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depends not only on good infection but also on the quality of the
miRNA sequence used and on suffi cient time between the virus
injection and testing to allow for protein degradation. When using
miRNA clones containing selection markers such as GFP, infected
neurons can easily be visualized. If native GFP fl uorescence is insuf-
fi ciently bright, and is often the case, immunohistochemistry with
anti-GFP antibodies can be employed. We routinely use mouse
monoclonal anti-GFP JL-8 antibody from Clontech, although
other antibodies such as Invitrogen's rabbit polyclonal and mono-
clonal anti-GFP antibodies can also be used. Immunohistochemistry
works best on the brain tissue from transcardially perfused animal.
If the brain area injected with the virus is large enough to provide
tissue for Western blot, then the degree of knockdown is best mea-
sured by Western blotting using antibodies to the protein of inter-
est. Simultaneously, the level of GFP expression is detected by
Western blot providing a measure of infection effi ciency. This is
also the best way to test for the off-target effects such as unwanted
knockdown of closely related proteins. This is a very likely possibil-
ity, since targeting only requires complementarity between the tar-
get and the seed sequence of 2-7 nt of the siRNA [ 17 ] that might
be present in other proteins. If the experiment requires knock-
down restricted to small brain nuclei, then only the detection of
infected neurons is possible but not the measurement of the actual
knockdown. In this case, selection and proper testing of miRNAs
is particularly important, since post hoc knockdown control is
not possible. It might be useful to inject larger amount of selected
miRNA-containing viruses fi rst into a larger brain area simply
for testing purposes and measure the actual degree of in vivo
knockdown.
In addition to the preexperiment testing of miRNA viruses, it
is prudent to collect the brain tissue following the large-scale
experiment and test for the actually achieved knockdown of the
target protein in each animal , by Western blot if possible. The tar-
geting of the virus injection, the infection effi cacy, and perhaps,
other factors important for the knockdown effi cacy (such as the
individual expression levels of miRNA-processing enzymes or pro-
teins involved in degradation of the target protein) vary from
animal to animal causing differences in the degree of the protein
knockdown achieved with lentivirally delivered miRNAs. Having
the data on the individual knockdown levels would undoubtedly
aid in the data interpretation, perhaps, providing the foundation
for the exclusion of some animals with minimal or undetectable
knockdown from the fi nal analysis, or allowing for the correlation
to be established between the degree of knockdown and observed
effects. This is particularly important in the situation when the
knockdown of the gene in question turns out to have no behav-
ioral or molecular effects. Otherwise, it would be impossible to
prove that the result is not due to a failure to achieve knockdown,
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