Target Discovery and Validation Reviews and Protocols

Transgenic Models 179
Fig. 6. Vector to identify specific siRNAs. The targeted sequence is introduced before
IRES, which is followed by luciferase gene. The siRNA cleaves the target, which suppresses
expression of the luciferase gene.
complex depends on the structure and the stability of the siRNA (77–79). Recent
studies have shown that the Dicer complex generates 21- to 23-bp siRNA, but
that these active RNAs are more efficiently generated when the precursors contains
26 to 27 bp (76,80,81). The structure of efficient siRNAs cannot be completely
predicted. Moreover, the efficiency of siRNAs is dependent not only on
their structures but also of the local structure of the targeted mRNA (82). siRNAs
may generate side effects. Their sequence may recognize several sequences
unspecifically (83). The systematic search of possible multiple target sequences
in a genome must be preferably achieved to design a specific siRNA. However,
in some cases, siRNAs may induce interferon and inflammatory cytokines (84).
Some siRNA may be derived from genomic sequences. Regions of genomes
that are transcribed in the sense and antisense orientation are more numerous
than imagined thus far. This phenomenon may generate multiple siRNAs having
important functions in the control of gene expression (85,86). Transgenes
that are integrated in an uncontrolled manner also may be transcribed in both
orientations and generate functional siRNAs having unpredictable side effects.
Interestingly, recent studies showed that a RNA fragments resulting from intron
degradation might generate functional siRNAs and microRNAs (87,88). This
finding indicates that single- or double-stranded sequences targeting an mRNA
may be added to introns of RNA polymerase II-dependent vectors. This
approach offers additional possibilities to use siRNA in transgenic models.
Finally, it should be mentioned that siRNA could be generated from genomic
microRNAs.
3.2.3. Use of siRNA to Inhibit Transcription
Studies carried in yeast, plants, and vertebrates indicate that siRNAs induce
targeted modifications of histones, leading to a local DNA methylation, an inactivation
of the promoters, and silencing of the corresponding genes. This phenomenon
known as transcriptional gene silencing (TGS) also has been observed
in human cells (89,90). Interestingly, these epigenetic mechanisms, which