Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 205

9. 293T cells adhere poorly to plastic, so several fl asks should be
tested to determine the optimal culture conditions. We use
Falcon Petri dishes and Corning fl asks.
10. The reproducibility of LV production can be increased by
generating large batches of plasmids, checking their quality,
and then storing them in aliquots (1 mg/ml) to prevent the
need for repeat freezing and thawing. One option is to sub-
contract plasmid production (packaging, VSV-G and MOK-G
envelopes, Rev).
11. The relative proportions of the various plasmids are optimized
and must be respected to achieve high production yields. For
LVs pseudotyped with the MOK-G envelope, the best results
were obtained by doubling the quantity of the envelope plas-
mid during transfection.
12. CaCl 2 aliquots are freshly prepared and not refrozen. The mix-
ture should be opalescent. If a coarse precipitate formed, it
should be broken down by vigorous shaking. Production
yields are higher when the DNA-CaCl 2 complex is deposited
on HBS rather than the reverse.
13. Harvest is possible 24 h, 48 h, or 72 h posttransfection, but
the best yields are obtained 48 h posttransfection.
14. Rotor and ultracentrifuge are cooled at 4 °C before use.
15. p24 values concentrations of 50-300 ng/μl l are usually

obtained for LV-MOK-G (supernatant from 293T cells, non-
concentrated LV) and of 100-1,000 ng/μl

μ

l for LV-VSV-G.
16. A relative transducing unit can be estimated from the p24
titer. This conversion factor, established by D. Trono for
LV-VSV-G, is based on the fact that there are approximately
10 4 physical particles per pictogram of p24. The estimated TU
is thus of the order of 10 5 TU/ng p24.
17. Increasing formaldehyde concentration causes the cells to
autofl uoresce.
18. Cells are maintained in the dark at 4 °C until FACS analysis.

μ

19. For calculation, only dilutions yielding from 1 to 20 % positive
cells should be considered.
20. Use Dumont forceps (numbers 4 and 5) to withdraw the
meninges and extract the brain. Place the brain in a large Petri
dish on ice.
21. Place the dorsal face of the brain towards you: cut out the
cortex (striatum visible from above), withdraw the thalamus
(the white ball at the center of the striatum), and separate the
hemisphere. Then, place the external face of the brain towards
you and remove the dura mater. Take off as much cortex as
possible.