Biology Reference
In-Depth Information
10. In the vast majority of cases coinfection with cell lysate and
helper virus at the same time allows to amplify the HC-Ad vec-
tor of choice. However, we found for a HC-Ad vector express-
ing the
EBNA-1
gene from Epstein-Barr virus, which is
suspected to interfere with Ad vector replication, that only
infecting the cells with helper virus 6 h prior to infection with
the cell lysate allowed for amplifi cation of HC-AdEBNA-1 to
high titers [
9
]. If you suspect your transgene to interfere with
Ad replication it may be worth to try this or a similar infection
schedule throughout the whole amplifi cation/production
process.
11. Check the dishes infected with lysates obtained from transfec-
tion/coinfection of pHC-AdEGFP for EGFP expression 24 h
after infection. There should at least be 5-10 % EGFP positive
cells in your dish. If the number of EGFP positive cells is sig-
nifi cantly lower your rescue step was ineffi cient and should be
repeated. Do not be mislead by this low number of EGFP pos-
itive cells (compared to initial transfection). This refl ects the
low effi ciency with which HC-Ad vectors can be rescued from
linearized plasmids. Importantly, in all subsequent amplifi ca-
tion steps there should be an at least tenfold increase in titer.
12. During HC-Ad vector production large quantities of HC-Ad
vector and helper virus genomes are produced. Note that the
helper virus genomes are replicated but not packaged into viri-
ons due to excision of the packaging signal by the cre-
recombinase. Thus, the presence of helper virus genomes in
the genomic DNA isolates is not indicative for a helper virus
contamination. However, the isolation of genomic DNA from
producer cells at the time point of harvesting, digestion of the
DNA with an appropriate restriction enzyme, and analysis by
agarose gel electrophoresis/ethidium bromide staining allows
to estimate the success of vector amplifi cation.
13. The number of amplifi cation steps required for successful
HC-Ad vector generation ranges from 4 to 7 and is infl uenced
by a wide variety of parameters including the HC-Ad vector
genome size (ideally between 30 and 35 kb), the toxicity of the
transgene expressed from the vector, and the presence of
repetitive DNA elements in the HC-Ad vector (which should
be avoided). However, if more than seven serial amplifi cation
steps are required it is likely that the fi nal vector preparation
will contain a high helper virus contamination and/or rear-
ranged HC-Ad vector genomes. In that case optimizing each
single amplifi cation step by varying the amounts of helper virus
and cell lysate added is strongly recommended.
14. Typical yields of HC-Ad vector allow to purify the vector pro-
duced from 2 to 4E8 cells (lysate in 4 mL) in one Beckman
