Biology Reference
In-Depth Information
typically yield one additional band, which is not present in the
digestion of the HC-Ad vector DNA and comprises the plas-
mid backbone. All other bands should be visible in both the
plasmid control and the HC-Ad vector genome. If the diges-
tion of the HC-Ad vector genome reveals additional bands,
which are not present in the plasmid control this is a hint for
either a high helper virus contamination or rearranged vector
genomes. In that case a more sensitive Southern blot analysis is
recommended.
20. Measuring the OD at 260 nm after rupture of the particles by
SDS is the easiest way to determine the physical particle titer of
a HC-Ad vector preparation. It should be noted that the accu-
racy of this method very strongly depends on the purity of the
vector preparation. Any contamination with nucleic acids or
protein will make the titer appear higher than it is. We there-
fore recommend to routinely analyze the purity of HC-Ad vec-
tor preparations by SDS-PAGE and subsequent silver staining
of the gels. Loading of 1E10 particles is suffi cient to visualize
hexon (100 kDa), penton base, IIIa, and fi ber (60-65 kDa)
capsid proteins. Any additional bands in the silver-stained gel,
which cannot be attributed to vector capsid/core proteins
indicate impurities. In that case the vector should be re-banded
with a CsCl step gradient. To obtain reliable titers by OD260
it is also important to not exceed the fi nal SDS concentration
of 0.1 % because at higher concentrations SDS starts to form
micelles, which infl uence the absorbance.
21. Large-scale HC-Ad vector preparations can be used to coinfect
fresh producer cells with the HC-Ad vector and helper virus to
obtain another large-scale preparation. However, due to the
fact that a contamination with mutated helper virus or rear-
ranged HC-Ad vector can occur and may overgrow the actual
HC-Ad vector. Therefore, it is advised to use one fully charac-
terized HC-Ad vector preparation as a master stock instead of
generating grand- and grand-grand-daughter preparations
over several generations.
22. The contamination of HC-Ad vector preparations with helper
virus can vary, but should be at least below 1 % and preferably
below 0.1 %. If helper virus contaminations are above 1 % it is
advised to adjust both the amount of helper virus and the
amount of lysate added during the serial amplifi cation steps
and the fi nal production step. Again, it should be noted that
only helper virus preparations whose sequence of the fl oxed
packaging signal is fully confi rmed should be used.
