Caenorhabditis elegans

The yeast model system has a number of limitations in its uses for human drug target discovery, including its evolutionary distance from humans and the fact that it is a single-celled organism. Thus the nematode worm, a simple metazoan with several complex organ systems, has become useful as a model for discovering types of drug targets that are not likely to be present in yeast. Caenorhabditis elegans was pioneered as a model system by Sydney Brenner for the purpose of studying development in a genetically tractable metazoan; this work was honored with the Nobel Prize in Medicine in 20 02.68 In addition to a small size (<1 mm), short generation time (3 days), and general ease of handling, nematodes have the ability to reproduce as hermaphrodites, facilitating genetic analyses, especially the production of homozygous mutants. Furthermore, the small number of somatic cells (959), optically clear body, and invariant cell lineage pattern allows for mapping of the entire set of cell divisions that go into making the adult animal, which in turn allows for easy interpretation of the cause of developmental defects in mutant strains. Worms have proven especially useful in basic research on cell-cell signaling, apoptosis, neurogenesis, and aging. In terms of target discovery, worms have been used to identify the target of a nematicide used to treat river blindness,69 to identify targets of volatile general anesthetics,70 to identify genetic modifiers of behavioral responses to alcohol,71 and to screen for small molecules that suppress muscle degeneration in a muscular dystrophy model.72

The C. elegans genome was the first completed sequence of a multicellular organism; approximately one-third of its 20 000 genes have close homologs in humans.73 Many of the genomics resources available in the yeast system are also available in worms, publicly accessible from a central database website called wormbase.74 Resources unique to the worm system include complete anatomies of the entire set of somatic cells, as well as a complete connectivity map of the neural cells. Some of the pioneering work in developing large protein interaction maps was conducted in worms,75,76 and a consortium is in the processing of knocking out every gene.77

Perhaps the most important tool for conducting functional genomics studies in multicellular eukaryotes is RNA interference (RNAi) technology. A key advance in development of RNAi came with the observation that injection of double-stranded RNA (dsRNA) efficiently and specifically knocked down cognate mRNA levels in worms.78 Although similar gene silencing phenomena had been previously described in plants and filamentous fungi, this was the first indication that the mechanism might involve enzymatic processes and that a generalized gene knockdown technology based in RNA interference could be developed for metazoans. Subsequent studies revealed a common mechanism for RNAi in eukaryotes: long dsRNA is processed into 21-23 bp siRNAby the RNase Ill-like enzyme Dicer. These siRNAs are recognized by the RNA-induced silencing complex (RISC), which interacts with the homologous mRNA resulting in its degradation and thus downregulation of the respective protein.79

Technological advances in the study of worms improved delivery of the interfering RNA; worms can either be soaked in a solution of dsRNA or simply fed bacteria overexpressing target dsRNA to cause efficient mRNA knockdown.80,81 The so-called feeding libraries are a very cost-effective and reproducible means of standardizing the RNAi platform, as the bacterial clones can be frozen and expanded at will. This type of library was used for some of the first large-scale RNAi screens in any organism, wherein basic growth, reproduction, morphology, and behavior phenotypes were assessed after application of dsRNAs covering approximately 90% of the genome.82,83 A compendium of the phenotype data from these and other functional screens is maintained at the wormbase website.74 Since worms have been studied extensively by classical genetics, this system allows for a rigorous assessment of the false negative rate in RNAi studies; unfortunately, for about 30% of known essential genes and about 60% of genes with known postembryonic developmental phenotypes, the dsRNA feeding approach gave no observable phenotype.83 This problem should eventually be alleviated by enhancements of RNAi efficacy, such as using a mutant strain that is hypersensitive to RNAi-mediated knockdown.84

One example of the power of whole-genome functional screens for target identification using feeding libraries comes in the area of obesity research; by using a live stain for fat droplets, Nile red, genes involved in storage and mobilization of fat reserves could be identified by RNAi.85 From a whole-genome feeding library, dsRNAs targeting approximately 300 genes exhibited a significant and specific fat deposition phenotype in this assay. Further testing of these dsRNA clones on mutants defective in known players of fat metabolism, such as the insulin and serotonin signaling pathways, allowed for pathway mapping of the targets and resulted in identification of potentially novel targets that affect fat regulation independently of the known pathways. This type of mixed approach, using genomics technologies to identify novel potential targets and using mutants derived from classical genetics studies to validate and characterize them, is extremely rapid and cost-effective in nematodes.

Was this article helpful?

0 0
Delicious Diabetic Recipes

Delicious Diabetic Recipes

This brilliant guide will teach you how to cook all those delicious recipes for people who have diabetes.

Get My Free Ebook

Post a comment