Follow approved safety protocol for working with lentivirus

Transcription

1 Production of High Titer Lentivirus Last Updated: 12/19/12 **IMPORTANT** Follow approved safety protocol for working with lentivirus NOTES: This protocol is for the production of high titer lentivirus. The protocol is written here for the production of 1 virus. The centrifuge has 6 rotor buckets, which can each hold approximately 36mL of media. This corresponds to 30, 10-cm plates total in order to fill the centrifuge. If you want to make 2 viruses, use 15 plates (3 rotor buckets) per virus. This protocol will yield virus that is 2 logs higher (maximum) than your unconcentrated virus. Larger transfer plasmids will produce lower titer virus. To know the highest titer possible with your plasmid, titer your unconcentrated supernatant (using the 293T cell based titer method). Anything that touches virus must be decontaminated with bleach or 95% EtOH. After working in the hood with virus, leave everything in the hood and decontaminate with UV light for at least 30 minutes. REAGENTS: 293T Growth Media 0.45 um Cellulose Acetate (CA) 150 ml Filter DMEM (Gibco) + 10% FBS (Heat Inactivated) Corning # NOTE: do NOT use antibiotics in the growth media Ultra-clear Centrifuge Tubes (1x3.5 in.) Beckman # PEI (Polysciences Inc. cat# ) 25 KDa Polyethylimine dissolved in dh2o DMEM/F12 (Hyclone) at a final concentration of 1 ug / ul w/v Packaging Plasmids: Serum Free Optimem pmdlg/prre Gibco #31985 pcmv-vsv-g prsv-rev *plasmid info and maps can be found at

2 PROTOCOL: Day 1 Day 2 Split 293T Cells for Transfection 1 Aspirate media from 6, >90% Confluent 10-cm Plates 2 Wash with 2ml PBS and aspirate 3 Add 1ml Trypsin and incubate at 37C for 1 min. 4 Pipette up and down to create single cell suspension and add to 4 ml Media containing 10% FBS to inactivate trypsin for each plate (i.e. 1:5 split) 5 Add 1 ml of cell suspension to 30 separate, 10cm plates containing 9mL 293T growth media 6 Mix cells and incubate at 37C + 5% CO2 for ~ 1 days Check cell health and transfect when the cells are ~90+% confluent 1 Remove cells from incubator to check condition and confluence Note: Cells should be >90% confluent. Wait until cells are at that density before transfection 2 Add PEI to Optimem for each plate to be transfected as indicated below. 3 Mix and incubate at RT for 5 min. 4 Make DNA mixes as indicated below per 10-cm plate: 5 Add DNA to PEI/Optimem, mix, and incubate at RT for min. 6 During incubation, aspirate and add 8mL fresh 37C 293T media to each plate for transfection 7 Add 1 ml transfection mixture dropwise to each 10 cm plate, mix, incubate o/n at 37C+5% CO2 (only have 1 stack of plates out of the incubator at a time. This keeps cells happier and makes is less likely to spill anything) NOTES: It is ideal for each plasmid to be 1ug/uL. This results in high transfection efficiency. Do not use plasmids lower than 500ng/uL. Also, it is best to mix PEI and optimem first before adding the DNA. This prevents precipitation of the DNA.

3 Number of plates for transfection: 30 PEI/Optimem Mix 1 plate x plates ul 30 Serum Free Optimem PEI 1 ug / ul In the formulas below, you may need to change the values in the highlighted boxes depending on your prep and the concentration of your plasmids. DNA Mix Plasmid [ ug / ul ] ug/plate ul/plate x total plates Transfer Plasmid Plasmid # Packaging Plasmid pmdlg/prre Envelope Plasmid pcmv-vsv-g RNA Export Plasmid prsv-rev Day 3 Day 4 Change Media on Transfected Cells 1 Check cells under microscope for flurescence to verify transfection was efficient NOTE: transfection efficiency should be at least 30%. 2 Aspirate media from transfected cells and disinfect aspirated media in 10% bleach 3 Add 8 ml fresh pre-warmed 293T media to transfected cells 4 incubate o/n at 37C + 5% CO2 Harvest Virus (48hr. Post transfection) NOTE: Everything should be at 4oC during harvest (i.e. always keep tubes/buckets on ice, all centrifuge steps should be at 4oC, ect ) 1 Collect supernatant from transfected plates and place in 50mL conical tubes ON ICE 2 Add 8 ml fresh pre-warmed 293T media to transfected cells and incubate o/n at 37C + 5% CO2

4 NOTES: 3 Centrifuge virus containing supernatant at 3500 RPM for 10 min. at 4C to pellet cell debris 4 Filter supernatant through a sterile 0.45 um PES or cellulose acetate filter. 5 Fill and balance ultracentrifuge tubes and spin at 25K RPM for 1 hr. 45 min. at 4oC Use ultra-clear centrifuge tubes to line the inside of the buckets. Use SW28 rotor in Beckman Optima LE 80K ultracentrifuge Tubes MUST BE nearly full. If left unfilled, they will collapse. 6 Decant supernatant into 10% Bleach *only decant 1 tube at a time to prevent virus being exposed to the air* 7 Resuspend pellet in 80 ul naked DMEM/F12 for infecting PMECs or base media for specific cells/cell lines. 8 Incubate overnight at 4oC. Cover tubes to prevent evaporation. Tip: ultra-clear centrifuge tubes fit perfectly inside a 50mL conical tube. This prevents evaporation and prevents any possible spill. Note: Try to avoid creating bubbles when resuspending virus. Additionally, scrape the visible pellet with your pipette tip while pipetting in order to fully resuspend the pellet It is also critical that the pellet is never left without media. i.e. do NOT dry the pellet Day 5 Harvest Virus (72hr. Post transfection) NOTE: Everything should be at 4oC during harvest (i.e. always keep tubes/buckets on ice, all centrifuge steps should be at 4oC, ect ) 1 Collect supernatant from transfected plates and place in 50mL conical tubes ON ICE 2 Add 2 ml of bleach to empty plates to kill cells and leftover virus 3 Centrifuge virus containing supernatant at 3500 RPM for 10 min. at 4C to pellet cell debris 4 Filter supernatant through a sterile 0.45 um PES or cellulose acetate filter. 5 Fill and balance ultracentrifuge tubes and spin at 25K RPM for 1 hr. 45 min. at 4oC Use ultra-clear centrifuge tubes to line the inside of the buckets. Use SW28 rotor in Beckman Optima LE 80K ultracentrifuge 6 Decant supernatant into 10% Bleach

5 *only decant 1 tube at a time to prevent virus being exposed to the air* 7 Resuspend pellet in 80 ul of suspended virus from Day 4. 8 Incubate 2 hours at 4oC. Cover tubes to prevent evaporation. Tip: ultra-clear centrifuge tubes fit perfectly inside a 50mL conical tube. This prevents evaporation and prevents any possible spill. Note: Try to avoid creating bubbles when resuspending virus. Additionally, scrape the visible pellet with your pipette tip while pipetting in order to fully resuspend the pellet It is also critical that the pellet is never left without media. i.e. do NOT dry the pellet 9 Aliquot virus into centrifuge tubes and freeze at - 80 for future use. Note: The volume of each aliquot will depend on how much virus you need to use for each experiment. After freeze/thaw you can expect to loose ~half of your viral particles. i.e. Small aliquots will be important if you will be infecting cell lines and larger aliquots will work better for primary cells that require high MOI. OPTIONAL To enhance viral titer, you may want to use sodium butyrate (Sigma) during the transfection and subsequent media changes. To do so, add 4mM final concentration sodium butyrate to your media. Filter sterilize before use. Use sodium butyrate- containing media during transfection and in subsequent media changes.