Recombinant Adenovirus in Neurobiology - Viral Vector Approaches in Neurobiology and Brain Diseases - page 15

that cell type. Alternatively, the shuttle vectors without the CMV
promoter allow for the addition of a cell-type-selective promoter
and, thus, express the gene of interest in specifi c type of cells. We
have successfully used promoters for 5-HT 1A receptors, 5-HT 2C
receptors, and GDA67 to control the expression of the gene of
interest in select neurons [ 5 , 6 ]. The drawback to the approach is
that the cell-type-selective promoters are usually less effi cient
resulting in low expression levels of the gene of interest.
Two approaches can be used to reduce gene expression,
antisense sequences and siRNA. Luo et al. [ 15 ] developed vectors
for identifi cation and insertion of siRNA for a gene of interest,
which can be then be easily used for recombination with the
AdEasy1 system. Since siRNA only binds to its target mRNA, it
selectively knocks down the expression of the gene of interest.
However, because not all of the siRNA candidates identifi ed by
software are able to knockdown gene expression, candidate siR-
NAs can be tested fi rst using SOS-HUS vectors with ex vivo sys-
tems. Moreover, it is necessary to generate a few siRNA viruses
[ 2 - 4 ] to ensure knockdown in vivo. Additionally, mismatch con-
trol sequences for the siRNA need to be prepared, resulting in the
need for generation of four to eight recombinant adenoviruses for
the knockdown one gene. On the other hand, the antisense approach
may be less selective, especially, if the CMV promoter is used. Since,
the specifi c antisense sequence that inhibits the gene expression is
usually not available, we used full sequence of coding region con-
trolled by cell-type-selective promoter. In this case, an adenovirus
with shuttle vector alone was used as a control. Thus, the antisense
approach requires less work for development of the virus. From
our experience, the effi ciency of knockdown is not signifi cantly dif-
ferent between the antisense and siRNA approaches.

As Fig. 1 shows, the procedure for generation of recombinant
adenovirus includes cloning the gene of interest, constructing a
shuttle vector and recombining the shuttle vector into an AdEasy1
vector. The detailed protocol for the procedures can be found in
several resources [ 12 - 14 ]. In this chapter, we briefl y discuss these
procedures.

3.2 Generation
of Recombinant
Adenovirus

Cloning sequences of interest gene : since all of the techniques used in
this step are common molecular techniques, multiple approaches
can be used to obtain the clones. Here, we describe a common
procedure to clone sequences required.

3.2.1 Constructing
Shuttle Vectors

Constructing pShuttle- and
pAdTrack-Based Shuttle
Vectors 1. Amplifi cation of the sequence using PCR: the coding region of

the gene of interest can be amplifi ed from cDNA, whereas the
promoter region should be amplifi ed from DNA. Several tips
for primer design are listed in Note 2 .