Biology Reference
In-Depth Information
Conventional methods for producing small volumes of viral
vectors involve culturing cells in stationary, adherent cultures. The
main protocols of this small-scale production method are pre-
sented below, while methods for large-scale production of Ad viral
vectors can be found in the literature. Recombinant adenoviruses
obtained at the end of the protocol are ready to be used in experi-
mental procedures involving cells or laboratory animals, as well as
a starting material for subsequent rounds of larger amplifi cations.
1. Twenty-four hours before transfection, plate 10 6 HEK-293
cells/well in a six-well cell culture plate with DMEM + 10 %
FBS (Fetal Bovine Serum). Three wells are needed for each
adenovirus vector, one of them as control.
2. Digest 100
g of recombinant adenoviral plasmid with 15 U of
Pac I for 5 h to separate the adenovirus genome from the
remaining plasmid sequences.
3. Precipitate digested DNA with 0.1 volume of sodium acetate
and 2.5 volumes of ethanol and resuspend in 100
μ
μ
L sterile TE
buffer.
4. Perform a standard transfection using 6
g of Pac I digested
plasmid per 10 6 HEK293 cells ( see Note 7 ).
5. Incubate at 37 °C and 5 % CO 2 for 3 days ( see Note 8 ).
6. Scrape/harvest medium and cells. Freeze at −80 °C and thaw
at 37 °C three times to lyse the cells and release the
adenoviruses.
7. Centrifuge at room temperature for 5 min and 1,200 × g and
keep the supernatant (crude lysate). Discard the pellet.
8. Use all the obtained crude lysate to infect 7 × 10 6 HEK293
cells (at 70-80 % confl uency) in a 10-cm plate, in a fi nal vol-
ume of 8 mL, with DMEM + 2 % FBS.
9. Incubate at 37 °C and 5 % CO 2 until general cytopathic effect
is observed (usually between 4 and 9 days).
10. Harvest medium and cells. Freeze-thaw three times to release
adenovirus from cells.
11. Centrifuge at room temperature for 5 min and 1,200 × g .
Discard the pellet and keep the supernatant (crude lysate).
12. Use 1 mL of the crude lysate obtained in step 11 to infect a
total 3.5 × 10 8 HEK293 cells in twenty 15-cm plates (70-80 %
confl uency), in a fi nal volume of 14 mL/plate with DMEM + 2 %
FBS.
13. Incubate at 37 °C and 5 % CO 2 until general cytopathic effect
is observed ( see Note 9 ).
μ
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