|pLVX-Puro||lentiviral backbone; puromycin resistance|
|psPAX2||2nd generation lentiviral packaging vector|
Opti-MEM (Life Technologies).
HEK medium: DMEM supplemented with 10% FBS and antibiotics.
- For a 10 mM PEI solution, weigh (for accuracy) 10 g of polyethylenimine (PEI; Sigma #408727).
- Add 10 ml of water and vortex to mix.
- Add 12M HCl to neutralise. You will need about 12 ml of HCl, added 1 ml at a time. Check the pH using indicator strips.
- Make to a final volume of 41.2 ml with water. Vortex to mix.
- Aliquot and freeze at -80°C.
Lentiviral particle production
- Plate HEK 293T cells in a 10 cm dish to be 80-90% confluent after 24 h.
- Mix 1µl of PEI with 5 ml of Opti-MEM.
- Mix 3.33 ml of Opti-MEM with 26.64 µg of pLVX-Puro, 6.66 µg of pMD2.G and 20 µg of psPAX2.
- Mix the DNA solution with 3.33 ml of the PEI solution and incubate for 20 min at RT.
- Wash cells with Opti-MEM and then add the PEI/DNA mix.
- Return the cells to the incubator for 4 h.
- Replace the transfection medium with fresh HEK medium and return to the incubator.
- Remove the medium after 48 h. Centrifuge at 3000 x g for 20 min and then filter the supernatant through a 0.45 µm syringe filter. This is the viral supernatant.
- The viral supernatant can either be use immediately or frozen at -20°C (short-term, or -80°C long-term).
Endothelial cell transduction
- Replace medium with viral supernatant and incubate for 6 h. We use undiluted supernatant for most transductions; however, you may need to dilute the supernatant for some highly-expressed genes.
- Replace the viral supernatant with fresh endothelial cell medium.
- For low-expressing constructs, we do a second viral transduction the next day, following the same protocol (incubations with virus for longer than 6 h do not improve infection).
Puromycin selection isn’t necessary, but will quickly select for transduced cells if your expression is not homogenous. We allow the cells 24 h after viral infection and then use 2 µg/ml puromycin in the medium to select. You should see most of the cell death overnight, with full selection after 4-5 days.