Lentiviral Packaging

LENTIVIRUS PRODUCTION

AO’s – Modified from Naldini’s lab

Lentiviral particles are produced in 293T cells by simultaneous transfection of 3 system plasmids (ENV, REV, PACK) plus a transfer plasmid carrying the transgene of interest (TRANSFER). Transfected cells release viral particles in the supernatant, which is then concentrated by ultracentrifugation.

The limiting factor for the procedure is the volume of supernatant that can be centrifuged. Allow at least two 15cm dishes per type of virus. From these you will get about 50 µL of concentrated virus, enough for 2-3 transductions.

All procedure MUST be carried out in a P2 contained facility according to the institute regulations on class II agents.

1. TRANSFECTION

- Maintain 293T cells in IMDM, 10% (heat inactivated) FBS, Penicillin (100U/ml), Streptomycin (100U/ml) and Glutamine (22.5 ml final volume). Use cells until passage 12, then thaw a new aliquot. Never grow to confluence!

- Seed 293T cells in 15cm dishes:

- 18x10⁶ cells, approximately 8 h before transfection

- or 9x 10⁶ cells, approximately 24 h before transfection

- Add fresh medium 2 h before transfection, 22.5 mL final volume. This is done to boost cell cycle progression, as transfected DNA will cross the nuclear membrane only during mitosis.

- Prepare plasmid mix as follow (recipe for one 15cm plate):

- ENV plasmid pMD2.VSV-g 9 µg

- PACKAGING plasmid pMDLg/p.RRE 12.5 µg

- REV plasmid pILVV01 6.25 µg

- TRANSFER vector 25 µg

- Make up the plasmid mix to a final volume of 1125 with 0.1X TE/dH₂O (2:1).

- Finally, add 125μl of 2.5M CaCl₂, and mix immediately.

- Leave the mix at RT for 5’

- Form the Ca-Pi precipitate by drop wise addition of 1300 µL of 2X HBS solution to the 1250μl DNA-TE-CaCl₂ mixture, while vortexing at full speed.

- Add the precipitate to the 293T cells immediately after the addition of the 2x HBS. Gently swirl the plate to mix.

- It is possible to transfect two plates at the same time to save time.

- Incubate with CaPi precipitate for 12-14 h

- Replace with 19 mL of fresh medium containing 1 µM Sodium Butyrate

- Collect supernatant after 30 h

- Filter through 0.22µm and either store O/N at 4°C or concentrate straight away

- Optional: add 16 mL for a second collection at 48 h

1.1 TRANSFECTION REAGENTS

2X HBS: 281mM NaCl, 100mM HEPES,

1.5mM Na₂HPO₄, pH 7.12

0.22μM filtered, store at –20°C or –80°C

2.5M CaCl₂: Sigma tissue culture grade

0.22μM filtered, and store at –20°C.

0.1X TE buffer: 10mM Tris (pH 8.0),

1mM EDTA (pH 8.0)

diluted 1:10 with sdH₂O

0.22μM filtered, store at 4°C

dH₂O (endotoxin-free): Sigma tissue culture grade, W-3500.

2. VIRUS CONCENTRATION

- Transfer filtered supernatant to Beckman n.326826 tubes (25x89mm). Tube capacity is 38 mL.

- Spin 2 h at 20,000 rpm at 20ºC in Beckman-Coulter Optima L90K Ultracentrifuge, using SW32 Ti rotor

- Discard supernatant

- Resuspend the pellet in sterile PBS so that is concentrated 500 fold. Example: if you centrifuged 36 mL of supernatant, resuspend pellet in 72 µL of PBS.

- Leave 30 min at RT

- Mix well and recover all virus.

- Optional: leave on the wheel to homogenise for 30-60 min

- Make 10-20µL aliquots and store at -20ºC

(NB: each freeze/thaw cycle will halve the virus titre)