Virus Production and Administration

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 was titered to 10¹²viral genomes (vgs) and resuspended to a volume of 100µl with sterile PBS.

Mice were anesthetized using 2% isoflurane, and virus was injected retro-orbitally.