10/24/2018

Protocol for Transient Virus Production in 293T Cells and Viral Infections

  1. Precipitate DNA before transfection
  2. 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)
  1. Transfect 293T cells with FuGene and DNAs
  2. Split 293T cells into p150s and grow until 50-80% confluent
  3. Refeed with 20ml media within 1 day before transfection
  4. 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.
  1. Viral Harvest
  2. Harvest 4 times, twice daily (am and pm), beginning 24 hours after transfection
  3. 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
  4. Refeed each plate with 20ml fresh media, slowly adding media along the side of the plate so that cells do not detach
  5. Store virus supernatant at -80C until all harvests are complete
  1. Virus Concentration/Infection of Neonatal/Human Foreskin Keratinocytes
  2. 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
  3. Thaw frozen virus aliquots in 37C water bath
  4. 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)
  5. Spin in SW28 rotor at 16K, 4C, 90 min in ultracentrifuge
  6. After spinning carefully remove tops of tubes and aspirate most of media
  7. Leave 500ul media in tube to assure viral pellet is not disturbed
  8. 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)
  1. Expansion of Infected Cells
  2. 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
  3. If infected HFKS use:
  4. 0.5 ug/ml puromycin (pBABE/puro resistant)
  5. 50 ug/ml G418 (LXSN/neo resistant)
  6. 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.