Recombinant Adenovirus in Neurobiology - Viral Vector Approaches in Neurobiology and Brain Diseases - page 12

Recently, a modifi ed AdEasy system has been used to express
small interfering RNAs (siRNAs) for silencing of a gene of interest.
Luo et al. developed an AdEasy-based siRNA expression system
[ 15 ]. Using a screening vector, one can fi rst identify siRNAs that
are able to inhibit the expression of the target gene. Once siRNAs
are identifi ed, they can be inserted in a shuttle vector as a DNA
oligo-nucleotide cassette that can be recombined into AdEasy viral
vector. This AdEasy-based siRNA system provides a high-effi ciency
siRNA deliver system, especially, for in vivo studies. Furthermore,
using DNA oligonucleotides as siRNAs is much cheaper than the
cost for synthesis of siRNAs.
Since the protocol for generation of recombinant adenovirus
has been discussed extensively [ 12 - 14 ], in this chapter, we focus
on the applications with AdEasy-1-based systems in neurobiologi-
cal research.

2 Materials

2.1 Cloning System

RT-PCR can be used to amplify the sequence of the gene of inter-
est. Common cloning techniques can be used to insert the sequence
into the shuttle vector.

2.2 AdEasy-1
System

Shuttle Vectors

Shuttle vectors are used for constructing the target sequences so that it
can be recombined into adenoviral vectors. The shuttle vectors contain
two fragments of the viral backbone that are located in the both sides of
the multiple cloning site (MCS) (right and left arms). These right and left
arms facilitate the recombination of the shuttle vector into the viral vec-
tor. There are four shuttle vectors available in AdEasy-1 system: pShut-
tle, pShuttle-CMV, pAdTrack, and pAdTrack-CMV. The pAdTrack and
pAdTrack-CMV contain a GFP gene that is controlled by a CMV pro-
moter (cytomegalovirus promoter). Thus, the recombinant adenovirus
prepared with these shuttle vectors expresses GFP, which assists in visu-
alization of viral expression. The pShuttle-CMV and pAdTrack-CMV
contain a CMV promoter at 5

′ ′

of the MCS and a SV40 poly A tail at 3

of the MCS, resulting in CMV promoter-controlled expression of the
gene of interest.

AdEasy-1 Vector

The adenoviral vector is used to generate adenovirus after recombina-
tion with shuttle vector. The E1 and E3 domains of the adenovirus are
deleted in AdEasy-1. Thus, reproduction of the recombinant adenovi-
rus requires a cell line containing an E1 domain such as HEK 293 and
291cells.