Target Discovery and Validation Reviews and Protocols

138 Matsumoto, Miyagishi, and Taira
10. Add equal amount of 4X sample buffer (final concentration 2X) to the proteins
and boil at 99°C for 3 min.
11. Size fractionate the proteins (30 µg/well) on 12% sodium dodecyl sulfatepolyacrylamide
gel electrophoresis (200 V; 1 h at room temperature).
12. After electrophoresis, transfer proteins to an Immobilon-PSQ membrane (15 V;
overnight at 4°C) using Bio-Rad Mini-Protein II blotting system.
13. Block membranes with 5% skim milk for 1 h.
14. Wash the membrane with TBS-Tween three times.
15. Incubate with primary antibody in 0.5% skim milk/TBS-Tween for 1 h at room
temperature. Here, we suppose that antibodies against the identified targeted genes
are commercially available.
16. Wash the membranes with TBS-Tween three times.
17. Incubate with secondary antibody in 0.5% skim milk/TBS-Tween for 1 h at room
temperature.
18. Chemiluminescence reagents detect reactive immunoreactive complexes.
To validate the experimental design and selection assays, include some positive
and negative controls, in particular when the signaling proteins involved in the
investigated pathway are known (see Note 8). Here, we have targeted dsRNAdependent
protein kinase R (PKR). The sequences of siRNA expression vectors
targeted against two different target sites of PKR transcripts were as follows: site
1, 5′-TAA TGA ATC AAT CAA TTC ATA-3′ and site 2, 5′-AAG ACT AAC TGT
AAA TTA TGA-3′. Moreover, two negative controls also were included: NC1, targeting
firefly luciferase (5′-ATG AAA CGA TAT GGG CTG-3′) and NC2 targeting
Renilla luciferase (5′-GTA GCG CGG TGT ATT ATA CCA-3′). As shown in
Fig. 4A, both siRNA expression vectors targeting PKR suppressed expression of
endogenous PKR. Notably, inhibition of PKR inhibited dsRNA-induced apoptosis
(Fig. 4B, C). Compared with the positive control, S1 to S4 clones also inhibited
dsRNA-induced apoptosis (Fig. 4D) (see Note 9).
Notably, the construction of large siRNA expression libraries against all
human genes is a big task. These libraries should enable us to identify a number
of known and novel functional genes. Scientists are making a great first step
in functional genome analysis, and people will soon enjoy the unraveled mystery
of cells (17–35).
4. Notes
1. To identify potential off-target effects (33) and nonspecific suppressive effects of
siRNA expression vectors, or both, for each gene, at least two sites should be targeted.
Two active siRNAs against the same gene should show the same phenotype.
2. In designing the oligonucleotides, we recommend introducing multiple C-to-T (or
A-to-G) mutations in the sense strand, avoiding near the 5′-sense terminal end.
When constructing hairpin-type siRNA expression vectors, some technical problems
may occur, such as sequence difficulty and high rate of mutation in hairpin