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Lentivirus production (3rd generation)

Mammalian cell culture · viral vectors

3rd-gen, self-inactivating lentivirus produced by transient transfection of HEK293T. BSL-2+ work — confirm institutional approval before starting. Always handle in a dedicated BSC, decontaminate with 10% bleach.

Plasmids needed

ComponentPlasmid (Addgene)µg per 10 cm dish
Transfer plasmid (your insert)(custom; pCDH, pLVX, pLentiCRISPR, etc.)5
Gag/PolpMDLg/pRRE (#12251)2.5
RevpRSV-Rev (#12253)1.5
EnvelopepMD2.G (VSV-G) (#12259)1.5

Total ~10 µg DNA per 10 cm dish. Use endotoxin-free preps.

Day 0: plate cells

  1. Plate 4 × 10⁶ healthy HEK293T per 10 cm dish in 10 mL DMEM-10. Aim for ~70% confluence next day.

Day 1: transfect

  1. Use PEI (cheapest) or Lipofectamine 3000.
  2. Mix the four plasmids above per dish. Combine with 1 mL OptiMEM + 30 µL PEI.
  3. Incubate 15–20 min, add to dish dropwise. Rock to distribute.
  4. ~6 h post-transfection, swap medium to 10 mL DMEM-10 + 1 mM sodium butyrate (optional — boosts viral output ~2×).

Day 2 & 3: harvest

  1. Collect medium at 48 h post-transfection. Replace with 10 mL fresh DMEM-10.
  2. Collect again at 72 h. Pool both harvests = ~20 mL viral supernatant.
  3. Spin 500 × g, 5 min, 4 °C, to pellet floating cells.
  4. Filter through a 0.45 µm low-protein-binding filter (PVDF or PES — never nitrocellulose).
  5. Use directly OR concentrate by ultracentrifugation (25,000 rpm × 2 h at 4 °C in SW28 rotor) over a 20% sucrose cushion.
  6. Resuspend pellet in 200–500 µL HBSS or DMEM. Aliquot 50–100 µL into cryovials. Snap-freeze on dry ice. Store at −80 °C.
  7. Avoid freeze-thaw — each cycle drops titre ~30%.

Transduction (general)

  1. Plate target cells in a 6-well at ~30% confluence.
  2. Add 100–500 µL viral stock + polybrene to 8 µg/mL final.
  3. Centrifuge plate at 800 × g, 32 °C, 90 min ("spinoculation") for tough lines.
  4. Replace medium 24 h later. Begin selection (puromycin etc.) at 48–72 h.

Safety