In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 248

TLX tabletop ultracentrifuge. The resulting pellet is resuspended
in sterile saline giving ~1,000-fold concentration over the original
supernatant. The viral suspension is divided into aliquots suffi cient
to inject one animal and stored at −80 °C until used for the injec-
tion into the brain. Several companies (Millipore and Cell Biolabs)
now sell lentivirus purifi cation kits that rely on virus-binding resin
and dialysis rather than ultracentrifugation to purify and concen-
trate lentiviruses.

To study the effect of silencing of the gene of interest, purifi ed and
concentrated lentiviruses containing miRNAs of choice, as well as
negative control miRNA, are injected in the brain using standard
stereotaxic surgery technique as other lentiviruses. Following sur-
gery, the animals are allowed to recover for a period of time before
their behavior of other experimental measure can be assessed.
Lentiviruses are known to induce rapid protein expression, so the
expression of EGFP is detectable 3-5 days postsurgery. However,
to downregulate an endogenous protein a longer period of time
might be necessary. Indeed, miRNAs induce mRNA degradation
and/or translational suppression, but the protein that has already
been produced still remains. Obviously, the time required to
achieve a measurable protein downregulation depends on the pro-
tein stability: the longer the protein's half-life, the longer time is
needed to decrease its concentration in the brain following the
miRNA expression. We previously employed 5-day postinjection
waiting period to achieve measurable GRK6 downregulation [ 68 ],
but GRK6 appears to be a short-lived protein. With more stable
proteins, longer times may be required. Now, we routinely wait
10-day postinjection before assessing the effects of the miRNA-
mediated knockdown.
It is very useful to evaluate the degree of the miRNA-mediated
knockdown in living animals before embarking on a large-scale
experiment. We have found that although testing in cell culture
predicts well relative activities of miRNA sequences, the absolute
degree of knockdown in vivo can only be evaluated in vivo.
Furthermore, the timing issue (how long to wait after the virus
injection) can only be worked out in vivo, since cultured cells
operate on a totally different time scale. Finally, likely off-target
effects are also best evaluated in the target brain tissue rather than
in cultured cells that may not express the proteins closely related to
the one targeted. It is important to compare the effects of active
miRNAs with that of the negative control miRNA in vivo.
When evaluating the effectiveness of the miRNA-containing
virus, two elements must be considered, the number of infected
neurons and degree of reduction in the concentration of the pro-
tein of interest. These parameters are not entirely independent, of
course, but they do need to be considered separately for correct
troubleshooting. The effectiveness of the protein knockdown

3.3 Detecting
Infected Cells in the
Brain and Evaluating
the Degree of
Knockdown