In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 253

the viral batch. The EGFP expression is indicative of the con-
centration of infectious viral particles in a batch. From previous
experience, we know how infectious the virus should be to
allow for high infection effi cacy in the brain. If a virus batch
does not produce a required level of EGFP expression in vitro,
we consider it not good enough for the brain injection.
3. Storage of the virus is an important consideration. We prefer
to use freshly prepared viral batches with minimal storage
time, although when the lentivirus is stored at −80 °C without
been thawed, it preserves its activity for many months. We
aliquot the concentrated virus into single-use aliquots, so the
virus does not undergo multiple freeze-thaw cycles.
4. The second common reason for low level of infection in the
brain is poor virus injection technique. The brain injection
requires highly concentrated viral preparation that can become
quite viscous. We practice a very slow injection technique and
leave the needle in place for at least 10 min allowing the viral
particles to spread from the injection site. If the miRNA virus
is equipped with a marker, it is important to evaluate the level
of infection in preliminary experiments by immunohistochem-
istry to adjust the technique, the targeting, or the injected
volume, if necessary.
5. There is also a possibility that the time between the virus injec-
tion and testing was insuffi cient for the protein concentration
to measurably decrease. However, unless a protein of interest
is unusually stable, a good result should be achieved with
5-10-day postinjection delay.

6 Conclusions

The repertoire of miRNAs and lentiviral vectors available today
commercially and from different laboratories is broad enough for
the use of gene silencing mediated by lentivirally delivered miRNA
in a variety of experimental situations. Loss of function is a stan-
dard approach to elucidate the functional role of a protein, and it
has been and is widely employed in in vitro experiments in cultured
cells. However, in vivo loss-of-function is mostly used in the form
of gene knockout. Gene knockout is a powerful technique because
it affords complete elimination of the protein of interest, but it has
its drawbacks. The most obvious one is the loss of the gene from
conception and consequent developmental effects that are not
always possible to separate from the acute functional effects.
Additionally, removal of a gene in development sometimes results
in embryonic lethality. Another downside is the loss of the gene in
all tissues and cells in the organism. These problems are obviated
with the use of conditional and tissue-specifi c knockouts. However,