TITLE: Lentivirus Production
AUTHORS: Addgene the Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for Lentivirus production. To see the full abstract and additional resources, visit the </span><a href="https://www.addgene.org/protocols/lentivirus-production/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Sample Data</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bky36.png" /></div></div>

[STEPS]
?. [Seeding cells]
Seed 293T packaging cells at 3.8×106 cells per plate in DMEM complete in 10 cm tissue culture plates.
?. [Seeding cells]
Incubate the cells at , 5% CO2 for ~.
37 °C
?. [Transfection]
Gently aspirate media, add  fresh DMEM complete containing  cloroquine diphosphate and incubate ~.
10 ml
For   of DMEM complete, add   of   chloroquine diphosphate.
?. [Transfection]
Prepare a mixture of the 3 transfection plasmids:  AB1ReagentAmount per 10 cm dish*2psPAX21.3 pmol3pMD2.G0.72 pmol4Transfer Plasmid*1.64 pmol5OptiPro SFM to total volume500 μL
AB1ReagentAmount per 10 cm dish*2psPAX21.3 pmol3pMD2.G0.72 pmol4Transfer Plasmid*1.64 pmol5OptiPro SFM to total volume500 μL
*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 negativeE. colistrain such as NEB stable.
?. [Transfection]
Dilute the above  mixture into  PEI-OptiPro SFM with enough PEI such that the ratio of μg DNA:μg PEI is 1:3 ( total per 10 cm dish).Using transfer plasmid pHAGE TRE dCas9-KRAB (total ug of plasmid DNA - 27.8 μg), this would be  of 1 mg/mL PEI in  of OptiPro SFM per 10 cm dish.
500 µl
500 µl
1000 µl
83.4 µl
416.6 µl
*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.
?. [Transfection]
ABC1Ratio of DNA:PEIAmount of DNA (μg)Volume of 1 mg/mL PEI (μL)21:118.918.931:218.937.841:318.956.751:418.975.661:518.994.571:618.9113.4
ABC1Ratio of DNA:PEIAmount of DNA (μg)Volume of 1 mg/mL PEI (μL)21:118.918.931:218.937.841:318.956.751:418.975.661:518.994.571:618.9113.4
?. [Transfection]
Gently add the diluted PEI to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture for 15-30 min at room temperature.
?. [Transfection]
Gently add the diluted PEI to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube.
?. [Transfection]
Incubate the mixture  -  at .
0 Room temperature
?. [Transfection]
Carefully transfer the transfection mix to the Lenti-X 293T packaging cells.
Add the transfection mix dropwise being careful not to dislodge the cells.
?. [Transfection]
Incubate the cells for , or until the following morning.
?. [Transfection]
The following morning, carefully aspirate the media. Replace the media with  of DMEM complete.
15 ml
?. [Transfection]
Incubate the cells.
?. [Harvest Virus]
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   between harvest.
?. [Harvest Virus]
Centrifuge the viral supernatant at ~ for  to pellet any packaging cells that were collected during harvesting.
?. [Harvest Virus]
Filter supernatant through a 0.45 μm PES filter.
?. [Harvest Virus]
The viral supernatant can be stored at  for several hours but should be aliquotted and snap frozen in liquid nitrogen and stored at  as soon as possible to avoid loss of titer.
4 °C
-80 °C