In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 247

with Invitrogen miRNAs shows that all predesigned as well as
custom-designed miRNAs demonstrate some activity in cultured
cells, but the degree of knockdown is quite variable. Our strategy
has been to select two most active miRNA and chain them. This
invariably increases the degree of knockdown.
Additionally, when using the Invitrogen BLOCK-iT™
HiPerform™ Lentiviral Pol II miR RNAi Expression Systems, a
choice of the promoter has to be made. Even if the planned experi-
ment does not involve the use of cell-specifi c promoter, since the
system is promoterless, either CMV or EF1 has to be chosen. The

choice of the promoter will have an effect on the silencing effi cacy.
We have successfully used EF1

α

to knockdown G protein-coupled
receptor kinase (GRK) GRK6 isoform in the rat brain [ 68 ]. The
comparison of the CMV and EF1

α

promoters to silence another
GRK isoform, GRK3, demonstrated higher EGFP expression and
higher knockdown effi cacy with the CMV in cultured cells (Fig. 4b )
and higher GFP expression in vivo (Fig. 4c ). High EGFP expres-
sion is convenient for detecting infected cells both in vitro and in
vivo, and since EGFP expression in cocistronic with miRNA, the
amount of expressed EGFP correlates with the amount of pro-
duced pre-miRNA and, supposedly, knockdown effi cacy. However,
EF1

α

might offer advantages in long-term experiments and/or in
cells where CMV is likely to be silenced.

α

3.2 Preparing the
Virus for the Injection
into the Brain

After selecting the active miRNA sequences, the next step is the
virus production. The virus production and purifi cation procedure
for lentiviruses containing miRNA is the same as for lentiviruses
encoding proteins for ectopic expression. Many companies (Open
Biosystems, Invitrogen, Biosettia, and Systems Biosciences) now
sell not only prepared lentiviral vectors with the miRNA already
cloned in but also premade and purifi ed lentiviruses. The lentivirus
is produced by cotransfecting the packaging cells with the lentiviral
vector encoding miRNA with the packaging mix containing two or
three plasmids encoding viral proteins needed for packaging of
infectious viral particles. Forty-eight to seventy-two hours after
transfection, the viral supernatant containing lentiviral particles is
collected. It is possible to collect the virus-containing supernatant
twice, fi rst at 48 h, then add fresh media, and collect again at 72 h.
Such procedure will increase not only the total yield of viral parti-
cles but also the total supernatant volume to be dealt with. The
supernatant should be cleared by fi ltering through 0.45 m fi lter

(we use Millex-HV, Millipore) and then can be used to determine
the viral titer or to infect cultured cells.
For injection of the virus into the brain, the virus needs to be
purifi ed and concentrated. The most common method to purify/
concentrate the lentivirus is by ultracentrifugation. We routinely
do so by sequential centrifugation at 90,000 g, fi rst using Beckman
Optima L-90 K ultracentrifuge (SW-32Ti rotor) and then Optima

μ