Biology Reference
In-Depth Information
cells by 10 ng of purifi ed Ad-BAC using 2,500 V, 200 W, and
25
F in Gene Pulser (or equivalent electroporation
equipment).
18. To propagate electrocompetent
E. coli
DH10B carrying pBA5-
FRT plate out frozen stocks on LB plates containing 25
μ
g/
mL chloramphenicol and incubate them at 37 °C overnight or
pick a colony from the plated retransformation (
step 2
) and
carry out the
steps 5-11
of Subheading
3.3
using LB contain-
ing 25
μ
g/mL chloramphenicol at 37 °C for all cultures.
19. The pCP20 Flp expression plasmid is available from the cor-
responding author upon request. Do not co-transform the
Ad-BAC with the pCP20 because the pCP20 also contains a
chloramphenicol resistance marker. Always introduce the
Ad-BAC fi rst and overtransform this intermediate with pCP20.
20. pCP20 is maintained in
E. coli
by a temperature sensitive origin
of replication, which is only active at lower culture tempera-
tures (30-33 °C) and expresses the Flp recombinase under the
control of the temperature sensitive lambda
cI857
repressor,
which is induced at higher temperature (42-43 °C). This
design provides a very useful conditional Flp expression sys-
tem, which can be maintained in suppressed state (30-33 °C)
and as soon as it is induced (42-43 °C) the plasmid is also
automatically cleared from
E. coli
[
13
]. Alternatively, custom
electrocompetent Flp ready pBA5-FRT preparations (Sirion
Biotech) can also be used. In this case proceed with
step 13
.
21. Insertion of donor plasmid to the BAC acceptor by this Flp/
FTR system is very effi cient and results in virtually 100 % posi-
tive clones. Sometimes even two copies of the donor plasmid
are inserted into the FRT site of the BAC acceptor. If the
donor plasmid is inserted into the genome end (like in the
pO6A5-CMV/pBA5-FRT approach described here) this does
not harm the rescue because the extra copy is simply cut out by
Pac
I activation of the Ad-BAC for rescue (
see
Subheading
3.4
and Fig.
2a
). However, if internal acceptor FRT sites are tar-
geted, care should be taken to sort out double insertions. This
requires an appropriate restriction analysis of 6-12 clones,
which can be carried out after small-scale BAC DNA prepara-
tions. If two single insertions are identifi ed, the procedure can
continue with the
step 16
of Subheading
3.3
as described.
22. 293 cells as well as other E1 complementing cells can be used
for reconstitution of BACs derived from species C and some
serotypes from other species [
1
,
4
-
6
]. 293 cells are robust and
we recommend using them because endotoxin load may be
higher than usual after BAC DNA preparations propagation of
high copy plasmids. Special complementing cell line may be
required for reconstitution of some BAC-derived Ad from
other species [
3
].
μ
