Lentiviral transduction of endothelial cells

Plasmids

plasmid description
pLVX-Puro lentiviral backbone; puromycin resistance
pMD2.G VSV-G coat
psPAX2 2nd generation lentiviral packaging vector

Materials

Opti-MEM (Life Technologies).
HEK medium: DMEM supplemented with 10% FBS and antibiotics.

Preparing PEI

  1. For a 10 mM PEI solution, weigh (for accuracy) 10 g of polyethylenimine (PEI; Sigma #408727).
  2. Add 10 ml of water and vortex to mix.
  3. 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.
  4. Make to a final volume of 41.2 ml with water.  Vortex to mix.
  5. Aliquot and freeze at -80°C.

Lentiviral particle production

  1. Plate HEK 293T cells in a 10 cm dish to be 80-90% confluent after 24 h.
  2. Mix 1µl of PEI with 5 ml of Opti-MEM.
  3. 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.
  4. Mix the DNA solution with 3.33 ml of the PEI solution and incubate for 20 min at RT.
  5. Wash cells with Opti-MEM and then add the PEI/DNA mix.
  6. Return the cells to the incubator for 4 h.
  7. Replace the transfection medium with fresh HEK medium and return to the incubator.
  8. 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.
  9. The viral supernatant can either be use immediately or frozen at -20°C (short-term, or -80°C long-term).

Endothelial cell transduction

  1. 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.
  2. Replace the viral supernatant with fresh endothelial cell medium.
  3. 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

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.

2 responses to “Lentiviral transduction of endothelial cells

  1. Thank you for posting your protocol. I’m currently trying to do lentiviral transduction of HUVEC and use puromycin for selection. Most of the cell death does occur overnight as you mention. Do you change the media (+puromycin) in 24 hours from addition of puromycin or wait until the 48-72 hour mark? Thanks and regards.

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    • I think it depends how long you want to keep them. When we do lentiviral transduction with an immortalised EC line, we leave them in puro for 72h and that definitely increases the level and homogeneity of expression. With the HUVEC, you probably want to get on with your experiments, so 24h is probably the best compromise. One other thing – if your expression is not good in HUVEC with 24h puro (or if the rate of killing is very high) then you might want to try doing two rounds of viral infection before selection. The virus isn’t very stable in TC conditions so two rounds makes a big difference (just doing a longer infection makes no difference in our experience). Good luck!

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