Biology Reference
In-Depth Information
8. One hour after infection with helper virus add 3 mL of fresh,
supplemented, pre-warmed medium (the fi nal volume should
be 5 mL).
9. 48 h after transfection the cells should show a complete cyto-
pathic effect (CPE) characterized by cell rounding and detach-
ment from the dish (
see
Note 6
). Harvest the cells in their
medium by pipetting up and down or scraping.
10. Spin down cells at 400 ×
g
for 10 min. Aspirate and discard
supernatant. Resuspend the cells in 2 mL PBS. Snap-freeze the
cell suspension in liquid nitrogen and store at −80 °C until
further use (
see
Note 7
).
Upon initial transfection only small amounts of HC-Ad vector are
produced by the 116 cells (
see
Note 1
). These small amounts are
serially amplifi ed by repeatedly coinfecting fresh 116 cells with
purifi ed helper virus and cell lysates containing the HC-Ad vector.
As a rule of thumb the HC-Ad titers should increase at least tenfold
in each amplifi cation step (
see
Note 8
).
3.3 HC-Ad Vector
Serial Amplifi cation
1. Release vector particles from the cells in the suspension
obtained in
step 10
of the rescue protocol by three repeated
freeze-thaw cycles in liquid nitrogen and a water bath (37 °C).
Gently swirl the tubes with the cell suspensions while freezing
and thawing.
2. Replace the medium of a 60-mm dish with 90 % confl uent 116
cells seeded 2 days before with 2 mL of fresh, supplemented
medium.
3. Infect cells with 0.5 mL of the freeze-thaw lysate obtained in
step 1
of the serial amplifi cation protocol (
see
Note 9
). Coinfect
the cells with 15-20 MOI of helper virus (
see
Note 10
). One
hour after infection fi ll up to 5 mL with supplemented medium
(
see
Note 11
). Supplement the remaining cell lysate with glyc-
erol to a fi nal concentration of 10 % (w/v) prior to storage at
−80 °C.
4. 48 h after infection the cells should show a complete cyto-
pathic effect (CPE) characterized by cell rounding and detach-
ment from the dish. Harvest the cells in their medium by
pipetting up and down or scraping.
5. Spin down cells at 400 ×
g
for 10 min. Aspirate and discard
supernatant. Resuspend the cells in 2 mL PBS. Snap-freeze the
cell suspension in liquid nitrogen and store at −80 °C until
further use.
3.3.1 First HC-Ad Vector
Amplifi cation Step
1. Repeat
steps 1
-
5
.
3.3.2 Second HC-Ad
Vector Amplifi cation
