Capsid Modification Strategies for Detargeting Adenoviral Vectors - Adenovirus: Methods and Protocols - page 49

2.1.2 Generation of an
Ad5 Penton Base Shuttle
Plasmid

We use the following oligonucleotides fl anking the penton base
gene and homologous regions. PCR of this region yields a 4.6 kb
fragment containing the entire penton base sequence, fl anked by
1.9 Kb (left) and 1 Kb (right) regions of homology from the Ad5
genome.

1. Oligonucleotide 1 (Forward): 5 ′ ATATGACGAGGACGAT

.
2. Oligonucleotide 2 (Reverse) 5

GAGTACG 3 ′

′ CGCCGTACACCTCATCA

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

TACAC 3 ′

We use the following oligonucleotides to introduce an aspartic acid
(D) to glutamic acid (E) mutation (5

2.1.3 Incorporation of
Integrin Binding Mutations ′ CGCGGCGAC 3 ′ > 5 ′

CGCGGCGAA 3 ) within the penton base RGD sequence by site

directed mutagenesis of the 1.3 Kb SexA I- Asc I fragment of the
penton shuttle vector.

′

1. Oligonucleotide 3 (penton forward) 5 ′ ACCCGTGTGTACC

.
2. Oligonucleotide 4 (penton reverse) 5

TGGTGGACAACAA 3 ′

′ TTTCACTGACGGT

.
Primers for introducing the D > E mutation (Underlined
sequences correspond to the mutation site.).
3. Oligonucleotide 5 (Forward): 5

GGTGATGGTGGG 3 ′

′ ATTCGCGGC GAA ACCT

.
4. Oligonucleotide 6 (Reverse): 5

TTGCCTG 3 ′

′ GGCAAAGGT TTC GCCGCG

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

AATGG 3 ′

We have previously described [ 25 ] the strategy we have used for
generating a hexon shuttle plasmid, where the hexon gene is ampli-
fi ed to incorporate an additional fl anking 1 Kb DNA from the
unique BamH I and AsiS I restriction sites present in the Ad5 genome,
using the oligonucleotides 5

2.1.4 Generation of Ad5
Hexon Shuttle Plasmid

′ CTCCTTATTCCACTGATCGCC 3 ′

(forward) and 5 (reverse).

Within the hexon gene is a unique Nde I site, which facilitates clon-
ing strategies.

′ ATCTGATCTCCGACAAGAGCG 3 ′

Oligonucleotides fl anking the region of interest (Ad5 region
between Nde I and BamH I).

2.1.5 Swapping HVR7
from Ad26 into the Ad5
Hexon Gene

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

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

CCAATGAAACCATGTTACGG 3 ′

′

TCGTCCATGGGATCCACC 3 ′ .