Dissection of Genetic Pathways in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 130

biological processes often have no observable phenotypes when individually
mutated ( Wagner, 2000 ). Although an enhancer screen can overcome some redun-
dancy, this approach does not always exhaust its multiple layers. RNAi-mediated
forward genetic screens can also bypass substantial redundancy and lethality by
causing partial loss of gene function; however, RNAi has its own limitations. RNAi
phenotypes are often variable in penetrance and RNAi is ineffective in neurons
( Tavernarakis et al., 2000; Tewari et al., 2004 ). To find genes that are not easily
identified through forward genetic screens, functional genomic and systems-level
approaches that complement conventional genetic screens can be used.
B. Functional Genomic Approaches to Identify Components of Pathways
The completion of sequencing C. elegans, D. Melanogaster, and H. sapiens
genomes along with rapidly evolving high-throughput techniques have changed
the methodological ways biologists study gene function and dissect genetic path-
ways. The genome-sequencing project in C. elegans revealed a significant number of
novel genes with unknown function. Undoubtedly, these genes are involved in a wide
variety of biological functions. To decipher their function, traditional forward
genetic screening still remains useful but now functional genomic studies can also
be utilized. Functional genomics uses high-throughput approaches, such as genome-
wide RNAi, DNA microarray, Serial Analysis of Gene Expression (SAGE), cis-
regulatory analysis, yeast-two-hybrid/yeast-one-hybrid techniques, and mass spec-
trometry, to acquire information about genome-wide patterns of gene expression,
protein-DNA interactions, and protein-protein interactions. By analyzing this infor-
mation, biologists can begin to elucidate the organization and regulation of genetic
pathways at a global level. Nevertheless, during analysis, experimental validation
using conventional single-gene genetic approaches, including genetic perturbation
analysis and reverse genetic approaches, is indispensable for confirming results
obtained from functional genomic studies. The combination of functional genomic
approaches with conventional methods has emerged as an effective way to gain a
more complete understanding of the gene networks that guide biological processes
(see also reviews by Grant and Wilkinson, 2003; Kim, 2001; Piano et al., 2006 ).
1. Genome-Wide RNAi Screens
RNAi is an endogenous cellular process during which double-stranded RNA
(dsRNA) complementary to sequences of target messenger RNAs (mRNA) mediates
degradation of these mRNAs, resulting in reduction of expression of corresponding
genes ( Boutros and Ahringer, 2008 ). Since RNAi was discovered ( Fire et al., 1998 ),
it has rapidly been adopted as an experimental means to silence expression of genes
in a range of organisms ( Boutros and Ahringer, 2008; Gilsdorf et al., 2010 ). In
C. elegans, RNAi assays can be conveniently carried out by feeding worms with
bacteria expressing dsRNA constructs ( Ahringer, 2006 ), soaking them in nematode
growth media containing these bacteria ( Lehner et al., 2006 ), or injecting dsRNA