Reporter design and virus production

Virus Production

Day 0: plate 800,000 293Ts per well of a 6-well plate. Make sure 293Ts are not overgrown (<90% confluent) on the day of plating.

Day 1: transfect cells

• PEI (polyethylenimine) transfections can be set up in medium containing pen/strep.
• Warm up an aliquot of OPTI-MEM.
• Warm up an aliquot of PEI (stored in 4C) to dissolve the precipitate.
• in a sterile eppendorf, combine 0.9 ug of viral backbone vector (miniprep quality is ok) and a mix of helper plasmids. For retrovirus packaging: gag/pol + VSV-G at the mass ratio of 5:1:1 of backbone : gag/pol : VSV-G (= 1 uL of RetroPack ). For lentivirus packaging: CMV-RaII + Tat16 + HgPM2 + VSV-G at the mass ratio of 5:1:1:1:2 of backbone : CMV-RaII : Tat16 : HgPM2 : VSV-G ( = 1 uL of LentiPack ).
• Add 100 ul OPTI-MEM and mix by pipetting.
• Add PEI to eppendorfs, at a 4:1 volume-to-total DNA mass ratio (= 5 uL for retrovirus, 7.2 uL for lentivirus).
• Mix by flicking the tubes a few times. Do not vortex.
• Incubate at room temperature for 10 min.
• Add to cells dropwise.
• Change media 5 hours later.

Day 3: harvest viral supernatant

• For filtering the supernatant, use 3 mL syringes and 0.45 micron filter attachments (virus will not pass through 0.22 micron filter).
• Remove the plunger and place the syringe (with filter attached), on top of a 15 mL conical tube.
• Pipet viral supernatant into the barrel of the syringe.
• Reinsert the plunger and push the supernatant through filter.
• Aliquot virus into cryovials and store in -80C long-term. Virus can be stored for 2-3 days in 4C without losing potency.
• Use 10% bleach to disinfect syringes, pipets and plates before placing them into trash.