 2024
Intravenous (tail vein) injection of AAV into rat
- 1University of Melbourne
- SPARCTech. support email: info@neuinfo.org
Protocol Citation: Janet R Keast, Nicole Wiedmann, Peregrine B Osborne 2024. Intravenous (tail vein) injection of AAV into rat. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrdbbpgmk/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: November 01, 2021
Last Modified: August 28, 2024
Protocol Integer ID: 54682
Keywords: surgery, tail vein injection, rat, virus, AAV
Funders Acknowledgement:
NIH SPARC
Grant ID: OT2OD023872
Abstract
This protocol is used to administer adeno-associated virus (AAV) in male and female rats. This procedure is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation, including methods of anesthesia, surgical environment, and post-operative monitoring and care.
Guidelines
All containers that are in contact with virus waste need to be properly labeled as biohazard waste, and disinfected appropriately following institutional safety requirements.
Virus stock needs to be placed on ice during surgeries.
For virus transport make sure to follow institutional safety requirements, e.g., double-containers are required for safe transport of biohazards (e.g. Eppendorf tube placed into 50ml Falcon tube).
Materials
IsofluraneZoetisCatalog #10015516 1 ml syringes (or U-100 Insulin Syringe)BD BiosciencesCatalog #329461 0.9% Saline SolutionThermo FisherCatalog #BO0334B 70% Ethanol 10% Bleach AAV-PHP.S:hSyn1-tdTomatoaddgene Sterile gauze pads (1 pad per patient)Contributed by users Biohazard disposal containerContributed by users Virkon™ S 5O tablets/bottleFisher ScientificCatalog #NC9821357
Preparation
Preparation
Determine the dose of virus to administer per rat. Divide the dose (viral genomes (vg)) by the titre (vg/ml) to calculate the volume of virus needed to inject one rat.
Viral dosage can be determined using: 
AAV-viraldosage-calculations.xlsx
AAV-viraldosage-calculations.xlsx In a screw-cap vial, prepare the solution to be administered. Add virus, and dilute with sterile 0.9% saline to a total 150 µl/rat. Briefly vortex each vial for 1 - 2 s before use. Transport the virus on ice once it is ready for injection.
Surgery
Surgery
Anesthetise animal (2.5% isoflurane in oxygen, or as required for maintenance).
While the rat is being induced, load a U-100 insulin syringe with virus. Remove the dead space in the syringe barrel by gently ejecting the virus back into the tube such that air bubbles are expelled. Load the syringe again and repeat the procedure until no bubbles remain in the barrel.
Place animal on heated pad.
Apply 70% Ethanol wipes to tail and turn 45º for a visible presentation of tail vein. If unable to visualise the vein, warm the area.
Introduce a U-100 insulin syringe into the tail vein. Withdraw the barrel slightly to confirm location in the vein. Gently inject the virus.
Apply light pressure over the injection site for approximately 30 s to prevent backflow.
Return rat to housing cage and monitor for any evidence of swelling or complications at the injection site. Continue to monitor over the period of incubation for adverse reactions.
Tissue Harvesting
Tissue Harvesting
Anesthetise and fix animals by intra-cardiac perfusion, then remove tissues of interest for further study.