Biology Reference
In-Depth Information
3. Purify the DNA band using a commercially available gel extrac-
tion kit (
see
Note 1
).
4. Clone the purifi ed PCR product into a plasmid, which does
not contain the restriction sites that will be used in future clon-
ing steps. For cloning steps, we recommend the use of
Strataclone Blunt PCR cloning kit.
5. Sequence the entire fragment to confi rm it does not contain
any mutations.
The penton shuttle plasmid is ready for genetic manipulation
to incorporate specifi c modifi cations.
In order to introduce the relevant DNA point mutation (5
′
GAC
3.1.2 Incorporation
of Point Mutations
into the Penton Gene
3
) to encode a RG
D
-RG
E
mutation within the Ad5
penton base protein, that effi ciently ablates interactions with
α
′
—5
′
GAA 3
′
3/5 integrins, we perform PCR based site-directed
mutagenesis.
v
β
1. Using the penton base shuttle plasmid as the backbone, per-
form separate PCR reactions using oligonucleotide 3 and 6,
and oligonucleotides 4 and 5.
2. Gel purify resultant fragments using commercially available
kits.
3. Using equimolar amounts of the purifi ed PCR products,
amplify the full 1.3 Kb
Sex
AI-
Asc
I fragment, and sub clone
back into the penton shuttle vector.
4. Sequence the insert to confi rm presence of the correct modifi -
cation, and check no other mutations have been introduced.
5. Linearize the penton shuttle vector using
Eco
RI, and the Ad5
backbone cosmid with
Pme
I.
6. Perform homologous recombination to rescue the full Ad
genome incorporating the desired mutation in the penton base
protein, using BJ5183 electroporation competent cells, and
purify genome using commercially available kits.
7. Rescue recombinant adenoviral vectors using permissive cells
(e.g. 293 cells) as described previously (
see
Notes 2
and
3
).
The adenovirus hexon protein, the most abundant of the capsid
proteins, once considered to have only a passive, structural role in
virus structure and function, has been of renewed and signifi cant
interest in recent years as roles have emerged for both liver
targeting, through interplay with blood coagulation factors, and
immunogenicity, through the interaction with neutralizing anti-
bodies. Therefore, strategies for modulation of the exposed hyper-
variable regions (HVRs) are likely to be important both for
reducing the host mediated innate anti viral response, as well as
enabling rational targeting strategies through elimination of
3.1.3 Generation of an
Ad5 Hexon Shuttle Plasmid
