Jul 10, 2024
Adeno-associated virus (AAV) production and administration
- 1Duke University
Document Citation: Shiyi Wang 2024. Adeno-associated virus (AAV) production and administration. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jyynl1b/v1
License: This is an open access document 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
Created: May 23, 2023
Last Modified: July 10, 2024
Document Integer ID: 82329
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
1. HEK293T cells were transfected with 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).
2. Three days after transfection, cells were collected in 15 mM NaCl, 5 mM Tris-HCl, pH 8.5, and lysed with repeat freeze-thaw cycles followed by treatment with Benzonase (Novagen 70664) at 37°C for 30 minutes.
3. Lysed cells were pelleted by centrifugation, and the supernatant, containing AAVs, was applied to an Optiprep density gradient (Sigma D1556, 15%, 25%, 40%, and 60%) and centrifuged at 67,000 rpm using a Beckman Ti-70 rotor for 1 hour.
4. The AAV-enriched fraction was isolated from between 40% and 60% iodixanol solution and concentrated by repeated washes with sterile PBS in an Amicon Ultra-15 filtration unit (NMWL: 100 kDa, Millipore UFC910008) to a final volume of ∼100 μl and aliquoted for storage at −80°C.
5. 9-week-old WT or LRRK2 G2019Ski/kimice placed in a stereotaxic frame were anesthetized through inhalation of 1.5% isofluorane gas.
6. 10 μl of purified AAVs having a titer of ∼1 × 1012GC/ml was introduced into the mouse brain intravenously by injection into the retro-orbital sinus.
7. After 3 weeks at 12-week-old, mice were anesthetized with 200 mg/kg Tribromoethanol (Avertin) and transcardially perfused with TBS/Heparin and 4% paraformaldehyde (PFA) at room temperature (RT).
8. Harvested brains were post-fixed overnight in 4% PFA, cryoprotected in 30% sucrose, and the brain blocks were prepared with O.C.T. (TissueTek) to store at −80°C.
9. 30 µm thick brain sections were obtained through cryosectioning using a Leica CM3050S (Leica, Germany) vibratome and stored in a mixture of TBS and glycerol at −20°C for further free-float antibody staining procedures.