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Recombinant Lentivirus Production Protocol


Lentivirus is classified as retroviridae. The pre-integration complex of the lentivirus nuclear protein has the characteristics of phagocytosis...

Recombinant Lentivirus Production Protocol

Lentivirus is classified as retroviridae. The pre-integration complex of the lentivirus nuclear protein has the characteristics of phagocytosis, and the viral genome translocates to the nucleus, so that it can infect and replicate in the non-mitotic cells. This feature makes it an ideal tool to introduce a gene into mammalian or other animal cells.

Transient transfection method is now widely used for generation of recombinant lentivirus. Envelope plasmid, packaging plasmid and recombinant lentivirus plasmid cotransfect 293T cells to get producing cells.

The experimental process of recombinant lentivirus production is as follows.

  1. Obtain the target genes by PCR.
  2. Choose the corresponding lentivirus vector plasmid according to your requests.
  3. Construct recombinant lentivirus plasmid.
  4. Identify the insert gene sequence, and then extract recombinant lentivirus plasmids.
  5. Seed 293T packaging cells at 1.3-1.5 X 105 cells/ml (6 ml per plate) in Seeding media (DMEM + 10% FBS without Penicillin / Streptomycin) in culture plates. Incubate cells for 24 h (37 °C, 5% CO2), or until the following afternoon. After ~24 h, the cells should be ~70% confluent.
  6. Transfect packaging cells:
    1. Prepare a mixture of the 3 transfection plasmids:
      • 900 ng Packaging plasmid (expressing Gag and Pol Protein)
      • 100 ng Envolop plasmid (expressing Env Protein)
      • 1 μg recombinant 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.
  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 (DMEMD + 30% FBS + 1x Penicillin / Streptomycin) 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

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.

Notes:

FBS serves as a stabilizer during long term storage of virus. The bovine serum albumin in FBS preserves the virus by preventing ice Krystal formation. This reduces loss of virus infectivity after freeze and thaw cycles. Other stabilizers can be used, such as sucrose.

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