In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 250

but rules out a role of the gene of interest in this particular behav-
ior or molecular mechanism. If the infected brain area is too small
to enable knockdown detection by Western blot, immunohisto-
chemical detection of infected neurons followed by quantitative or
semiquantitative assessment of the number of such neurons in each
animal can serve as an adequate substitute.

4 Anticipated Results

An experiment employing miRNA-mediated knockdown using
lentiviral delivery is illustrated in Fig. 5 . In this study [ 68 ], a
chained miRNA construct containing two miRNA sequences com-
plementary to the N-terminal and C-terminal portions of the
kinase domain (see [ 69 ] for review of the GRK6 structure and
function) expressed under control of EF1

promoter was used to
downregulate GRK6 expression in the dopamine-depleted stria-
tum in the 6-hydroxydopamine model of Parkinson's disease. The
rat miRNA sequences were designed using Invitrogen's
BLOCK-iT™ RNAi Designer and tested in vitro in HEK293 cells
transfected with the full-length rat GRK6 as described above.
Easily transfectable Rat1 cells of rat origin were also used for test-
ing, although the expression of endogenous GRK6 in these cells
was low. All three sequences originally designed showed some
activity in cultured cells, and two most active were chained in the
viral destination vector.
The injection of miRNA lentiviruses into the brain of experi-
mental animals resulted in a widespread infection of medium spiny
neurons that can be easily detected via visualization of a cocistroni-
cally expressed EGFP marker by GFP immunohistochemistry
(Fig. 5a ). The degree of the GRK6 knockdown was determined by
Western blotting. Figure 5b, d illustrate the expression of GRK6A
and GRK6B splicing variants in the striatum injected with the neg-
ative control lentivirus in comparison with the striatum injected
with the GRK6 miRNA lentivirus. The GRK6 miRNA lentivirus
injection resulted in ~ 40 % reduction in the concentration of
GRK6A (Fig. 5c ). The knockdown rate is similar to that achieved
in another study utilizing the miRNA BLOCK-iT Lentiviral Pol II
miR RNAi expression system to knockdown RGS7 in the mouse
striatum [ 70 ]. This degree of knockdown refl ects the effi ciency of
infection as well as the effi cacy of knockdown in infected cells.
Since the virus obviously did not infect all striatal neurons, the
Western blot data underestimate the degree of knockdown achieved
in infected cells. Additionally, the concentration of another GRK6
splicing variant, GRK6B, was also reduced to the same degree
(Fig. 5e ). This is not surprising, since GRK6A and B differ in the
C terminus, whereas miRNA targeted the kinase domain identical
in both proteins. Importantly, the GRK6 knockdown achieved via

α