Biology Reference
In-Depth Information
3.7.2 Last Viral
Amplification Step
in 293S Cells
Last amplification step of the chimeric adenovirus should be
performed in 293F cells (
see
Note 16
).
1. Seed 10
6
293F cells/mL in 1 L shake flasks (400 mL working
volume) in Infection Medium.
2. Add polybrene to a final concentration of 9 μg/mL.
3. Infect cell cultures by adding the cell lysate from
step 9
of the
previous amplification procedure.
4. Change the cell culture media to fresh media at 4-6 h post-
infection by centrifugation at 180 ×
g
for 5 min (
see
Note 17
).
5. Harvest cells at the optimal harvest time found in
Subheading
3.5
(56 h post-infection for HAdV-5/40S) and
centrifuge for 5 min at 180 ×
g
.
6. Resuspend the cell pellet in 20 mL of supernatant and store at
−80 °C.
7. Optional step: If the virus genome carries the Death Protein
(ADP) gene, the supernatant should be concentrated down to
20 mL using a Midjet system.
8. Lyse the cell pellet by three freeze/thaw cycles. Remove cell
debris by centrifugation for 5 min at 1,150 ×
g
.
9. Purify the crude viral stock following Subheading
3.2
.
4
Notes
1. Before digesting, check if the adenoviral sequence has internal
Pac
If cleavage sites. If this is not the case, use another restric-
tion enzyme to digest the bacterial sequences.
2. It is recommended to use a control plate transfected with an
irrelevant plasmid to test the PEI's toxicity.
3. Use a control plate to compare the cytopathic effect.
4. It is recommended to use a noninfected plate as control.
5. As freeze/thaw cycles affect the stability of the vectors, we
recommend to aliquot vectors in small volumes (e.g., 10, 50,
and 100 μL aliquots).
6. To calculate the titer by “end point dilution” do not count the
number of infected cells because in the positive wells is possible
to find a high number of infected cells if waiting for more than
one replicative cycle.
7. When using a different resuspension buffer, add SDS to a final
concentration of 0.1 % to disrupt the capsids.
8. The OD
260
must be within the lineal range of your spectropho-
tometer. If not, repeat the previous steps with different viral
dilutions.
