Lentivirus production for primary neuron transduction
Itika Saha, F. Ulrich Hartl, Mark S. Hipp, Miguel Da Silva Padilha, Irina Dudanova
Abstract
This protocol describes the production of lentiviruses to transduce mouse primary neurons and has to be performed in a biosafety level 2 laboratory
Steps
Expand HEK293T cells (LentiX 293T cell line, Takara) for lentiviral packaging to 70-85% confluency in DMEM Glutamax (+4.5 g/L D-glucose, - pyruvate) supplemented with 10% Fetal Bovine Serum (FBS)(Sigma), 1% G418 (Gibco), 1% Non-Essential Amino Acids (Thermo Fisher) and 1% HEPES (Biomol).
For lentiviral production, plate cells (~ 4.8 x 106 ) in a three-layered 525 cm2 flask (Falcon).
On the following day, transfect cells with 59.52 μg expression plasmid, 35.2 μg packaging plasmid psPAX2 (RRID:Addgene_12260) and 20.48 μg envelope plasmid pVsVg (gift from Dieter Edbauer) using 345.6 μl TransIT-Lenti transfection reagent (Mirus) in 9.6 mL DMEM without FBS.
Incubate transfection mix for 20 min at room temperature and exchange the cell medium in the meantime.
Add 10 mL of transfection mix to the flask, followed by incubation overnight.
Exchange the medium on the following day.
After 48-52 h, collect culture medium containing the viral particles and centrifuge for 10 min at 1,200 x g.
Filter the supernatant through 0.45 μm pore size filters using 50 mL syringes and add 20 mL Lenti-X concentrator (Takara) to filtered supernatant.
Incubate overnight at 4 °C and centrifuge samples at 1,500 x g for 45 min at 4 °C.
Remove the supernatant and resuspend the lentivirus pellet in 150 μL TBS-5 buffer (50 mM Tris-HCl pH 7.8, 130 mM NaCl, 10 mM KCl, 5 mM MgCl2).
After aliquoting, store lentivirus at -80 °C.
Thaw virus preparation immediately before adding to freshly prepared neuronal culture medium.
Remove a fifth of the medium from cultured neurons and add the equivalent volume of virus-containing medium.
