Biology Reference
In-Depth Information
3. Add the total volume of the cell pellet (18 mL) in one SW32
tube and the precipitated solution from the supernatant in
another SW32 tube on the top of the gradient and equilibrate
tubes with 1×-PBS.
4. Centrifuge for 1 h 42 min at 125,500 ×
g
at 18 °C in a Beckman
SW32 rotor with maximum brake.
5. Vector appears as an opaque band at the interface between
1.25 g/mL and 1.4 g/mL CsCl. Collect the band by piercing
the SW32 tube about 1 cm below the band using a 2 mL
syringe loaded with a 18G needle.
6. Add 5 mL of 1.32 g/mL CsCl solution in one SW40 centri-
fuge tube and fill a second SW40 tube with 5 mL of the
1.32 g/mL CsCl solution.
7. Add the recovered vector band from the previous step on the
top of the 1.32 g/mL CsCl solution. Equilibrate with 1×-PBS.
8. Centrifuge for 22 h at 155,000 ×
g
18 °C in a Beckman SW40
rotor with maximum brake.
9. Vector appears as opaque band in the middle of the tube.
Collect the band as above in less than 2 mL of volume.
10. For each band, prepare ten eppendorf tubes to collect the viral
fractions.
11. Pass through PD-10 column, 25 mL of 1× PBS Ca
2+
/Mg
2+
to
remove the preservation solution and equilibrate the column.
Discard the flow-through.
12. Add the total volume of the collected band from
step 9
.
Discard the flow-through.
13. Add 500 μL of 1× PBS Ca
2+
/Mg
2+
to the PD-10 column and
collect the flow-through in an eppendorf tube.
14. Repeat the previous step until 10 fractions per band are
collected.
15. Add glycerol to a final concentration of 10 % to each fraction
(approx. 55 μL per fraction).
16. Store the purified viral fractions at −80 °C.
3.3 Measurement
of Viral Infectivity
The infectivity of a viral particle is the capability to enter into a cell
and express its genome. The infectivity depends on multiple factors
such as the cell line, its passage, or the properties of the viral particle
by itself, and it can be altered in a new chimeric adenovirus. Usually,
the infectivity of the Human Adenovirus type 5 in HEK-293 cells
ranges between 1/10 and 1/100 (IU/vg; Infection Units per viral
genomes). Although the viral backbone may be almost identical,
the infectivity of chimeric adenoviruses may differ significantly
from HAdV-5. Thus, HAdV-5/40 has a poor IU/vg ratio
(between 1/100 and 1/600) [
9
].
