Nov 02, 2021

Public workspaceVirus Production and Administration

DOI
dx.doi.org/10.17504/protocols.io.bzn6p5he
  • 1California Institute of Technology
  • Mazmanian Lab
Jessica Griffiths
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DOI: dx.doi.org/10.17504/protocols.io.bzn6p5he
Protocol Citation: Sarkis Mazmanian, Bryan Yoo, Jessica Griffiths 2021. Virus Production and Administration. protocols.io https://dx.doi.org/10.17504/protocols.io.bzn6p5he
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 02, 2021
Last Modified: November 02, 2021
Protocol Integer ID: 54718
Funders Acknowledgements:
Emerald Foundation
Grant ID: n/a
National Institutes of Health
Grant ID: GM007616 and DK078938
Department of Defense
Grant ID: PD160030
Center for Microbial and Environmental Interactions (CEMI)
Grant ID: n/a
Aligning Science Across Parkinson’s
Grant ID: ASAP-000375
Aligning Science Across Parkinson's
Grant ID: ASAP-020495
Abstract
Protocol for virus production and administration to mice in Yoo et al 2021
Virus Production
Virus Production
Virus was produced using methods described in Challis et. al. (Challis et al., 2019)
Cells were grown in DMEM + Glutamax + Pyruvate (Gibco, Gaithersburg, MD-Stock# 10569-010) + 5% FBS + non-essential amino acids (Gibco, Gaithersburg, MD-Stock# 11140-050), penicillin-streptomycin (Gibco, Gaithersburg, MD-Stock# 15070-063).
Briefly, human embryonic kidney (HEK293T) cells were triple-transfected with pUCmini-iCAP-AAV-PHP.S, pHelper Plasmid, and one of the following pAAV genomes (CAG-NLS-GFP, hSYN1-tDTomato, GFAP-tDTomato, hSYN1-mRuby2, hSYN1-DiO-mRuby2, hSYN1-mNeonGreen, hSYN1-DiO-mNeonGreen, hSYN1-mTurquoise2, hSYN1-DiO-mTurquoise2, hSYN1-DiO-hM3Dq-mRuby2, CAG-GCaMP6F) in a DPBS + polyethylenimine (PEI)
Virus is precipitated from cells and supernatant with an 8% PEG solution (wt/vol), and purified by ultracentrifugation using 15%, 25%, 40%, 60% stacked iodixanol gradients.
Virus Administration
Virus Administration
Virus was titered to 1012 viral genomes (vgs) and resuspended to a volume of 100µl with sterile PBS.
Mice were anesthetized using 2% isoflurane, and virus was injected retro-orbitally.