Biology Reference
In-Depth Information
3.1.5 Generating Ad5
Containing HVR 5
from Ad26
To generate Ad5 vectors containing the HVR5 from Ad26, we
used a similar strategy to above.
1. Using an Ad26 genome as a template, generate Ad26 HVR5
fragment (fragment 1; 69 bp) using oligonucleotides 13 and
16
2. Using the Ad5 cosmid (or hexon shuttle plasmid), amplify an
Ad5 hexon fragment (fragment 2; 116 bp) from the
Nde
I site
until HVR5 using oligonucleotides 7 and 14.
3. Using the Ad5 cosmid (or hexon shuttle plasmid), amplify an
Ad5 hexon fragment (fragment 3; 1,898 bp) from HVR5 until
the
Bam
H I site using oligonucleotides 15 and 8.
4. By PCR, fuse together fragments 1 (69 bp) and fragment 2
(116 bp) using primers 7 and 14 resulting in a fragment of
168 bp (fragment 4).
5. By PCR, fuse fragment 4 (168 bp) and fragment 3 (1,898 bp)
using oligos 7 and 8 resulting in a hexon fragment of 2,050 bp
containing the HVR5 of Ad26 (
see
Note 4
).
6. Using the restriction endonucleases
Bam
HI and
Nde
I, sub-
clone the generated fragment back into the hexon shuttle
plasmid.
7. Sequence the insert to confi rm presence of the correct modifi -
cation, and to check no other mutations have been introduced
by the PCR procedures.
8. For homologous recombination, linearize the hexon shuttle
vector using
Asi
SI, and rescue the full length genome using
BJ5183 electroporation competent cells, as described previ-
ously (
see
Note 3
).
9. Rescue recombinant adenoviral vectors using permissive cells
(e.g. 293 cells) as described previously (
see
Note 3
).
1. Denature 5 × 10
10
vp (gauged by microBCA assay for total viral
protein) of adenovirus in as small a volume as possible (ideally
less than 50
3.2 Adenoviral QC
Using Silver Staining
and NanoSight™
μ
l) in denaturing buffer by heating at 95-100 °C
for 10 min.
2. Subject denatured adenovirus to electrophoresis on a 9 % poly-
acrylamide gel for approximately 2-3 h, until the viral proteins/
ladder reach the end of the gel (
see
Note 5
).
3. Carefully remove the gel from the casing, wash with dH
2
O,
place into plastic box, and stain a commercial silver stain kit
following the manufacturer's instructions.
4. Following destaining to reveal banding pattern, compare
banding pattern with positive control to assess for any defects
in packaging of modifi ed Ad vectors compared to wild type
capsid confi guration (
see
Fig.
2
).
3.2.1 Silver Staining
