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Fig. 4
Testing of the miRNA clones. (
a
) The activity of predesigned miRNA sequences for arrestin3 from
Invitrogen tested in HEK293 cells cotransfected with the full-length mouse arrestin3 and the lentiviral clone
encoding miRNAs.
Left lanes
show the purifi ed arrestin3 standards, ng/lane. Cells in 24-well plates were
cotransfected with 0.2
g/well of mouse arrestin3 DNA and varying amounts of DNA of the miRNA clones
(indicated above the lanes as
μ
g DNA/well). The total amount of DNA per well was equalized by adding the
empty miRNA. The samples were collected 48-h posttransfection and analyzed by Western blot. Arrestin3
(
upper panel
) was detected with rabbit polyclonal antiarrestin3 antibody we characterized previously [
72
], GFP
(
middle panel
)—with mouse monoclonal anti-GFP antibody (Clontech), and actin (
lower panel
)—with mouse
monoclonal antiactin antibody (Millipore). The expression of GFP increases proportionally to the amount of
DNA. The level of actin serves as loading control. Note that one of the predesigned miRNA sequences, #523962,
is much more effective than the second #523693 sequence in reducing the arrestin3 expression. Also note
that when high amounts of DNA are used, negative control also caused some decrease in the arrsetin3 expres-
sion. Therefore, the range of DNA concentrations of interest is 0.05-0.2
μ
g/well. In this range, #523693 shows
minimal activity, whereas #523962 demonstrates robust knockdown of the arrestin3 expression. (
b
) The activ-
ity of the predesigned miRNA sequences for the rat G protein-coupled receptor kinase 3 (GRK3) isoform from
Invitrogen tested in HEK293 cells cotransfected with the full-length rat GRK3 (0.2
μ
μ
g DNA/well) and varying
amounts of DNA (indicated above the lanes in
or CMV promot-
ers encoding the miRNA. The experiment was conducted in 24-well plates as described in (
a
). Note a more
effective knockdown of GRK3 by the clone with the CMV promoter, which correlates with higher GFP expres-
sion. (
c
) The expression of GFP in the striatum of rats infected with the lentiviruses encoding the GRK3 miRNA
under control of EF1
μ
g DNA/lane) of the lentiviral clones with EF1
α
was injected into one hemisphere and the CMV virus—into
the other hemisphere. Striatal samples were analyzed by Western blot with mouse monoclonal anti-GFP anti-
body (Clontech). Note signifi cantly high GFP expression induced by the CMV promoter
α
or CMV promoters. The EF1
α
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