Protocol for Transient Virus Production in 293T Cells
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
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.
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