Biology Reference
In-Depth Information
14. If a verifi ed intermediate is used from previous experiments, it
is enough to plate out 100
L of the water control. A few colo-
nies can be found in the control plate since the counter selec-
tion is never 100 % effi cient. However, if there is not at least
fi vefold difference in favor to the main reaction, contamination
of the intermediate stock must be considered.
15. pO6A5-CMV is a small plasmid vector with a basic CMV
promoter-based transcription unit for expression of the cloned
genes in mammalian cells either after transfection of the plasmid
itself or transferring it into the Ad vector. The left ITR and the
packaging signal of the Ad5 (in front of the CMV promoter) will
provide the left end of the rescued viral genome after recombina-
tion with the BAC acceptor. It also contains the R6Kgamma origin
of replication [
15
], which is dependent on the presence of the
pir
locus and therefore cannot be maintained in normal laboratory
E.
coli
strains.
E. coli
strain PIR1, which expresses the
pi
protein
encoded by the
pir
gene in trans, is required for cloning the gene
of interest in pO6A5-CMV and propagation of its recombinants.
This plasmid carries a 34 bp FRT site for integration
(5
μ
).
The PIR1 strain is available from Invitrogen and the pO6A5-CMV
from Sirion Biotech.
′
-GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3
′
16. When constructing normal fi rst generation vectors by Flp
recombination, the Ad genome is usually deleted from the left
end to the end of the E1B gene in the BAC acceptor. Instead,
a 48 bp at the FRT site (5
-GAAGTTCCTATTCCGAAGTTC
CTATTCTCTAGAAAGTATAGGAACTTC-3
′
) is inserted,
allowing insertion of donor plasmids carrying 34 bp FRT sites
(
see
Note 15
). In this design the donor will provide the left-
most region of the vector genome with ITR and the packaging
signal after recombination. Thus the BAC acceptor cannot be
rescued to virus. This setting can be found in the pBA5-FRT
exemplifi ed here, which is available from Sirion Biotech. The
FRT site can be inserted virtually into any place of the vector
genome by recombineering (Subheadings
3.1
and
3.2
) in
order to customize the expression locus. In this case, however,
the pO6A5-CMV should also be modifi ed, for example
sequences corresponding to the ITR and the packaging signal
should be removed from the donor plasmid used for internal
insertions.
17. To propagate electrocompetent
E. coli
DH10B plate out fro-
zen stocks of DH10B (Invitrogen) cells on LB plates and incu-
bate them overnight at 37 °C and carry out the
steps 5-11
of
Subheading
3.3
using LB without antibiotics at 37 °C for all
cultures. Alternatively custom electrocompetent DH10B
preparations can also be used. Electroporate the competent
′
