Recombinant Adenovirus in Neurobiology - Viral Vector Approaches in Neurobiology and Brain Diseases - page 21

(continued)

The injector is then moved to desired anterior/posterior and medial/
lateral coordinates. A hole (two holes for bilateral injection) aligned
with the coordinates is drilled. After being fi lled with the dialyzed viral
solution, the injector(s) (not guide cannulas) is inserted into the brain
at the dorsal/ventral coordinate. The dialyzed adenovirus is then
injected into the brain region (usually 1

l/
min. The needle is left in the place for additional 20 min after the injec-
tion to allow the viral solution time to penetrate the surrounding tissue
and reduce the amount of solution entering needle track. The injector
is then removed and the incision is closed ( see Note 7 ).

μ l/site) with a rate of 0.5 μ

Verifi cation of the Effects of Recombinant Adenovirus

The viral infection can be verifi ed by GFP or RFP (siRNA) expression.
The recombinant adenovirus-induced alteration of gene expression can
be evaluated by western blot or functional assays such as a receptor
binding assay. In our studies, we inject virus unilaterally and collect the
GFP (or RFP)-positive region by punching the tissue out from thick
coronal cryostat cut sections of the brain ( see Note 8 ). Tissue from a
site contralateral site to the GFP-positive region is used as a control.
Alternatively, coronal sections of the brain can be collected for autora-
diography of receptor binding or immunohistochemistry assays. A time
course and a dose response examinations of the effects of recombinant
adenovirus are usually conducted to determine the optimal time for gene
manipulation and optimal injection volume of recombinant adenovirus.

3.4 Expected
Outcomes

From our experience, the viral-induced gene expression can be
observed 3 days after injection and reached a maximum 5-7 days after
the injection. Although higher injection volumes of recombinant ade-
novirus produce higher effects of gene manipulation, it also causes
higher toxic effects. We usually use 1-2 l of high-titer virus, depend-

ing on the size of the region of interest and the titer of the virus.
Using recombinant adenovirus with a sense gene sequence,
the expression of the gene of interest can be increased up to three-
to fi vefolds over endogenous protein levels (control). On the other
hand, recombinant adenovirus with antisense or siRNAs can reduce
the gene expression to more than 50 % when the tissue punched
from viral-infected area is measured relative to contralateral
control. However, in experiments in which recombinant adenovi-
ruses are bilaterally injected and tissue in the target region is col-
lected, knockdown rate for a group of animal is usually about
30-40 % relative to a control viral group. This could be due to the
variation of injection between the animals and even between both
sites of the brain regions, which masks the reduction of gene
expression induced by adenovirus ( see Note 9 ).

μ