Development of Cell-Type-Specific Viral Vectors to Tease Apart the Neural Circuitry that Contributes to Drug Addiction - Viral Vector Approaches in Neurobiology and Brain Diseases - page 153

Fig. 1 Neuropeptide promoters provide long-lasting viral-mediated gene expression in HSV viral vectors. Green
fl uorescent protein (GFP) expression in the striatum is strong at 10 days ( left panel ) and 21 days ( right panel )
postviral infusion when the enkephalin promoter is used to drive gene expression. We have noted that at least
three times more GFP-positive neurons are observed when we perform immunohistochemistry for the GFP
rather than visualize direct fl uorescence; however, direct GFP fl uorescence is convenient and suffi cient for
confi rming the anatomical accuracy of transgene expression in experiments. Scale bars represent 50 μ m

even be preferable to heavy overexpression with viral promoters in
many cases.
Once the viral vectors are packaged and suffi cient titers have
been obtained (at least 10 8 infective units/ml for HSV), it is next
necessary to confi rm that the viral vectors are cell-type specifi c.
Prior to in vivo work, experiments can be conducted in cell lines
that use the promoter of interest as well as those that do not (these
provide a negative control). For these experiments, cells are
infected with a virus that expresses either a fl uorescent protein
under your promoter of interest (such as GFP) or a tagged trans-
gene (such as a hemagglutinin [HA]-tag). If gene expression is
only driven by your promoter of interest, then the fl uorescent pro-
tein will be visualized in cells that normally express genes that use
that promoter but not in the negative control cell line. If a viral
vector containing a tagged transgene is used, then immunocyto-
chemistry must be performed on the infected cells. These experi-
ments can also be conducted prior to packaging of the viral vectors
using transfection methods. Cell specifi city will also need to be
confi rmed in vivo using the methods described below.
Using viral vectors offers the opportunity to utilize molecular
manipulations even in species not usually associated with transgenic
strategies (i.e., not just mice). Furthermore, it is possible to use the
same manipulation in several strains or even multiple species; how-
ever, it is necessary to confi rm that the viral vector will infect the
target cells and that promoter sequence used in the viral vector is
compatible with the desired cell type in the selected species/strain.
This is especially important when using a cell-type-specifi c pro-
moter. We often use a combination of immunohistochemistry for