Biology Reference
In-Depth Information
4
Notes
1. It is recommended to include a transfection control with a
linearized HC-Ad vector plasmid that expresses a reporter
gene like
EGFP
or
ß-gal
.
EGFP
is preferable since it allows for
determining transfection effi ciencies using a conventional fl uo-
rescence microscope and does not require staining procedures.
The transfection effi ciency should be >60 %. Attempts to rescue
and amplify HC-Ad vectors from their corresponding plasmid
at low transfection rates (<40 %) frequently result in a large
number of serial amplifi cations and, importantly, high helper
virus contaminations and/or vector genome rearrangements.
High transfection rates in the rescue step are required, because
only few copies of the transfected plasmid will undergo replica-
tion and be packaged into capsids for reasons that are not yet
fully understood.
2. Purifi cation of the linearized HC-Ad vector plasmid can sig-
nifi cantly increase transfection effi ciencies and phenol/chloro-
form extraction typically results in higher yields and higher
quality DNA compared to commercially available spin
columns.
3. When performing agarose gel electrophoresis/ethidium bro-
mide staining with the linearized HC-Ad vector plasmid, care-
fully check if there is DNA remaining in the pocket of the gel.
DNA remaining in the loading pocket indicates that the purity
of the HC-Ad vector DNA is not suffi cient for transfection. In
that case phenol/chloroform extraction should be repeated.
4. One hour incubation of the polyplexes with the cells allows for
adherence of the polyplexes to the cell surface. The infection
with helper virus at that time will signifi cantly increase trans-
fection rates since the concomitant uptake of polyplex and
helper virus increases the effi ciency of endosomal escape of the
polyplexes and reprograms the cytoskeleton for transport to
the nuclear pores.
5. The infectious titer of the helper virus should not be
determined by a plaque assay, since this method is diffi cult to
reproduce. Plaque formation requires a series of events after
transduction of a cell and is thus not representative for the
infectious titer of the helper virus. Consistently pfu titers are
typically fi ve- to tenfold below the infectious titer determined
by DNA-based methods. We recommend to determine the
infectious titer either by qPCR or by a DNA-based slot-blot
procedure [
25
,
26
].
6. The optimal CPE is characterized by >90 % of the cells being
rounded and detached from the dish in clusters. If more than
10 % of the cells are not rounded and still attached to the dish
