Jul 11, 2024
Adeno-associated virus (AAV) production and administration
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Adeno-associated virus (AAV) production and administration. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l623b5gqe/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: July 11, 2024
Last Modified: July 11, 2024
Protocol Integer ID: 103196
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Adeno-associated virus (AAV) production and administration
**Transfection of HEK293T Cells**
- Transfect HEK293T cells with the following plasmids: pAd-DELTA F6, serotype plasmid AAV PHP.eB, and AAV plasmid (pZac2.1-GfaABC1D-Ezrin-BioID2-HA or pZac2.1-GfaABC1D-Ezrin T567A-BioID2-HA).
**Cell Lysis and AAV Purification**
- Three days after transfection, collect cells in a lysis buffer (15 mM NaCl, 5 mM Tris-HCl, pH 8.5).
- Perform repeat freeze-thaw cycles followed by treatment with Benzonase at 37°C for 30 minutes.
- Centrifuge lysed cells to pellet debris, and collect the supernatant containing AAVs.
**Optiprep Density Gradient Centrifugation**
- Apply the supernatant containing AAVs to an Optiprep density gradient (15%, 25%, 40%, and 60% iodixanol).
- Centrifuge at 67,000 rpm using a Beckman Ti-70 rotor for 1 hour.
- Collect the AAV-enriched fraction from between the 40% and 60% iodixanol layers.
**AAV Concentration**
- Concentrate the AAV fraction by repeated washes with sterile PBS using an Amicon Ultra-15 filtration unit (100 kDa NMWL) to a final volume of approximately 100 μl.
- Aliquot the concentrated AAVs for storage at −80°C.
**Mouse Anesthesia and Surgery**
- Anesthetize 9-week-old WT or LRRK2 G2019Ski/ki mice using 1.5% isoflurane gas in a stereotaxic frame.
**Intravenous AAV Injection**
- Inject 10 μl of purified AAVs (titer of ~1 x 10^12 GC/ml) into the mouse brain intravenously by injection into the retro-orbital sinus.
**Post-Injection Care and Tissue Preparation**
- Allow mice to recover for 3 weeks until they reach 12 weeks of age.
- Anesthetize mice with 200 mg/kg Tribromoethanol (Avertin) and perform transcardial perfusion with TBS/Heparin followed by 4% paraformaldehyde (PFA) at room temperature (RT).
**Brain Processing**
- Post-fix harvested brains overnight in 4% PFA, then cryoprotect in 30% sucrose.
- Embed brain blocks in O.C.T. (TissueTek) and store at −80°C.
**Cryosectioning**
- Cut 30 µm thick brain sections using a Leica CM3050S vibratome.
- Store sections in a mixture of TBS and glycerol at −20°C for subsequent antibody staining procedures.
Notes:
- Ensure all procedures are performed in accordance with institutional guidelines for animal care and use.
- Maintain sterile conditions during AAV preparation and injection procedures to prevent contamination.
- Document all experimental details including AAV titers, injection volumes, and storage conditions for reproducibility.