Lenti virus production with HEK293T#

Note

This theme is still under active development

Cell lines#

HEK293T

Reagent#

  • Cell

  • DMEM (high glucose without L-glutamine)

  • Fetal bovine serum

  • TrypLE Express

  • Penicillin/Streptomycin (P/S)

  • Cell cutlure medium

  • PEI, 1 mg/mL

  • 0.22 μm polyethersulfone (PES) filter

  • 0.45 μm PES filter

  • psPAX2 (Addgene, #12260)

  • pMD2.G (Addgene, #12259)

  • pRSV-REV (Addgene, #12253)

  • pMDLg/pRRE (Addgene, #12251)

  • Transfecr plasmid

Culture medium : DMEM +10% FBS + 1%P/S

Reagent Volume
DMEM 450 ml
FBS 50 ml
P/S 5 ml
Total 505 ml

Protocols#

Thawing cells from freezing cells#

  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. Prepare a mixture of the 3 transfection plasmids:

2nd generation lenti virus : 3 plasmid system

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

3rd generation lenti virus : 4 plasmid system

Reagent Amount per 10 cm dish
pMDLg/pRRE 1.3 pmol
pRSV-REV 0.72 pmol
pMD2.G 0.72 pmol
Transfer Plasmid* 1.64 pmol
OptiPro SFM to total volume 500 μL

Important

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.

  1. 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).

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 75.6
1:5 18.9 94.5
1:6 18.9 113.4

  1. Gently add the diluted PEI to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube.

  2. Incubate the mixture 15-20 min at room temperature.

  3. Carefully transfer the transfection mix to the Lenti-X 293T packaging cells. Add the transfection mix dropwise being careful not to dislodge the cells.

  4. Incubate the cells for 18 hours, or until the following morning. The following morning, carefully aspirate the media. Replace the media with 15 mL of DMEM complete or OptiPro SFM.

  5. Incubate the cells.

  6. 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.

  7. Centrifuge the viral supernatant at ~500 x g for 5 minutes to pellet any packaging cells that were collected during harvesting.

  8. Filter supernatant through a 0.45 μm PES filter.

  9. 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.