Target Discovery and Validation Reviews and Protocols

178 Houdebine
systems. Moreover, stable clones expressing the siRNA and tested individually
should be tested to evaluate properly the siRNA efficacy. Alternatively, transfection
of siRNA synthesized in vitro or chemically may be transfected with
high efficiency into cells and give a good prediction of the siRNA potency.
The U6- or H1-siRNA constructs are expressed in transgenic mice when they
are integrated into lentiviral vectors (65). A vector designed to be devoid of
introns to loose its cap and its polyA tail and containing a RNA polymerase II
promoter has been constructed. The RNA synthesized by this vector cannot
leave the nucleus. The RNA which can be a long double RNA molecule is
degraded in the nucleus and the 21–23 bp fragments migrate to the cytoplasm
where they can act as siRNAs. No long double-stranded RNA is in cytoplasm,
and the interferon response is not induced. This vector made it possible the generation
of mice in which ski gene was knocked down. Mice showed essentially
the same biological properties as those obtained after ski gene knockout (66).
A vector containing a murine CMV early gene promoter, no intron, a minimal
transcription terminator, and a region coding for a siRNA was able to generate
an active siRNA (67). A vector containing the ecdysone regulatory system
proved able to synthesize siRNA in a controlled manner (68). Vectors based on
the use of the tetracycline-dependent system may be used as well. Ideally, others
vectors dependent on RNA polymerase II promoters remain to be found.
They would offer the most flexible system to control siRNA synthesis in transgenic
animals. These vectors must express siRNA at a high level. Indeed, in
plants and in lower invertebrates such as C. elegans, siRNAs are autoamplified,
whereas this mechanism is lost in other species, such as Drosophila, and in
vertebrates (64).
Experimental data have shown that many different siRNAs can be designed
to target an mRNA, but only some of them seem active. The active siRNA
sequence can be searched empirically, by using siRNA libraries (69,70). Such
siRNA screening should be performed with appropriate vectors. The vector
shown in Fig. 6 contains the target sequence followed by IRES and a luciferase
reporter gene. The target sequence may or my not be an open reading frame.
The IRES enables ribosomes to start translation of the following cistron irrespective
of the sequence of the proceeding cistron (61). This vector can be
cotransfected plasmid coding for hairpin siRNAs. The level of luciferase in
cells should reflect the inhibitory effect of the designed siRNA. The same type
of vector may be used for screening siRNA libraries.
A systematic comparison of the active and inactive siRNAs showed that those
siRNAs are the most frequently efficient are rich in GC and rich in AU in the 5′P
and 3′OH end, respectively, of the sequence similar to that which is in the target.
A U must preferably be present in position 10 of the siRNA (71–76). Both
strands of a siRNA may target mRNAs. The choice of the strand by the RISC