Recombinant Adenovirus in Neurobiology - Viral Vector Approaches in Neurobiology and Brain Diseases - page 19

RFP positive) are observed 24 h later. The number of GFP-positive
cells is counted in the well with highest viral dilution. The titer of
the virus can be calculated as:

# of infected cells × viral dilution i nnthewell× 1 000 ,

Infective viral particles / ml =

10

3.3 Manipulation
of Gene Expression in
the Brain Using
Recombinant
Adenovirus

Once generated, the high-titer recombinant adenovirus can be
used to manipulate gene expression in vivo and ex vivo. In this
chapter, we focus on the approach to alter the expression of a
target gene in specifi c brain regions using recombinant
adenovirus.

Preparation for Adenovirus Injection

High-titer adenovirus is collected by CsCl gradient centrifugation and
stored in a high-salt glycerol solution that can cause tissue damage.
Therefore, it is necessary to remove the remaining CsCl, high salt and
glycerol before injection into the brain. On the day of injection, the
viral stock solution is dialyzed by transferring the viral stock into the
bottom of a slide-A-Lyzer mini dialysis unit (10,000 MWCO, PIERCE)
that is then placed in a fl otation device (regular vial fl oating plates can
also be used). The dialysis units are fl oated in saline (500-1,000 ml)
with slow speed stirring at 4 °C for >30 min. Collect the viral solution
(the volume may be slightly increased) into a clean vial and further
dilute 1:1 with saline. The viral solution is ready to use and is placed in
ice ( see Note 6 ).

Assembling the Stereotaxic Injector

To inject recombinant adenovirus, it is essential to control the speed of
injection so that the viral solution can diffuse into tissue and reduce the
amount that backs up into the needle track. Thus, we use a syringe
pump to control the rate of injection. An internal injector (33 gage,
C315I, Plastics One Inc., Roanoke, VA) is connected to a 25- l

Hamilton syringe with PE50 tubing. The syringe is placed on an injec-
tion pump to control the injection rate. The injector is held by a 26-gage
guide cannula with a tubing length of 2 mm below the pedestal, which
is then mounted on the stereotaxic device (Fig. 2). Before injection, the
PE50 tubing is fi lled with ddH 2 O to reduce resistant. An air bubble is
left at the injector end to separate recombinant adenovirus from
ddH 2 O. For bilateral injection, two internal injectors are held by a dou-
ble guide cannula (Fig. 2a). An alternative is to directly inject through
a 10-

μ

l Hamilton syringe that is held by a microsyringe pump with four
channel microcontroller (Fig. 2b).

μ