In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 241

Lentiviruses tend to yield modest levels of transgene expres-
sion while effectively transducing neurons. This makes the
lentivirus-mediated expression of shRNA or miRNA an optimal
choice for silencing genes of interest in the brain of living animals.
The in vivo knockdown technique is challenging and is still rarely
used, although it is a powerful tool for the study of the gene role
in the normal brain function and in neural diseases. This chapter
provides a detailed up-to-date description of the use of lentiviral
vectors to achieve miRNA-mediated gene silencing in the brain of
living animals.

2 Material and Commercial Sources

The virus-mediated miRNA knockdown in vivo experiment
requires active miRNA sequence(s) cloned into an appropriate viral
vector suitable for the production of infectious lentiviral particles.
Predesigned and validated miRNA as well as native for many
human or mouse genes as along with lentiviral vectors adapted for
miRNA expression are available commercially from many sources
such as Invitrogen, Open Biosystems, and Systems Biosciences.
Several less known other companies, such as Biosettia, ABM, and
Capital Biosciences, also offer lentiviral vectors for miRNA clon-
ing. Invitrogen was the fi rst company to offer the miRNA-based
lentiviral knockdown technology, and it still offers the widest
choice of miRNAs and lentiviral vector systems to suit various
experimental needs.
The basic miRNA lentiviral vector drives the miRNA transcrip-
tion by RNA polymerase II under control of CMV promoter
(Fig. 3 ). An alternative choice of the promoter is EF1

2.1 Composition
of the Basic Lentiviral
Vector

(translation
elongation factor alpha) that also drives RNA polymerase
II-dependent transcription. This is a mammalian promoter (human
is often used) that, unlike viral promoters such as CMV or SV40
that can be silenced in mammalian cells over time, ensures long-
term expression. Most vectors contain a selection marker allowing
for labeling of infected cells in the brain. The most frequent choice
is some version of GFP, although other fl uorescent markers such as
RFP/Puro fusion are also used. In some commercial vectors, GFP
or other marker is expressed under the control of the same pro-
moter that drives the miRNA expression (one promoter system). As
a result, GFP is actually transcribed as a part of pri-miRNA (Fig. 3 ).
In the Invitrogen's vectors, GFP is located immediately down-
stream of the promoter before the miRNA. MiRNA is removed by
Drosha during miRNA processing, liberating GFP mRNA for
translation initiation at 5

α

end. Alternatively, one promoter system
can include a marker under control of IRES (internal ribosomal
entry site) after miRNA. In this case, both miRNA and the marker
are transcribed as pri-miRNA, miRNA is excised by Drosha, and

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