Target Discovery and Validation Reviews and Protocols

132 Matsumoto, Miyagishi, and Taira
Fig. 1. Schematic of the mechanism of RNA interference.
efficiently induce the cleavage of corresponding mRNAs (7). Since then, RNAi
has received attention as a breakthrough technology for silencing specific genes
in mammalian cells. However, the efficiency of transfection of synthesized
siRNAs into human cells depends on cell types. Moreover, the RNAi effect
seems to be sustained only for short time (8). To overcome this limitation, several
groups have developed vectors encoding hairpin RNAs, leading to the
expression of siRNA in cells and sustained gene silencing (9–14). Applications
of RNAi-based screens were initially applied to Caenorhabditis elegans
(15,16). Similarly, some recent articles highlight the potential of siRNA libraries
for target discovery (17–34). We have screened siRNA library to identify components
of the dsRNA-induced apoptotic pathway. The siRNA library contains
700 vectors targeting 241 genes directly or indirectly involved in apoptosis.
2. Materials
2.1. Construction of siRNA Expression Vectors
1. pcPUR hU6 vector (iGENE Therapeutic, Inc., Tsukuba, Japan, http://www.iGENEtherapeutics.co.jp).
This plasmid contains the human U6 promoter and BspMI
cloning sites (Fig. 2; [11]).