Using Viral Vectors to Study the Memory Trace in Mice - Viral Vector Approaches in Neurobiology and Brain Diseases - page 136

3.1.1 Overview of HSV-1
Gene Delivery System

The system developed by Dr. Neve is based on the determination
that a minimal number of sequences, namely an origin of DNA
replication and a packaging site, are suffi cient for a DNA plasmid
to be replicated and packaged into HSV particles using appropriate
proteins that are supplied by a “helper” virus (strain 5dl1.2 ). The
amplicon contains no other viral genes and the 5dl1.2 helper virus
itself is replication defective due to a deletion in the gene for
ICP27. Therefore, each component individually is unable to form
a replication competent virus and can only combine to form a virus
for gene delivery when they are in a cell type that provides the
missing ICP27 protein (e.g., Vero-derived “2-2” cell line).
Recombinant viruses are singly infectious and replication defective
in all cell types that lack the HSV ICP27 gene. Expression of the
gene of interest is driven by the endogenous viral promoter, IE4/5,
or any other mammalian promoter that can be cloned into the
amplicon plasmid.

Amplicon plasmid DNA containing the gene of interest (e.g., GFP
fused to CREB or GFP alone) is transfected into 2-2 cells. One
day after transfection, 5dl1.2 helper virus is added and, after another
30-35 h, cells are subjected to osmotic lysis and the supernatant
(containing recombinant and helper virus) is collected. The super-
natant is then used to infect a fresh batch of 2-2 cells, thereby
amplifying the virus stock as well as reducing the concentration of
helper virus. This will be repeated twice more before collecting the
fi nal stock that will be subjected to a sucrose gradient and high-
speed centrifugation to concentrate and further purify the stock of
recombinant virus. The fi nal virus-containing solution is divided
into single use aliquots (5

3.1.2 Methodology for
Generating HSV Particles
for Gene Delivery

l) and stored at −80 °C, where they are
stable for at least 1 year. For infusion, aliquots can be thawed on ice
and kept there while the animal is being prepared.

μ

A typical HSV preparation as described above should yield any-
where from 10 7 to 10 9 infectious units per ml. For amplicon prepa-
rations containing fl uorescent genes such as GFP, titering can be
done in vitro by simply infecting cultured cells (e.g., HEK 293
cells) with increasing dilutions of virus and counting the numbers
of fl uorescent cells 24 h after infection. Titers less than 10 7 /ml
should not be used for behavioral experiments.
An important factor in obtaining a high-quality virus prepara-
tion is the amount of contaminating helper virus. Although we
have not observed any adverse effects of helper virus in our experi-
ments, as measured by comparing behavioral phenotypes of virus-
injected animals with wild-type controls as well as examining
neuronal morphology in infected areas of the brain, putative effects
of excessive amounts of helper virus cannot be discounted and
therefore helper virus titers in the fi nal preparation should be mea-
sured as described [ 6 ]. Since the generation time of helper viruses

3.1.3 Critical Factors in
Generating High-Quality
Preparations