Biology Reference
In-Depth Information
the helper virus amount was too low and the transfection/
coinfection should be repeated with an adjusted amount of
helper virus. If all cells are rounded and detached but the
majority is present as single cells (instead of clusters) the helper
virus amount was too high and the transfection/amplifi cation
should be repeated with an adjusted amount of helper virus.
For all steps of rescue and amplifi cation the optimal CPE
should occur within 40-48 h. If it occurs earlier or later the
amount of helper virus should be adjusted and the transfec-
tion/amplifi cation step should be repeated with an adjusted
amount of helper virus. For researches who are not or only
marginally familiar with high-titer production of fi rst-
generation adenovirus vectors it is strongly recommended to
learn to microscopically evaluate the completeness of a CPE by
infecting a series of dishes with 116 cells with increasing
amounts of helper virus and observing the (sometimes subtle)
morphological changes over time. In addition, for trouble
shooting it is advised to take photographs of the different
stages of CPE formation during HC-Ad vector amplifi cation.
Please note that while the microscopic evaluation of the CPE
is an indispensable tool for successful rescue and amplifi cation
of HC-Ad vectors, the appearance of the CPE may—in addi-
tion to the amounts of helper virus used—be infl uenced by the
transgene expressed from the HC-Ad vector to be amplifi ed.
7. Cells containing HC-Ad vector from the different rescue/
amplifi cation steps can be snap-frozen and stored at −80 °C
without cryoprotectant until further use. It is not recom-
mended to store lysates of the cells (i.e., lysates obtained by
repeated freeze-thaw cycles as in
steps 11
,
17
,
22
) for
prolonged periods at −80 °C without the addition of a cryo-
protectant (10 % w/v glycerol).
8. We recommend to amplify the HC-AdEGFP vector used as a
transfection control (
see
Note 1
) in parallel to the actual
HC-Ad vector to be produced, because for the HC-AdEGFP
vector the process of amplifi cation can easily be visualized
using a fl uorescence microscope. Monitoring the amplifi cation
process gives valuable hints on how to optimize the different
amplifi cation steps. However, it should be noted that vectors
with different transgenes (e.g., toxic transgenes or transgenes
that interfere with adenovirus replication) can behave differ-
ently during the amplifi cation process.
9. There is no need to clear the cell lysates by centrifugation after
freeze-thaw. Clearing the cell lysates may result in spinning
down vector which is attached to membrane fragments. Instead
of clearing the lysates take care to also use the cell debris in the
infection step. This holds true for all amplifi cation steps.
