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[Bio101] Lentivirus Production

Microbiology > Microbial biochemistry > RNA
Author: Nabila Aboulaich
3/5/2011, 14977 views, 5 Q&A

[Abstract] Lentivirus is a common tool used to introduce a gene into mammalian or other animal cells.This protocol is to produce lentivirus stocks from hairpin-pLKO.1 plasmid.

Materials and Reagents

  1. Hairpin-pLKO.1 vector (Open Biosystems).
  2. Packaging vectors: 2nd generation packaging plasmids containing gag, pol, and rev genes (pCMV-dR8.91 or pCMV-dR8.74psPAX2) and envelop plasmid (pMD2.G) (Addgene).
  3.  Lipofectamine 2000 (Invitrogen).
  4. OptiMEM serum free media (Invitrogen).
  5. 293T packaging cells (ATCC)
  6. DMEM (Invitrogen).
  7. Fetal Bovine Serum (Invitrogen).
  8. Pen/Strep (Invitrogen).
  9. 6 cm cell culture plates (Fisher).
  10. 45 μm filter (Fisher).

Equipment

  1. Tissue Culture Incubator
  2. Centrifuge

Procedure

  1. Seed 293T packaging cells at 1.3-1.5 X 105 cells/ml (6 ml per plate) in Seeding media in 6 cm tissue culture plates.
  2. Incubate cells for 24 h (37 °C, 5% CO2), or until the following afternoon. After ~24 h, the cells should be ~70% confluent.
  3. Transfect packaging cells:
    1. Prepare a mixture of the 3 transfection plasmids:
      900 ng Packaging plasmid
      100 ng Envolop plasmid
      1 μg pLKO.1 plasmid
      10-30 μl OptiMEM media
    2. Dilute Lipofectamine 2,000 with OptiMEM: 10 μl Lipofectamine + 90 μl OptiMEM. Add the Lipofectamine reagent dropwise and mix by swirling the tip or gently flicking the tube (do not mix by pipetting or vortexing). Incubate 5 min at room temperature.
    3. Add the 3 plasmid mix dropwise to the diluted Lipofectamine reagent and mix by swirling the tip or gently flicking the tube.
    4. Incubate the transfection mix for 20 - 30 min at room temperature.
    5. Carefully transfer the transfection mix to the packaging cells in Seeding media. The packaging cells can be sensitive to perturbation - take care not to dislodge the cells from the plate. The total volume of transfection mix should be 100 to 125 μl per plate.
  4. Incubate cells for 18 h (37 °C, 5% CO2), or until the following morning.
  5. Change media to remove the transfection reagent and replace with 6 ml Harvest media for viral harvests.
  6. Incubate cells for 24 h (37 °C, 5% CO2).
  7. Harvest media containing lentivirus at ~40 hours post-transfection. Transfer media to a storage tube. Replace with 6 ml Harvest media.
  8. Repeat viral harvesting every 12-24 h and replace with 6 ml Harvest media. Viral titer tends to decrease in later harvests; typically collect a total of 2-3 time points. After the final harvest, discard the packaging cells. The viral harvests may be pooled as desired.
  9. Spin the media containing virus at 1,250 rpm for 5 min to pellet any packaging cells that were collected during harvesting. Pass the supernatant through 45 μm filter and transfer to a sterile storage tube.
  10. Virus may be stored at 4 °C for short periods (hours to days), but should be frozen at -20 °C or -80 °C for long-term storage. To reduce the number of freeze/thaw cycles, aliquot large-scale virus preps to smaller storage tubes prior to long-term storage.

Recipes

  1. Seeding media: DMEM + 10% FBS without Pen/Strep
  2. Harvest media: DMEMD + 30% FBS + 1x Pen/Strep


How to cite this protocol: (2011). Lentivirus Production . Bio-protocol Bio101: e39. http://www.bio-protocol.org/e39



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