Lentiviral Vectors as Research Tools in Neurobiology: Design and Production - Viral Vector Approaches in Neurobiology and Brain Diseases - page 6

4 Methods

4.1 LVs Production
(see Notes 1 and 2)

1. Seed and incubate 9×10 6 HEK 293T cells [ATCC, CRL-11268]
in 150 mm dishes, approximately 24 h before transfection.
The medium used is DMEM GlutaMax containing 10 % FBS,
1× Pen/Strept. Use low passage cells (not more than P12-15)
and do not ever let cells grow to confluence.
2. Change medium 2 h before transfection: IMDM supplemented
with 10 % FBS, 1× Pen/Strept, 1× glutamine (22 ml final
volume).
3. Prepare the plasmid DNA mix by adding together: 9 μg ENV
plasmid (VSV-G), 12.5 μg packaging plasmid (pMDLg/pRRE
or CMV R8.74), 6.25 μg of pRSV-REV, and 32 μg of gene
transfer plasmid. The plasmid mix solution is made up to a final
volume of 1,125 μl with 0,1× TE Buffer (1× : 10 mM Tris
pH 8.0; 1 mM EDTA pH 8.0 in water). Finally, 125 μl of 2.5 M
CaCl 2 is added.
4. Leave the mix 15 min at room temperature.
5. The precipitate is formed by dropwise (Critical!) addition of
1,250 μl of 2× HBS (281 mM NaCl, 100 mM HEPES,
1,5 mM Na 2 PO 4 , pH 7.06-7.12) solution to the 1,250 μl
DNA-TE-CaCl 2 mixture from step 3 while vortexing at full
speed. The precipitate should be added to HEK 293T cell
immediately following the addition of the 2× HBS. High-
magnification microscopy of the cells should reveal a very small
granular precipitate of calcium phosphate and plasmid
DNA, initially above the cell monolayer and after incubation
in 37 °C incubator overnight, on the bottom of the plate in the
large spaces between the cells.
6. The precipitate should be allowed to stay on the cells for
14-16 h, after which the media should be replaced with fresh
medium (IMDM with 10 % FBS, 1× Pen/Strept, 1× glutamine
and 1 M sodium butyrate).
7. Collect the cell supernatants at 36 h after changing the
medium, filter (0.22 μm) and centrifuge at 20,000 rpm at
20 °C for 2 h (Beckman Ultracentrifuge, SW32Ti rotor).
8. Discard the supernatant and gently resuspend the pellet in
sterile PBS 1×.
9. Aliquot and store at −80 °C.

4.2 Titration
of the LVs

1. Seed and incubate 5×10 4 HEK 293 T cells in 35 mm dishes,
12-14 h before the infection.
2. Make serial dilution of the LV in the growing medium (DMEM
GlutaMAX, 10 % FBS, 1× Pen/Strept) and transduce the cells
with the desired dilutions in a final volume of 1 ml.