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protocolPageIcons_protocols_TJF_2016_6_10-01.png Lentivirus Production


Introduction

This protocol can be used to produce lentivirus from a lentiviral vector transfected into Lenti-X 293T cells using a polyethyenimine (PEI) transfection protocol. This procedure can be modified for alternative packaging cell lines or transfection reagents. Once produced, lentivirus can be used for a variety of downstream applications such as stable-cell line generation.

Last Upload: August 26, 2016

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Workflow Timeline

Day 0: Seed 293T packaging cells

Day 1 (pm): Transfect packaging cells

Day 2 (am): 18 hours post transfection. Remove media, replace with fresh media

Day 3-4 (am): Harvest virus

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Equipment

  • Biosafety cabinet
  • Pipetman
  • Pipettors
  • Incubator
  • pH meter
  • Stir plate
  • Magenetic Stir Bar

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Reagents

  • DMEM high glucose
  • L-alanyl-L-glutamine (or alternative stable glutamine)
  • Heat-inactivated FBS
  • Low serum medium such as Opti-MEM or Opti-Pro SFM
  • Chloroquine diphosphate, 25 µM
  • PEI, 1 mg/mL
  • Microcentrifuge tubes
  • 10 cm tissue culture dishes
  • Pipettes
  • Pipette tips
  • Hydrochloric acid
  • Sodium hydroxide
  • 0.22 μm polyethersulfone (PES) filter
  • 0.45 μm PES filter
  • Syringes for filtering

Reagent Preparation

  1. DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine
    • To a 500 mL bottle of DMEM high glucose, add 55 mL of heat inactivated FBS and 11 mL of 200 mM L-alanyl-L-glutamine. Store at 4 ℃.
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    *Pro-Tip* Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one suitable with your protocols. FBS can be purchased already heat inactivated or it can be inactivated in the lab by heating to 56 ℃ for 30 minutes.
  2. 25 mM chloroquine diphosphate
    • Dissolve 0.129 g of chloroquine diphosphate salt in 10 mL of sterile water.
    • Filter sterilize through a 0.22 μm filter.
    • Aliquot 50-100 μL and store at -20 ℃.
    • Aliquots can be thawed and stored at 4 ℃ prior to use. Thawed aliquots should be discarded after 1-2 months.
  3. 1 mg/mL PEI, linear MW 25,000 Da
    • Dissolve 100 mg of powder in 100 mL of deionized water.
    • While stirring, slowly add hydrochloric acid until the solution clears.
    • Check the pH of the solution.
    • Use hydrochloric acid or sodium hydroxide to adjust the pH to 7.0. Typically the solution will be basic and will need adjustment with hydrochloric acid first.
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    *Pro-Tip* The pH of this solution will drift rapidly upon addition of acid or base. Add only a few drops at a time, allow them to mix and recheck the pH to prevent over or undershooting the desired pH.
    • Allow the solution to mix for 10 min and then recheck the pH to ensure that it has not drifted.
    • Filter the solution through a 0.22 μm membrane.
    • Aliquot 500-1000 μL into sterile tubes.
    • Store the tubes at -80 ℃.
      • After thawing, the solution can be stored at 4 ℃ for up to 2 months. After 2 months, discard the tube and thaw a new working stock.
    • The optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI and for each cell line.

Considerations Before You Start

  • The health of the packaging cell line is critical for obtaining high viral titer.
  • 293T cells should be split 3 times a week:
    • Monday: Plate 1×106 cells in a T75 flask in 15 mL DMEM complete.
    • Wednesday: Plate 1×106 cells in a T75 flask in 15 mL DMEM complete.
    • Friday: Plate 8×105 cells in a T75 flask in 15 mL DMEM complete.
  • Do not add pen-strep to the media.
  • Use cells that are below passage 15 for viral production.

Procedure

  1. Seed 293T packaging cells at 3.8×106 cells per plate in DMEM complete in 10 cm tissue culture plates.
  2. Incubate the cells at 37 ℃, 5% CO2 for ~20 hours.
  3. Gently aspirate media, add 10 mL fresh DMEM complete containing 25 μM cloroquine diphosphate and incubate ~5 hours.
    • For 10 mL of DMEM complete, add 10 μL of 25 mM chloroquine diphosphate.
  4. Prepare a mixture of the 3 transfection plasmids:

    Reagent Amount per 10 cm dish*
    psPAX2 1.3 pmol
    pMD2.G 0.72 pmol
    Transfer Plasmid* 1.64 pmol
    OptiPro SFM to total volume 500 μL
    *Plasmid concentrations and ratios should be optimized for each transfer plasmid.
  5. Exclamation.jpg
    *Pro-Tip* Endotoxins can inhibit transfection, therefore, plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, the plasimd should also be propagated in an endonuclease negative E. coli strain such as NEB stable.
  6. Dilute the above 500 μL mixture into 500 μL PEI-OptiPro SFM with enough PEI such that the ratio of μg DNA:μg PEI is 1:3 (1000 μL total per 10 cm dish).

    • Using transfer plasmid pHAGE TRE dCas9-KRAB (total ug of plasmid DNA 27.8 μg), this would be 83.4 μL of 1 mg/mL PEI in 416.6 μL of OptiPro SFM per 10 cm dish.
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    *Pro-Tip* There can be batch to batch variation when making the PEI working stock, therefore the ratio of μg DNA:μg PEI needs to be empirically determined. Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after transfection to determine what ratio gives the highest percentage of GFP positive cells.
    • Refer to the table below for a possible range of ratios to test:

    Ratio of DNA:PEI μg of DNA μL of 1 mg/mL PEI
    1:1 18.9 18.9
    1:2 18.9 37.8
    1:3 18.9 56.7
    1:4 18.9 56.7
    1:5 18.9 94.5
    1:6 18.9 113.4
  7. Gently add the diluted PEI to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture 15-20 min at room temperature.
  8. Carefully transfer the transfection mix to the Lenti-X 293T packaging cells. Add the transfection mix dropwise being careful not to dislodge the cells.
  9. Incubate the cells for 18 hours, or until the following morning.
  10. The following morning, carefully aspirate the media. Replace the media with 15 mL of DMEM complete.
  11. Incubate the cells.
  12. Virus can be harvested at 48, 72, and 96 hours post transfection in individual harvests or a combined harvest where all the individual harvests are pooled. If pooling harvests, transfer the harvested media to a polypropylene storage tube and store at 4 ℃ between harvest.
  13. Centrifuge the viral supernatant at ~500 g for 5 minutes to pellet any packaging cells that were collected during harvesting.
  14. Filter supernatant through a 0.45 μm PES filter.
  15. The viral supernatant can be stored at 4 ℃ for several hours but should be aliquotted and snap frozen in liquid nitrogen and stored at -80 ℃ as soon as possible to avoid loss of titer.

Sample Data

DNA-PEI Ratio Test

Figure 1: Lenti-X 293T cells were transfected with the GFP-expression plasmid pRosetta using μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided high transfection efficiencies as measured by the highest proportion of GFP positive cells without limiting cell growth. Left panels: bright field images; right panels: GFP channel images.