UCSF

Lentivirus Production

Xinxin Liu, Updated August 2014

Lentivirus 3 Packaging plasmid Mix Preparation

pRSV

10 ug

Mix

Measure concentration

pMDL

10 ug

VSV

10 ug

pLenti‐Plasmid or PLKO.1‐Plasmid

Measure concentration

Transfection and harvest virus in 6 well plate

  1. 293 T packaging cells at 1.3‐1.5 X 105 cell/ml
  2. Incubate cells for 24h, the cells should be ‐70% confluent.
  3. Transfect packaging cells:
    1. Prepare a mixture of the transfection plasmids with OptiMEM 250ul/well: Mix Plasmid 1.8ug/well + PLKO.1‐plasmid 1.8ug/well or Plenti‐plasmid 1.8ug/well;
    2. Dilute Lipofecamine 2000 with OptiMEM 250ul/well;
    3. Mix the two and incubate the transfection mix for 20‐30min at room temperature;
    4. Transfer the transfection mix (500ul) to 293T cells in Seeding media.(2ml per well of 6 well plate)
    5. Incubate cells for 18h
    6. Change media to remove the transfection reagent and replace with Harvest Media (2‐4ml per well of 6 well plate) for viral harvests.
    7. Incubate cells for 24h, retrieve the media containing virus to a storage tube. Continue incubating cells with Harvest media (2‐4ml per well of 6 well plate).
    8. Repeat viral harvesting every 12‐24h and replace with Harvest media (2‐4ml per well of 6 well plate).
    9. Spin the media containing virus at 1250 rpm for 5 min. And pass the supernatant through 45um filter and transfer to a sterile storage tube.

6‐well Plate

Packaging Mix Plasmids 1.8ug

Mix 20‐30min

10ul Lipofectamine 2000
PLKO.1‐Plasmid or Plenti‐Plasmid 2ug 250ul OptiMEM Media
OptiMEM Media 250 ul    
Incubate 5min Incubate 5min
  • Seeding media: DMEM + 10%FBS without Pen/Strep (ordinary media also applicable)
  • Harvest media: DMEMD + 30% FBS + 1X Pen/Strep