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[Bio101] Lentivirus Production

Microbiology > Microbial biochemistry > RNA
Author: Nabila Aboulaich
3/5/2011, 15328 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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Anonymous

7/19/2011 10:15:52 PM  

why should one use the harvest media?

bio-protocol

7/21/2011 2:30:42 PM  

It is recommended to use Harvest media that contains more FBS than Seeding media to provide the 293T packaging cells with additional nutrients while producing the virus particles. Also antibiotics (Pen/Strep) are added to the Harvest media to avoid bacterial infection of packaging cells. It is not recommended to use antibiotics in Seeding media while transfecting the cells using Lipofectamine 2000 as this causes cell death.

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2/27/2012 7:55:19 PM  

how can i increase the titre of the virus.Will the helper plasmids ratio alteration help in boosting virus production.

Nabila

2/29/2012 6:50:24 AM  

This is a link to a paper where they study the factors affecting
lentivirus titer.
http://www.aaronlogan.com/downloads/Logan_infectivity.pdf
Clontech has a kit for lentivirus concentration that would increase the titer by 100 fold. Also ultracentrifugation can be used to concentarte lentivirus.

I hope this answers the question.

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Piers

4/2/2012 5:57:40 PM  

I've been wanting to know how long I can store the virus at 4 deg. I've seen that most people quote days, would a week be ok? Has anyone seen any decreases in titre of their virus when stored for this amount of time at this temperature?

I've only been storing my virus for this long at this temperature becuase I was producing quite a lot of virus and wanted to wait for production to finish before I concentrated the virus and then froze these down. I've heard that each freeze/thaw cycle may decrease titre/infectivity by 20-50% so wanted to avoid any freeze thaws until concentration and subsequent use.

Nabila

4/7/2012 1:59:52 PM  

Lentivirus stock can be stored at 4C for for up to 5 days. No significant effects on the titer have been reported at those conditions.

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1/23/2013 4:01:45 AM  

Can anybody please confirm that using lipofectamine is better that lentiviral transduction using PEI ? Thank u

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Megha

5/11/2013 5:41:45 AM  

Forgot to store the virus at 4degC. Is it stable at room temp for a few hours or do I need to rush to the lab? Any ideas?

Nabila

5/13/2013 2:28:53 PM  

I have not tested that. So I don't have a good answer for that. But I think that few hours storage at RT may result in loss of the virus titer. The only way to find out to is check the titer. Adenovirus however are more stable at RT than lentivirus (up to 72 h).

Hope that is helpful.

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