Lentiviral Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 47

1. Twenty four hours prior to transfection (day 1), plate each
150 mm cell culture dish with 1.4 × 10 7 HEK 293T cells at a
density of ~7.6 × 10 4 cells cm −2 in 16 ml complete DMEM and
incubate overnight in a standard tissue culture incubator.
2. The next day (day 2), 3 h prior to transfection, make and
warm to room temperature fresh TVM1 and TVM2 media.
Reagents required are listed in Table 3 .
3. Immediately before transfection prepare one Falcon tube per
virus and this time adjust the quantity of plasmids for single
dishes. Add to each Falcon tube 15 g of lentiviral vector plas-

mid pRRL-sin-cppt-CMV-GFP-WPRE, together with 15

ʼ

ʼ g

of pMD2-LgpRRE and 5 ʼ g of pRSV-Rev packaging plasmids

g of envelope plasmid DNA. Make up to a fi nal vol-
ume of 2.66 ml with TVM1.
4. Add 136.6

and 5.1 ʼ

ʼ l 1 M CaCl 2 in two stages as described in

Sect. 3.2.2 .
5. Incubate the reaction mixtures at room temperature for
10 min.
6. During the last 4 min of incubation examine the HEK 293T
and carefully remove the complete DMEM medium.
7. Add 10.9 ml of TVM2 media to each Falcon tube and mix by
pipetting up and down.
8. Immediately after mixing add 13.5 ml of the DNA/TVM1/
TVM2 transfection mixture to the dish, taking care not to
dislodge cells.
9. Return the dishes to the incubator.
10. Sixteen hours post-transfection (day 3) examine the cells
under a microscope and proceed as described in Sect. 3.2.2 .
11. Replace transfection mixture with 15 ml of low serum com-
plete DMEM (2 % NCS) (Table 4 ) supplemented with 10 mM
sodium butyrate, as described in Sect. 3.2.2 .
12. Thirty-six hours later (day 4) examine the cells under a micro-
scope. Most of them should still be attached and around
90-95 % should fl uoresce green. Harvest the viral
supernatants.
13. This time collect the supernatant in Falcon tubes and centri-
fuge for 5 min at 750 rpm to remove cell debris. Proceed to
fi ltration through a 0.45 m pore-sized fi lter.

14. Keep one 1 ml aliquot of the unconcentrated viral supernatant
and store at −80 °C.
15. From the rest of viral supernatant, which should be 14 ml,
transfer 7 ml into a sterile polypropylene ultracentrifugation
tube. Then insert a pipette all the way to the bottom of the
tube and gently underlay it by slowly expelling 2 ml of 20 %

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