Capsid Modification Strategies for Detargeting Adenoviral Vectors - Adenovirus: Methods and Protocols - page 50

3. Oligonucleotide 9 (Ad5 Ad26 HVR7 fusion forward-1) 5 ′

.
4. Oligonucleotide 10 (Ad5 Ad26 HVR7 fusion reverse-1) 5

TGCTTTCCACTGAATGGCACTGGAACCAATTCC 3 ′

′

.
5. Oligonucleotide 11 (Ad5 Ad26 HVR7 fusion forward-2) 5

AGTGCCATTCAGTGGAAAGCAGTAATTTGG 3 ′

′

.
6. Oligonucleotide 12 (Ad5 Ad26 HVR7 fusion reverse-2) 5

ACAAAACCAAATAAGAGTTGGAAATAATTTTGCC 3 ′

′

.
7. Oligonucleotides for sequencing (1 every 400 nucleotides).

AACTCTTATTTGGTTTTGTCTAGAAATTGCATCG 3 ′

1. Oligonucleotide 7 (Ad5 hexon forward Nde I) 5 ′

2.1.6 Swapping HVR5
from Ad26 into the Ad5
Hexon Gene

.
2. Oligonucleotide 8 (Ad5 hexon reverse BamH I) 5

CCAATGAAACCATGTTACGG 3 ′

′

.
3. Oligonucleotide 13 (Ad5 Ad26 HVR5 fusion forward-1) 5

TCGTCCATGGGATCCACC 3 ′

′

.
4. Oligonucleotide 14 (Ad5 Ad26 HVR5 fusion reverse-1) 5

CAATTTTTCGACGTCCCTGGCGGAAGTCC 3 ′

′

.
5. Oligonucleotide 15 (Ad5 Ad26 HVR5 fusion forward-2) 5

AGGGACGTCGAAAAATTGCATTTCCAC 3 ′

′

.
6. Oligonucleotide 16 (Ad5 Ad26 HVR5 fusion reverse-2) 5

AATACAAACCTAAAGTGGTATTGTACAGTG 3 ′

′

.
7. Oligonucleotides for sequencing (1 every 400 nucleotides).

CCACTTTAGGTTTGTATTCTTCCCCACTACC 3 ′

2.2 Additional QC
Checks for Adenoviral
Integrity Validation

Silver staining allows high resolution and rapid detection of both
core and capsid proteins within an adenovirus preparation, and has
the advantage of much greater sensitivity compared to Coomassie
staining. We have found that this procedure can often inform of
capsid packaging defi ciencies, where specifi c genetic alterations of
the capsid prove incompatible with assembly. We recommend
using the Pierce Silver Stain Kit, which gives good, reproducible
band staining.

2.2.1 Silver Staining

1. Gel Loading Buffer II (Life Technologies).
2. Denatured Adenovirus—typically we boil 5 × 10 10 viral parti-
cles for 10 min before subjecting to electrophoresis on a 9 %
SDS polyacrylamide gel.
3. Silver staining kit—Pierce Silver Stain Kit (Pierce, part #
24612).

We have recently begun employing NanoSight™ technology within
our lab for direct imaging of viral particles to establish viral prep
monodispersity and concentration (for details see http://www.
nanosight.co.uk ). Our NanoSight™ is the LM14 model.

2.2.2 Adenovirus
Concentration and
Monodispersity as
Monitored Using
NanoSight™