Target Discovery and Validation Reviews and Protocols

Transgenic Models 177
models, the blocking molecules (siRNAs, mRNAs, transdominant negative
proteins) should be encoded by transgenes, which can be regulated.
3.2.1. Roles of Small RNAs
Recent data show that cells contain a large number of small RNAs, which
have multiple functions: protein synthesis (tRNA and 5S rRNA), control of
chromatin structure, degradation of mRNA, inhibition of translation, control
of mRNA splicing, and other actions yet to be discovered (62).
3.2.2. Use of siRNAs to Inactivate mRNAs
siRNAs were discovered fortuitously a few years ago (63). Long doublestranded
RNAs are cleaved in 21- to 23-base pair (bp) fragments by an enzymatic
complex (Dicer). Another enzymatic complex, RNA-induced silencing
complex (RISC), targets the small RNAs to complementary mRNAs and induces
their degradation (64). In plants, this mechanism, known as posttranscriptional
gene silencing, acts as an antiviral system. Indeed, a number of viruses are in
double strand at some steps of their replication cycle. siRNAs are not encoded
by genomes. Using siRNAs implies that they are introduced into cells, which
may be achieved by adding synthetic siRNA to cells or whole organisms.
Alternatively, they can be endogenously expressed from appropriate vectors.
Biosynthesis of long double-stranded RNAs can be easily achieved with conventional
vectors by using RNA polymerase II promoters. Long doublestranded
RNAs induce interferon secretion, inhibition of protein synthesis, and
cell death in higher organisms. Long double-stranded RNAs can thus be used
in plants but not in most animals. They are used successfully in early animal
embryos because the interferon mechanism is functional at this stage. RNA
polymerase III vectors can synthesize small double-stranded RNAs at a high
rate in all cell types. Promoters from U6 and H1 genes are being used successfully
for this purpose. Synthetic DNA can direct the synthesis of a doublestranded
RNA containing a short loop between the two complementary strands
and ending in five uridines, acting as a termination signal for RNA polymerase
III. Partial degradation of the five Us generates RNAs ending with two overhanging
nucleotides required for siRNA action (64).
Gene constructs containing RNA polymerase III promoter followed by a
short sequence such as siRNA are highly expressed in cultured cells, even in
stable clones, but they are essentially silent in transgenic animals. The mechanism
of action of RNA polymerase III promoters is not fully understood, and
some important elements for in vivo expression may be lacking in the vectors
currently used. Alternative vectors are being used and studied. However, before
generating transgenic animals, it is preferable to determine which siRNAs
or microRNAs induce the most intense knockdown of the targeted gene in cell