2/12/2016
Protocol for Transient Virus Production in 293T Cells and Viral Infections
1. Precipitate DNA before transfection
 Aliquot DNAs, 32 ug/tube/each p150 plate of 293T cells:
 5 ug LTR/VSV-G (envelope protein, drive by HIV LTR)
 2 ug CMV/tat (HIV transactiv. protein, drives HIV LTR)
 10 ug pJK3 (encodes MoMLV gag and pol proteins)
 15 ug retroviral construct (i.e. LXSN, LXSN/APA-1)
 32 ug total
 Add 1/10 vol 10M AmmOAc (or 3 M NaOAc) and 2.5 vol 100% etoh
 Vortex briefly – should see fluffy white pellet
 Spin down at 14K, 15 min, 4C
 Remove supernatant carefully
 Wash once with 100ul 70% etoh to remove salts
 Spin down at 14K, 5 min, 4C
 Remove supernatant
 Speedvac dry DNA pellet, 5 min, medium heat
 Resuspend pellets in 50ul dH20
 Let sit for a few hours (assures DNA fully resuspended)
2. Transfect 293T cells with FuGene and DNAs
 Split 293T cells into p150s and grow until 50-80% confluent
 Refeed with 20ml media within 1 day before transfection
 For each plate to be transfected, prepare FuGene mix (1 DNA: 4 FuGene):
 Add 128ul FuGene to 672 ul serum free DME media. Flick to mix.
 Incubate for 5 min at RT.
 Add 800ul FuGene mix to resuspended DNA. Flick to mix.
 Incubate for 15 min at RT.
 Add 850ul mix to plate of 293T cells in a dropwise fashion. Swirl to mix.
3. Viral Harvest
 Harvest 4 times, twice daily (am and pm), beginning 24 hours after
transfection
 For each harvest, carefully remove supernatant and pool like-plates in 50ml
conical tubes, using 30ml syringes and 0.45uM syringe filters (yellow) to
filter supernatant
 Refeed each plate with 20ml fresh media, slowly adding media along the side
of the plate so that cells do not detach
 Store virus supernatant at -80C until all harvests are complete
2/12/2016
4. Virus Concentration/Infection of Neonatal/Human Foreskin Keratinocytes
 One day prior to infection, split NFKs into p100 plates. For each p150 of
293T virus (80ml total supernatant), plan on infecting one p100 plate
 Thaw frozen virus aliquots in 37C water bath
 Add exactly 38ml of viral supernatant to each sterile tube (80ml supernatant =
2 sterile tubes) and load into cleaned SW28 rotor. Assure each tube is balance
(using media to top off if needed)
 Spin in SW28 rotor at 16K, 4C, 90 min in ultracentrifuge
 After spinning carefully remove tops of tubes and aspirate most of media
 Leave 500ul media in tube to assure viral pellet is not disturbed
 Prepare a master mix of media/polybrene for infection cocktail. Per 2 sterile
tubes, mix:
 4ml media
 5ul 10 mg/ml polybrene (helps virus stick to cells) (1:1000 dilution)
 Add 2ml of master mix to each tube/viral pellet and let rest x2 min at RT
 (Aspirate media off of keratinocyte plates while resting)
 Mix master mix and pellet together by pipetting, and combine like-virus
together (4ml media + 5ul polybrene + (500ul x2) viral pellets = 5 ml total
volume)
 Add 5ml infection cocktail to each p100 plate
 Incubate for 2-3 hours at 37C
 Aspirate infection cocktail off plate
 Refeed plates with 10ml media with pen/strep added (100X stock)
5. Expansion of Infected Cells
 When cells become confluent (often 24 hours after infection), expand each
p100 plate to p150 plate, and 48 hours after infection start selection in media
 If infected HFKS use:
 0.5 ug/ml puromycin (pBABE/puro resistant)
 50 ug/ml G418 (LXSN/neo resistant)
 12 ug/ml hygromycin (LXSH/hyrgo resistant)
 If infected HeLas, HFFs, or other cells in DME+Ab media use:
 1.5 ug/ml puromycin
 1 mg/ml G418
 200ug/ml hygromycin
 Refeed all plates every 3 days and keep under selection
**LXSN cells usually come through selection quickly and may need to be split around
day 6-7. APA-1 infected cells come out very slowly and are usually ready to harvest
around day 10-11. Try to have LXSN cells at near same confluency when APA-1 cells
are ready to harvest, harvest on same day.