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慢病毒生产方案

发布时间:2019-12-27浏览:165次

Lentivirus Production

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).
  11. Equipment

  12. Tissue Culture Incubator
  13. Centrifuge

    Procedure

  14. 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.
  15. Incubate cells for 24 h (37 °C, 5% CO2), or until the following afternoon. After ~24 h, the cells should be ~70% confluent.
  16. Transfect packaging cells:
    1. Prepare a mixture of the 3 transfection plasmids:
      900 ng Packaging plasmid
      100 ng Envolop plasmid
    2. 1 μg pLKO.1 plasmid
      10-30 μl OptiMEM media
    3. 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.
    4. Add the 3 plasmid mix dropwise to the diluted Lipofectamine reagent and mix by swirling the tip or gently flicking the tube.
    5. Incubate the transfection mix for 20 - 30 min at room temperature.
    6. 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.
    7. Incubate cells for 18 h (37 °C, 5% CO2), or until the following morning.
    8. Change media to remove the transfection reagent and replace with 6 ml Harvest media for viral harvests.
    9. Incubate cells for 24 h (37 °C, 5% CO2).
    10. Harvest media containing lentivirus at ~40 hours post-transfection. Transfer media to a storage tube. Replace with 6 ml Harvest media.
    11. 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.
    12. 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.
    13. 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

    14. Seeding media: DMEM + 10% FBS without Pen/Strep
    15. Harvest media: DMEMD + 30% FBS + 1x Pen/Strep
    16. Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.

      How to cite: Aboulaich, N. (2011). Lentivirus Production . Bio-101: e39. DOI: 10.21769/BioProtoc.39.

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