Biology Reference
In-Depth Information
the choice of tissue-specifi c promoters remains limited, and most
of the time we cannot yet achieve a knockout restricted to a specifi c
brain structure or a nucleus. The time and expense associated with
these approaches also restrict their use to laboratories that can
afford them. Furthermore, Cre-lox recombination employed in
these cases never achieves complete removal of the target protein
in all target cells, negating, to a certain extent, the biggest advan-
tage of the knockout approach. Finally, the cost of producing and
breeding even conventional knockout animals is considerable,
which effectively limits the number of strains a laboratory can gen-
erate and keep. Until recently, knockouts were produced exclu-
sively in mice, which is another limiting factor for neurobiology.
Many animal models of disease have been developed in rats and
they are not always transferable to mice. Recently, knockout rats
have been introduced, which in itself is a good idea from the bio-
logical standpoint, but the cost of breeding these animals for
experiments will certainly be prohibitive. Thus, for practical pur-
poses, knockout technology is largely limited to mice.
The virus-mediated gene knockdown, on the other hand, is a
much cheaper and faster alternative to the genetic knockout.
A virus containing a miRNA or shRNA targeting specifi c gene can
be prepared in a few weeks starting from custom miRNA/shRNA
sequence. A multitude of commercial predesigned miRNAs, pre-
made viral clones, or even packaged viruses available makes the
process even easier and faster, obviating, to a certain degree, the
need for molecular/cell biology expertise in the lab. The miRNA-
containing virus can be injected into any area of the brain provid-
ing for a structure-specifi c knockdown. By using lentiviruses as
carriers, it is possible to restrict the knockdown to neurons leaving
glia essentially unaffected. Furthermore, there are no species limi-
tations. The gene knockdown mediated by the virally delivered
miRNA can be achieved in any species for which the sequence of
the target gene is known. This is particularly benefi cial for the
studies in nonhuman primate models of human neurological dis-
eases, since it is the only way such gene manipulation can be made
today in these species. The most important downside of this tech-
nique is that the gene silencing is never complete, and the degree
of the reduction in the protein concentration in vivo can be mod-
est. Furthermore, since the infection rate is never 100 %, the pro-
tein will not be downregulated in all cells in the brain region of
interest. Although optimization of the technique may improve the
degree of knockdown, it is unlikely to reach the level achievable
with the genetic knockout. On the other hand, the range of pro-
tein manipulation produced by miRNA-based knockdown is cer-
tainly more physiologically relevant than a complete removal of a
protein of interest. A biologically signifi cant effect resulting from a
modest gene knockdown might be indicative of an important role
of the gene in the biological process of interest. On the whole, in
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