Target Discovery and Validation Reviews and Protocols

180 Houdebine
contribute to generate heterochromatin and to inactivate transposons and retrovirus
integrated into genomes, induce a gene silencing that is transmitted to
daughter cells. TGS should therefore be a potent way to silence endogenous or
viral genes in transgenic models.
3.2.4. Use of MicroRNAs
Plant and animal genomes contain several hundred genes coding for 120-bp
RNAs known as microRNAs. These RNAs are processed by an enzymatic complex
(Drosha). The resulting short-hairpin RNAs (shRNAs) are processed by
Dicer and act as siRNAs to degrade the targeted mRNAs or as potent translation
inhibitors if they recognize sequences in the 3′ UTR of mRNAs (Fig. 5). It
should be noted that these small RNAs act as siRNA if they perfectly match
with the targeted mRNAs and as translation inhibitors if they form several mismatches
with their target (64,91). The microRNAs play an essential role in the
control of gene expression especially during development (92,93). They
are transcribed by RNA polymerase II-dependent promoters (94), and they can
be engineered to express recombinant small RNAs activity as siRNAs or
microRNAs (95). These observations offer many possibilities to generate transgenic
models expressing siRNAs or microRNAs. Interestingly, natural
microRNAs are sometimes present in the same transcription unit. A single RNA
polymerase II-dependent vector may thus express several microRNAs targeting
different mRNAs. It is interesting to note that several companies are providing
researchers with chemical compounds or kits optimized to synthesize siRNA
and to transfect them into cells (96,97).
3.3. Inhibition of Gene Expression at the Protein Level
Antibodies theoretically offer many possibilities to inhibit protein action.
Monoclonal antibodies can be expressed in transgenic animals to create lines
resistant to pathogens (98), and intrabodies can be expressed in transgenic animals.
Their targeting in cell compartments may be necessary to optimize their
action (99). This approach, although attractive, has not been often used. The
overexpression of a transdominant negative protein acting as a decoy or as a
competitive inhibitor is a natural mechanism of protein action. This concept has
been used in some cases to create models or to generate animals resistant to diseases.
Overexpressed mutated insulin receptor traps the hormone, which cannot
bind anymore to the wild receptor. This binding allows the generation of transgenic
mice suffering from type II diabetes (100). Similarly, transgenic mice
overexpressing the soluble part of the receptor for the Aujeszky disease virus
are protected against the infection (101). Transgenic pigs could be protected as
well, and this protection could reduce the frequency of the disease. The use
of transdominant negative proteins may be easy and efficient. Conventional