Model Organisms in Drug Discovery

TARGET IDENTIFICATION/TARGET VALIDATION STRATEGIES 141
(Clemens et al., 2000). With the availability of the whole genome sequence, it
is possible in cell culture to carry out genome-wide screens by RNAi and to
silence every single gene (K. Basler, personal communication). In Drosophila,
double-stranded RNA is delivered to the embryo by injection, therefore RNAi
screens have some of the same limitations as the screens for recessive
mutations in that they will only be able to detect the earliest function of a gene
during development. This problem can be overcome by generating an
inducible transgenic construct coding for the double-stranded RNA (Kennerdell
and Carthew, 2000).
The RNAi technique has been used successfully to study functionally
redundant genes. It is possible to silence simultaneously several genes by
injecting a mixture of double-stranded RNAs into a single animal. For
example, RNAi helped to identify the Wg receptors frizzled and frizzled 2
(Kennerdell and Carthew, 1998). In contrast to many other components of the
Wg pathway that were found as mutants with a Wg phenotype, the situation
for the Wg receptor was more complex because Wg has been shown to interact
with Fz and Fz2 proteins in cell culture. However, various mutations in fz
indicated that it plays no role in Wg signaling. For fz2, no mutation was
available at that time. Thus RNAi with either fz or fz2 alone had no effect, but
silencing both genes together produced embryonic defects that mimic the loss
of Wg function. This was the first demonstration that fz and fz2 act in the Wg
pathway and are functionally redundant. Here, the advantage of RNAi lays
not only in the ease with which a double mutant situation is created but also in
the fact that, by injecting double-stranded RNA into early embryos, both
maternal and zygotic mRNAs are degraded. In fact, many mutations,
particularly also in signaling pathways, do not show an embryonic phenotype
because there is sufficient maternal mRNA in the egg to support gene function
during the first 24 h of development. In the case of fz and fz2, RNAi was used
for epistasis analysis to confirm the function of these genes in the WNT
signaling pathway. Both Fz and Fz2 double-stranded RNA suppressed the
phenotype caused by overexpression of Wg but did not when the WNT
pathway was activated by the loss of GSK2/shaggy. This example
demonstrates how versatile RNAi is, even for genetically well-characterized
model organisms such as Drosophila.
After praising the method of RNAi interference, it is worth pointing out
some of its limitations. First, like all the non-genetic methods of gene
silencing, the degree to which gene function is inactivated by double-stranded
RNA (either by embryo injection or by transgene expression) is variable both
from animal to animal and within organisms. This variable penetrance and
expressivity makes it difficult to identify a consistent phenotype, particularly if
there is no clear indication of what to look for. For many genes that are
studied by RNAi, this is precisely the problem. After all, mutations in these
genes have not been identified in conventional genetic screens. Furthermore,