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into gonads of young adult hermaphrodites to obtain progeny with mutant pheno-
types. Essentially, RNAi can be used, instead of a mutagen, for variously designed
forward genetic screens already discussed. RNAi-mediated forward genetic screens
have some unique advantages. For instance, the identity of genes whose inactivation
causes phenotypes is immediately known, in contrast to the time-consuming cloning
of mutation-harboring genes from forward mutagenesis screens. RNAi also has
temporal flexibility. It can be applied to animals at different developmental stages
to avoid embryonic or larval lethality caused by inactivation of corresponding genes
at the early developmental stages. Moreover, RNAi usually results in reduction of
function of gene activity rather than complete loss, which allows effective investi-
gation of the roles of essential genes (
Kemphues, 2005
).
There are currently two RNAi feeding libraries available for C. elegans research.
One library constructed by the Vidal lab has 11,511 clones containing full-length
gene cDNAs that were cloned into a double T7 vector by the Gateway cloning
method (
Rual et al., 2004
). This library is commercially named the C. elegans
ORF-RNAi Collection V1.1, available through Open Biosystems. The other library
was constructed by the Ahringer lab and has 16,757 clones containing the genomic
sequences of genes (
Fraser et al., 2000; Kamath et al., 2003
). This library is
commercially available through Geneservice. With the availability of two RNAi
libraries, which together target 94% of C. elegans genes (
Ahringer, 2006
), RNAi
screens are often carried out at a genome-wide scale and more frequently in an
automated high-throughput fashion. For example, to identify genetic interactors with
the RTK/Ras/MAPK pathway,
Lehner et al. (2006)
performed an RNAi screen in the
background of loss-of-function mutations in the 12 known components of the RTK/
Ras/MAPK pathway (
Kamath et al., 2003
). To perform this screen in a high-
throughput manner, the RNAi was delivered in 96-well plates in which mutants
were soaked in a liquid containing RNAi feeding bacteria. They screened for RNAi
clones that produced synthetic lethality with any of these 12 known components.
Notably, 16 genes that had no previously reported roles in RTK/Ras/MAPK signal-
ing were found to genetically interact with two or more components of the pathway.
Nine out of these 16 genes were shown to regulate RTK/Ras/MAPK signaling during
vulval induction (see
Fig. 1
), perhaps the best-characterized function of this pathway.
This study highlights how high-throughput functional genomic approaches can
rapidly identify new components of a specific pathway.
The growing number of large-scale RNAi studies carried out in C. elegans have
produced a wealth of RNAi-induced phenotypic information. This information is
being deposited into online databases, such as Wormbase (
Harris et al., 2010; Rogers
et al., 2008
), RNAiDB (
Gunsalus et al., 2004
), and PhenoBank (
Sonnichsen et al.,
2005
), to facilitate gene function studies. For example, using these databases, our
group narrowed our search for genetic regulators of anchor cell invasion in a
sequential RNAi screen (
Matus et al., 2010
). Anchor cell invasion through basement
membranes, which mediates formation of uterine-vulval attachment in C. elegans,
has been used as a simple in vivo model for investigating cell invasion (
Sherwood,
2006; Sherwood et al., 2005; Ziel et al., 2009
). As a failure of anchor cell invasion
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