Biology Reference
In-Depth Information
4. Transfect 10
6
HEK-293 cells plated at 60-70 % of confluency
with 6 μg of
Pac
I-digested plasmid (
see
Note 2
).
5. After 3 days, scrape the cells. Freeze/thaw three times the
crude lysate to release adenoviral particles from the cells.
6. Centrifuge at 4,500 ×
g
for 5 min. Save the supernatant and
discard the pellet to remove the cell debris.
7. Seed one 10-cm plate with HEK-293 cells at 60-70 % of con-
fluency. Add 3 mL of DMEM 2 % FBS on the cells as well as all
the supernatant from the previous step (
see
Note 3
).
8. Check the cells during a period of 5-20 days until a 50 % of
CPE is observed. Harvest medium and cells and freeze/thaw
three times to release adenoviral particles from the cells.
9. Centrifuge at 4,500 ×
g
for 5 min. Save the supernatant and
discard the pellet to remove the cell debris.
10. Seed one 150-mm plate with HEK-293 cells at 60-70 % of
confluence (15 × 10
6
HEK-293 cells). Use DMEM 2 % FBS as
medium.
11. Add the supernatant from
step 9
to the cells.
12. Follow the cytopathic effect during the first 2-4 days. Unlike
the previous amplification step, the CPE should appear in a
period of 30-48 h post-infection. Nevertheless, if vector's pro-
ductivity or infectivity is poor, check the cells during a period
of 5-20 days until a 50 % of CPE is observed. Harvest medium
and cells, and freeze/thaw three times to release the adenovi-
rus from the cells.
13. Centrifuge at 4,500 ×
g
for 5 min. Keep the supernatant and
discard the pellet to remove the cell debris.
14. Seed 10 × 150-mm cell plate with HEK-293 cells at 60-70 % of
confluency. Use infection medium.
15. Distribute the supernatant from
step 13
among the ten plates.
16. Check the cells during a period of 2-4 days until a 50 % of CPE
is observed as previously describe. Harvest medium and cells.
17. Harvest the cells and resuspend the cell pellet in 18 mL of
supernatant. Freeze/thaw three times to release the adenovi-
rus from the cells.
18. Titrate the viral pre-stock (
see
Subheading
3.3
).
1. Add 10 mL of 1.25 g/mL CsCl solution in two SW32
centrifuge tubes.
2. Add carefully 10 mL of 1.40 g/mL CsCl solution at the bot-
tom of each SW32 centrifuge tubes to generate two density
phases. Take care to not disturb the gradient when removing
the pipette.
3.2 Purification
of Viral Pre-stock
