Optimized protocol for high titers
This protocol is to maximize the titers of lentivectors. It uses JetPRIME and Benzonase.
JetPRIME is to achieve maximum and reproducible transfection. In Switzerland, do not order via Polyplus, they will charge you with a super high price, plus shipping, plus customs fees. Totally obscene. Instead order from Brunschwig and ask for a quote. It may end up to a 50% discount compared to Polyplus Europe.
Benzonase treatment of the supernatant before filtration and PEG-IT precipitation is to break down the mesh of high-molecular chromosomal DNA released by the dying producer cells (high amounts of high MW DNA because the cells are highly transfected and thus die a lot). This high MW DNA has two major impacts on the downstream steps. First, it clogs the filters. Second, it traps the vector particles. By just shortly incubating with the Benzonase DNAse, you reduce the MW of the chromosomal DNA released by the dying cells, and hence make the filtration easier and yields much higher.
- Maintain 293T cells in D10 medium, in 10-cm tissue culture dish in a 37°C humidified incubator with a 5% CO2 atmosphere, and split them at ratio 1:10 using Trypsin/EDTA, three times per week (e.g., every Monday, Wednesday, and Friday). Frequent passages and keeping the 293T as individual cells will ensure high transfection efficiency.
- The day before the transfection, seed 3 dishes at 1.5 to 2.5 million cells per dish (10 cm). Cells must be approximately 1/2 to 2/3 confluent on the day of transfection.
- Incubate overnight in a 37°C humidified incubator with a 5% CO2 atmosphere.
- On the following day, co-transfect the cells according to the following recipe.
Envelope plasmid pCAG-VSVG 6 µg
Packaging plasmid psPAX2 12 µg
Vector plasmid pCLX-EF1-GFP (as control and example) 12 µg
- Mix the plasmids in a 2.2 ml Eppy and pipet up and down 20 times
- Add 1500 µl of JetPRIME Buffer, vortex 10 s and spin down
- Add 60 µl of JetPRIME reagent on the plasmid mix. Be careful with the DNA quantity, since if you have too much DNA and your JetPrime/DNA ratio drops below 2, your transfection efficiency will drop dramatically!!!
- Vortex and spin down.
- Leave the precipitate form at room temperature for 10-15 min at RT.
- For each TC100, add 500 µl of precipitate dropwise to the cells. Mix by gentle swirling.
- Place the dish overnight in a 37°C humidified incubator with a 5% CO2 atmosphere.
- In the morning the day after, aspirate the medium and replace with 10 ml of new pre-warmed D10 medium.
- Collect the supernatants after 2 days.
- Transfer and pool the supernatants from the identical 3 plates into one 50-ml centrifuge tube.
- Quick spin the cell debris if you notice a lot of floating cells
- Add 15 µl of Benzonase (at 200'000 Units/ml) to the 30 ml of supernatant, which makes a final concentration of 15x200/30 = 100 Units/ml, and incubate at 37°C for 1 hour.
- Filter the 30 ml of pooled supernatant with a 50 ml syringe connected to a 0.45 µm PVDF disk filter. Collect in a new 50-ml centrifuge tube.
- At that stage, you must have around 28 ml of unconcentrated supernatant.
- To this 28 ml of unconcentrated supernatant, add 8 ml of PEG-it, mix thoroughly but gently and without vortex and store at +4°C overnight and up to 4-5 days.
- Spin at 1500 g for 30 minutes.
- Aspirate as much supernatant as possible without disturbing the pellet.
- Add 200 µl of cold PBS on the pellet and gently resuspend without making bubbles. I pipet up and down 30 times.
- Aliquote in 20 µl aliquots and store at -80°C