Lentiviral Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 45

and 10 ml 1 M HEPES. Adjust to pH 7.9 by addition of 1 M
NaOH. Sterilize by fi ltration using a 0.22 ʼ m fi lter with 1 l

capacity and store at 4 °C ( see Note 4 ).
3. Immediately before transfection prepare four 50 ml Falcon
tubes and add to each 45 g of lentiviral vector plasmid pRRL-

sin-cppt-CMV-GFP-WPRE, together with 45

ʼ

ʼ g of pMD2-

LgpRRE and 15 ʼ g of pRSV-Rev packaging plasmids and

g of envelope plasmid DNA (This is a typical four plas-
mid co-transfection. In other protocols where six plasmids are
required for lentiviral production the quantity of plasmids can
be adjusted [ 65 ]).
4. Make each tube up to a fi nal volume of 8 ml with TVM1.
5. Add 410

15.3 ʼ

l 1 M CaCl 2 in two stages; fi rst drop in 5-6 drops
while mixing by fl icking or slightly vortexing and then add the
remaining CaCl 2 quickly without mixing.
6. Incubate the reaction mixtures at room temperature for 10 min.
7. During the last 4 min of incubation examine the HEK 293T in
the dishes and carefully remove the complete DMEM medium.
8. Add 32.8 ml of TVM2 medium to each Falcon tube and mix
by pipetting up and down.
9. Immediately after mixing add 13.5 ml of the DNA/TVM1/
TVM2 transfection mixture to each dish, taking care not to
dislodge the cells.
10. Return the dishes to the incubator in stacks of four (usually no
more than six).
11. Sixteen hours post-transfection (day 3) examine the cells
under a microscope. If a reporter gene encoding a fl uorescent
protein, such as green fl uorescent protein (GFP), is used under
the control of a constitutive promoter (such as CMV), exam-
ine the cells under a fl uorescence microscope for reporter gene
expression. More than 70 % of cells should fl uoresce green.
Proceed to viral production if 60-70 % of producer cells
express the reporter gene. If this is not the case, stop produc-
tion and start again.
12. Replace transfection mixture with low serum complete DMEM
(2 % NCS) (Table 4 ) supplemented with 10 mM sodium
butyrate. It is important the sodium butyrate is always added
fresh to the low serum medium. Return the dishes to the incu-
bator ( see Note 5 ).
13. Thirty-six hours later (day 4) examine the cells under the
microscope. Cells should be fused and multinucleated. This
morphological change is expected and does not affect the pro-
duction of lentiviral vectors. Most cells should still be attached
and around 90-95 % should fl uoresce green. To harvest the
viral supernatants, collect the supernatant from the 12 dishes

ʼ