Biology Reference
In-Depth Information
2.4 Chimeric
Adenoviral Production
from a Pre-stock
1. 150-mm petri dishes (20 plates).
2. HEK-293 cells.
3. Growing and infection medium as described in Subheading
2.3
.
4. CsCl solutions: 1.4 g/mL, 1.34 g/mL, and 1.25 g/mL in 1×
D-PBS.
5. Ultracentrifuge and rotors: Beckman Coulter Optima L90K or
L100XP. Swinging bucket rotors SW32 and SW40ti.
6. Centrifuge tubes for SW32 and SW40ti rotors.
7. 2 and 10 mL syringes, and 18G needles.
8. Glycerol, anhydride.
9. PD-10 columns Sephadex G-25.
10. 1× D-PBS Ca
2+
/Mg
2+
.
11. 96-Well plates.
12. Optional: Polybrene.
2.5 Production
in Suspension 211BS
Cell Cultures
1. Polycarbonate shake flasks, 125 mL and 1 L.
2. Infection Medium for suspension cells: Freestyle serum-free
medium (12338-018, Invitrogen) supplemented with 100 U/
mL Penicillin, 0.1 mg/mL Streptomycin, and 1 % Pluronics.
3. 211BS cells [
9
].
4. Polybrene.
5. Growing medium for suspension cells: SFMII medium (11686-
029, GIBCO) supplemented with 4 mM Glutamine, 100 U/
mL Penicillin, 0.1 mg/mL Streptomycin, and 1 % Pluronics.
6. Midjet System (GE Healthcare).
3
Methods
3.1 Generation
of Viral Pre-stock
It is highly recommended to perform a small pre-stock to generate
enough viruses for the following amplification steps. As general
trend, for each amplification step it is recommended to wait for
cytophatic effect (CPE) to harvest the infected cells. Finally, viral
infectivity, viral productivity, and viral cycle must be analyzed to
characterize the viral production.
1. Digest 100 μg of the recombinant adenoviral plasmid with 30
Units of
Pac
I restriction enzyme in a total volume of 100 μL
to remove the plasmid backbone of bacterial origin sequences.
Digest for 4 h at 37 °C (
see
Note 1
).
2. Add another 30 Units of
Pac
I to guarantee a complete diges-
tion. Digest 4 h at 37 °C.
3. Precipitate the digested DNA with 2 volumes of ethanol and
resuspend in 50 μL of sterile ddH
2
O.
