Lentivirus concentration solution

Mouse wild-type Sod1, Sod1^C58S, and Sod1^C147S (kindly provided by Dr. X.F.Steven Zheng) plasmids were cloned into the tet-inducible lentiviral vector pINDUCER20 (Addgene, Massachusetts, USA, 44012). For virus production, the Sod1 plasmids were co-transfected with psPAX2 (Addgene, 12260), and pMD2.G (Addgene, 12259) into HEK293T cells using FuGENE® 6 Transfection Reagent (Promega, Wisconsin, USA, E2691). Viral supernatants were collected 24 and 48 h after transfection by passage through a 0.45 mm filter, and concentrated with lentivirus concentration solution (OriGene Technologies, Maryland, USA, TR30025) according to the manufacturer’s instructions.
Organoids were dissociated into single cells and transfected with concentrated lentiviruses as described previously [61 ]. Briefly, concentrated viral supernatants were added to cells in 48-well plates, incubated at 37 °C for 3 h, collected, and reseeded in Matrigel-containing medium. Infected organoids were selected with G418 (Thermo Scientific, 10131035) at 72 h after viral transduction for 2 weeks.
To induce exogenous vector-, wild-type Sod1, Sod1^C58S, and Sod1^C147S expression in Sod1-deficient organoids, 1 μg ml⁻¹ doxycycline (Sigma-Aldrich, D9891) was added to the culture media on day 0 and supplemented with media changes at 2-day intervals.

To produce lentivirus, pLenti ^emGFP-Slc37a2, and pHIV-PV-VSVG (kind gift from Dr. Scott A. Fisher, The University of Western Australia) packaging plasmid were co-transfected into HEK293FT cells (R70007, Thermo Fisher) using TransIT-Lenti transfection reagent (Mirus) according to manufacturer’s instructions. 18 h post-transfection, media containing the transfection reagent was removed and replaced with fresh, high-BSA DMEM (Gibco) containing 10% FBS (Gibco) and 0.1 g/L bovine serum albumin (Sigma-Aldrich). Culture media containing lentivirus were collected 48–72 h post-transfection and concentrated using a lentivirus concentration solution (Origene) according to the manufacturer’s instructions. The lentiviral pellet obtained post concentration was resuspended in cold, sterile PBS, aliquoted, and stored at −80 °C until required.
Prior to transduction, lentiviral particles were titrated using the qPCR lentivirus titration kit (Applied Biological Materials Inc.) according to the manufacturer’s instructions. BMMs were transduced with lentiviral particles in the presence of 20 µg/ml protamine sulfate (Sigma-Aldrich) 24 h after the first RANKL (10 ng/ml, R&D Systems) stimulation of BMMs.