Biology Reference
In-Depth Information
2.4 Titration
of Recombinant
Adenovirus Using
Anti-Ad/Hexon
Staining
1. Dulbecco's Modifi ed Eagle's Medium (DMEM) supplemented
with 2 % fetal bovine serum (FBS) (PAA, ref. A15-151).
2. Primary Antibody anti-hexon 2Hx-2 from ATCC or similar
antibodies.
3. FITC or Alexa488-conjugated secondary antibody.
4. Fluorescence microscope.
2.5 Quantifi cation of
Adenovirus Particles
by Spectrophotometry
1. Lysis Solution: 0.1 % SDS, 1 mM EDTA in 10 mM Tris-Cl,
pH 7.4.
2. Heat block.
3. Spectrophotometer and cuvettes.
3
Methods
Cloning a gene of interest by homologous recombination in bac-
teria is a two-step procedure. In the fi rst step, the gene of interest
is cloned into a shuttle plasmid vector using standard molecular
biology methods. The shuttle plasmid contains two fragments of
adenovirus genomic sequence (usually 4-5 kb from the 5
end)
fl anking the multicloning site. The presence and orientation of the
gene of interest is confi rmed by restriction digestion analysis and/
or sequence analysis. The second step consists in introducing the
gene of interest into the adenovirus genome by homologous
recombination between the shuttle plasmid and a large backbone
plasmid. This backbone plasmid contains most of the adenovirus
genome, but lacks essential genes for virus propagation, usually, E1
genes. Rapid detection of positive recombinants is achieved by
antibiotic selection and restriction digestion analysis.
′
3.1 Adenovirus
Construction
by Homologous
Recombination
in Bacteria
In this protocol, the recombination between the shuttle plasmid
and the adenovirus genome contained in the backbone plasmid is
performed in the
E. coli
strain BJ5183. Positive recombinants are
selected by resistance to an antibiotic (
see
Fig.
1
).
1. Linearize the backbone plasmid (i.e., pKP1.4 [
7
,
9
] or similar)
with a restriction enzyme cutting in the insertion site, such as
Swa
I. Digestion should be made in two steps: fi rst, digest
1.5
3.1.1 Homologous
Recombination in Bacteria
g plasmid with 10 U of
Swa
I for 24 h. Then, add 10 U
more of
Swa
I and digest during seven additional hours
(
see
Note 1
).
2. Check background by transforming BJ5183 bacteria with
100 ng of digested plasmid. If the number of colonies obtained
is higher than fi ve, repeat
steps 1
and
2
. After verifi cation,
store in 200
μ
μ
L aliquots.
