In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 243

Finally, the lentiviral vector contains all the elements necessary
for the expression of miRNA/marker in mammalian cells, so it can
be simply transfected into cultured cells. This is quite useful for
testing the effi ciency of miRNAs and marker expression before
making the lentivirus. In addition to plasmids containing prede-
signed miRNAs or required for the miRNA subcloning, all com-
mercial lentiviral miRNA systems contain plasmids encoding viral
genes required for the packaging of viral particles and some also
include packaging cells modifi ed to stably express some of these
genes or otherwise modifi ed to enhance virus production. The
viral genes are distributed between 3 and 4 separate plasmids. This
is a safety measure standard in later generations of lentiviral systems
to prevent recombination and the possibility of production of
replication-competent virus. Obviously, there is a practical limit as
to the number of plasmids used, since they need to be cotrans-
fected into packaging cells with high effi ciency that drops with
increasing number of plasmids. Most current lentiviral systems use
three plasmids in addition to the lentiviral clone containing miRNA
(referred to as “four-plasmid system”). In this respect, the miRNA-
containing viruses are not different from lentiviruses used to over-
express proteins.

2.2 Lentiviral System
for miRNA Expression
from Invitrogen

The lentiviral miRNA system from Invitrogen deserves further dis-
cussion, because it offers the most fl exibility. Currently, Invitrogen
sells two miRNA lentiviral kits, the older BLOCK-iT™ and newer
BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression
Systems. Both systems come with or without cocistronic expres-
sion of EGFP (Emerald GFP) marker. The systems are similar in all
essential points. The major advantage of the HiPerform system is
the ability to choose a promoter between CMV and EF1

included
in the kit or even to incorporate any Pol II promoter selected by
the experimenter for tissue- or cell-specifi c miRNA expression.
The traditional BLOCK-iT™ is CMV based. Additionally, the
HiPerform lentiviral destination vector includes a mRNA stabiliz-
ing sequence (WPRE) and a nuclear import sequence (cPPT) that
help to increase the virus titers and improve virus-driven protein
expression in many cell lines. The miRNA cassette is assembled in
the pcDNA-based expression clone containing multiple cloning
sites and EGFP (when this marker is used) fl anked by attB1 and
attB2 recombination sites. The multiple cloning sites are arranged
in a way allowing for cloning of multiple miRNA sequences in a
row, or “chaining” (Fig. 3 ). MiRNAs for different regions of the
same gene can be chained to increase the knockdown effi ciency.
Alternatively, miRNA targeting different genes can be chained to
achieve simultaneous knockdown of multiple genes in the same
cell. The promoter, CMV or EF1

α

fl anked by attL4 and attR1
recombination sites, is supplied by a separate plasmid, pENTR™5

α

.
The promoter and EGFP-miRNA are transferred into the

′