Biology Reference
In-Depth Information
Table 1
293T cell seeding conditions for LV production
In vitro
experiments
In vivo experiments
in rodents
In vivo experiments in large
animals (nonhuman primates)
Viral batch for
Number of Petri dishes (10 cm) 6
20
Cell stack chambers
5 stacks
Table 2
Preparation of CaPO
2
precipitate for LV production
Plasmids
(
see
Note 10 and Note 11)
Quantity for one
Petri dish (
Quantity for one
stack chamber (
μ
g)
μ
g)
Packaging plasmid
13
150
Rev plasmid
3
35
Envelope plasmid
Vesicular stomatitis virus
3.75
43
G protein of the Mohola virus
7.25
79
Transfer plasmid
13
150
incubated at room temperature before transfection. For the
preparation of 1 ml of precipitate for a 10 cm Petri dish, the various
plasmids are mixed in a 1.5 ml tube, with 250
μ
l of deionized water
and then 250
l of 0.5 M CaCl
2
. The DNA-CaPO
2
solution should
be added drop-by-drop to a second tube containing 500
μ
l of 2×
HBS, with continual vortexing. Commercial HBS should be
favored to guarantee pH stability. A fi ne, opalescent precipitate is
obtained after a few minutes. The precipitate should be added in a
drop-wise manner to the cells 5-20 min later; the precipitate
should not be added more than 30 min later (
see
Note 12
). When
the volume of precipitate is large, we recommend generating bub-
bles in the HBS with a 2 ml pipette and adding the DNA-CaCl
2
complex drop by drop. The medium should be replaced 5 h after
transfection, and care is required to ensure that the cells are not
lost, because they adhere only weakly to the plate.
μ
The supernatant is harvested and concentrated 48 h after transfec-
tion (
see
Note 13
). An aliquot of supernatant is fi rst removed
for p24 determination in the nonconcentrated supernatant to
monitor production effi ciency. The supernatant is then passed
through a Stericup fi lter with 0.45
3.6.3 Day 4: Harvesting
and Concentration of
Lentiviral Vector
μ
m pores and centrifuged at
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