Recombinant Adenovirus in Neurobiology - Viral Vector Approaches in Neurobiology and Brain Diseases - page 16

Fig. 1 Flow chant for procedure of generating recombinant adenovirus. The left panel presents the procedure
for recombinant adenovirus containing sense or antisense sequences of the gene of interest. The right panel
presents the procedure for recombinant adenovirus with siRNA

2. The PCR products can be directly inserted into shuttle vector
after digestion with restriction enzymes. An alternative
approach is fi rst to insert the products into a PCR vector such
as TOPO pCR II. After a miniprep, correct colons can selected
by restriction digestion and sequencing. Although this
approach adds a cloning step, it provides plasmid for higher
effi cient digestion of the insert. Furthermore, most PCR
vectors contain common promoters for sequencing, so that
the insert can be sequenced at this step.

Ligation of insert into the shuttle vector : after digestion of insert and
shuttle vector with same restriction enzymes, the insert can be
ligated into the shuttle vector. When the pShuttle and pAd-track
vectors are used, a promoter and a polyA tail should also be ligated
into 5 of the target gene sequence, respectively. Confi rming

the expression of the gene of interest by transient transfection of
shuttle vectors into a cell line before further generation of recom-
binant adenovirus is recommended. As described in Note 2, the
expression of the gene of interest may require specifi c sequences,
such as noncoding regions, not only Kozak consensus sequences.

′ and 3 ′