Biology Reference
In-Depth Information
DNA prepared from early passage isolates providing a fast access to
molecular clones of virtually any Ad strain or variant including
mutants from the past or isolates with a high risk of changes induced
by adaptation to tissue culture [
1
,
4
]. As BAC technology appeared
to be very useful for fast and correct manipulation of Ad genomes,
most of the applications were adapted (back) to Ad5 as well [
5
-
7
].
Here we present the basic methodology to manipulate and
construct Ad-BACs by a modifi ed recombineering, which is one of
the most advanced technologies available for manipulating
recombinant DNA in
E. coli
by homologous recombination.
Recombineering was described as a two-step approach [
8
], in
which homologous recombination is mediated by the red system
of bacteriophage lambda and mutant selection is facilitated by the
galK
marker [
8
,
9
]. We slightly modifi ed this protocol combining
the
galK
marker with an antibiotic selection [
3
,
10
], which made
the procedure simpler for viral BACs (
see
Fig.
1a
). Since use and
also construction of recombinant Ad become an everyday protocol
in many molecular biology laboratories, we also included a simpli-
fi ed BAC-based protocol to construct fi rst generation recombinant
Ad vectors in a single step by Flp recombination. This protocol was
adapted from the originally described approach for construction
recombinant herpesviruses [
2
,
11
,
12
] and working for Ad5 BACs
with virtually 100 % effi ciency. The gene of interest is fi rst cloned
into a small donor plasmid with standard cloning techniques and
this construct is unifi ed with a BAC acceptor in
E. coli
by condi-
tional expression of Flp recombinase via their FRT sites [
11
,
13
].
2
Materials
1. Prepare LB medium, LB plates, and M9 medium according to
Sambrook and Russell [
14
]. Add 25
2.1 Bacteriological
Media for Propagation
and Mutagenesis
of BACs
μ
g/mL chloramphenicol
(for selection of BAC plasmid), 50
g/mL ampicillin (for
selection of pCP20 plasmid), and 25
μ
μ
g/mL kanamycin
(for selection of inserted markers).
2. 5× M36 salts: dissolve 10 g (NH
4
)
2
SO
4
, 68 g KH
2
PO
4
, 2.5 mg
FeSO
4
·7H
2
O in 900 mL deionized water and adjust the pH to
7 with KOH. Add deionized water to 1,000 mL and
autoclave.
3. MgSO
4
stock solution: prepare 1 M MgSO
4
·7H
2
O solution in
deionized water. Sterilize by fi ltration.
4.
D
-Biotin stock solution: prepare 1 mg/mL
D
-biotin solution in
deionized water. Sterilize by fi ltration.
5.
L
-Leucine stock solution: add
L
-leucine to deionized water to
10 mg/mL fi nal concentration, heat to 50 °C until leucine is
fully dissolved. Then, cool down the solution and fi lter sterilize.
