Lentivirus Production and Transduction Protocol

Virus Production

Note: use lipofectamine 2000 at a 1:2.5 DNA to lipofectamine ratio. The following protocol has been optimized for lipofectamine 2000 transfection in a 10 cm dish.

Materials:

HEK293T cells
DMEM media containing 10% FBS and glutamine (minus pen-strep)
Endotoxin free plasmid DNA: 4:3:1 ratio of plasmids – transfer:PAX2:MD2.G
1. Transfer plasmid (containing GOI or shRNA)
2. psPAX2 (plasmid containing gag, pol and rev genes)
3. pMD2.G (VSV-G expressing envelope plasmid)
Lipofectamine 2000
10 cm tissue culture dishes (or 60 mm dishes and 35 mm dishes/6-well)

1 day before transfection:

Note: plates can be coated with gelatin or lysine to enhance 293T attachment.

Seed 293T cells 18-24 hours before transfection to achieve ³ 90% confluency
a. Split 90-95% confluent cells 1:2-1:3 to achieve ~80-90% confluency
or
b. Seed 7 x10⁶ cells/10 cm dish, 2.2 x10⁶ cells/60 mm dish and 2.2 x10⁶ cells/35 mm dish

Day of transfection:

Follow transfection protocol for the reagent you are using or use the following protocol for Lipofectamine 2000 reagent. The protocol can be scaled down for a 60 mm dish or 35 mm dish.

Label two tubes – (1) lipo/plasmid name and (2) plasmid name only.
Add 500 ul Opti-MEM to both tubes.
Add the following amount of plasmid to the tube labeled with the plasmid name only (total 16 ug) and gently mix (60 mm dish).
1. 8.0 µg transfer plasmid
2. 6.0 µg psPAX2
3. 2.0 µg pMD2.G
Add 40ul lipofectamine (1:2.5 DNA:lipofectamine) to tube labeled lipo and gently mix.
Incubate for 5 minutes at room temperature.
Combine the two tubes and mix gently (add diluted plasmid to diluted lipo). Incubate at room temperature for 20 minutes.
Carefully add the 1ml DNA / Lipo complex to the dish using a P1000.
Note: 293T cells detach very easily from the plate. Take care in adding the complex mix. Tilt the plate and add complex to pooled media dropwise.
Swirl/rock the plate very gently to mix. Incubate the cells at 37º C and 5% C02.
After 6-24 hours, replace media.
Note: Slowly add media to side while tilting the dish. Less media can be added to concentrate the virus. For example, add 8 ml media to 10 cm instead of 10 ml.

Day 2 (48 hours) after transfection:

Collect virus-containing supernatant into 15 ml conical.
Centrifuge at 1000 x g for 10 minutes with centrifuge bucket covers for virus spinning then filter through 0.2 µm cellulose acetate or polysulfonic (low protein binding) syringe filter to remove cellular debris.
Label eight 1.5 ml tubes with virus name and date.
Aliquot 1 ml of viral supernatant into the 1.5 ml tubes.
Freeze aliquots at -80º C.
Discard the 293T cells.
Note: virus can be harvested earlier at 24 hours, but the titer will be reduced by ~3-10 fold.

Functional Virus Titer

Day before

1. Seed cell line into 2 6-well plates at about 60-70% confluency. Label wells: C, 1-10

Day of transduction

Prepare media containing 4 µg/ml polybrene.
Label 1.5 ml tubes with dilution number and virus name.
To tube 1, add 1.5 ml virus stock and 4 µg/ml polybrene.
Perform 3-fold serial dilutions of the virus stock as follows:
1. Add 1.0 ml of medium containing 4 ug/ml polybrene to each of 1.5 mltubes.
2. Transfer 500 µl from tube 1 to tube 2. Mix.
3. Transfer 500 µl from tube 2 to tube 3 and mix. Continue making serial dilutions by transferring 500 µl from each successive dilution into the next prepared tube.
Infect cells by adding 1 ml of each viral dilution to each appropriate well. The final polybrene concentration will be 4 µg/ml. Control well (C) will receive media with polybrene only.
Change media after 6-24 hours.
After 48 hours, subject the cells to selection using the concentrations that are optimal for your cell line and treatment length correspond to the antibiotic.
Upon selection, pick the well with < 20-30% cell survival.
Note: This ensures cells are not being infected with multiple copies of virus. i.e. 1 virus integration per cell.
Expand for polyclonal cells or pick isogenic clones.