Model Organisms in Drug Discovery

52 C. ELEGANS FUNCTIONAL GENOMICS IN DRUG DISCOVERY
C. elegans. Fluorescence activity in the gut is proportional to food uptake or
drinking, and hence pumping frequency. The assay can be used for both
genetic analysis and to screen for compounds that effect pathways involved in
pumping. In the following, we demonstrate the use of the assay in the
identification of genetic targets in the serotonergic pathway.
Design of C. elegans genetic screens
Genetic analysis has been the preferred tool for the study of genes and
proteins for nearly a century. In classical or forward genetics, the genome of a
model organism is randomly mutagenized. Mutants that exhibit the desired
phenotype are used to discover the identity of genes responsible for producing
the phenotype. The following simple procedure highlights some specific
aspects of a C. elegans genetic screen. A typical genetic screen uses the
mutagen ethylmethanesulfonate (EMS), which induces G/C to A/T transitions
and small deletions in genes. Hermaphrodites are incubated in 50 mM EMS
for 4 h in order to accumulate 10–20 mutations per genome. After treatment,
the worms are distributed on Petri dishes and left to grow for two generations,
resulting in homozygous mutants. The F2 progeny is scored for the desired
phenotype and isolated mutants are retested for the phenotype. The strains
can be conveniently preserved for long-term storage in liquid nitrogen.
Caenorhabditis elegans hermaphrodites produce homozygous offspring, thus a
simple F2 screen for recessive mutations can be completed within only two
weeks. Owing to the high mutation frequency, such a screening campaign
requires only 10 to 20 000 haploid genomes to recover a few mutants per gene.
Therefore, the mutant strain needs to be out-crossed several times, but this
step is also rapid and can be completed in less than a month.
Devgen has used the C. elegans ‘drinking assay’ to screen for mutations in
genes that enhance pumping activity and hence they are candidate genes
associated with serotonergic signaling. A set of mutant strains has been
isolated that exhibit a positive effect in the ‘drinking assay’, as measured by a
significant increase in dye uptake. Before we illustrate the process of the
positional cloning of one of these mutants, we shall describe a few examples of
more complex genetic screens.
Genetic screens commonly lead to the identification of three categories of
genes. The first category contains the genes that contribute directly to the
biological process of interest. Pertaining to the ‘drinking assay’, this category
would include the genes that, when mutated, directly increase the serotonergic
tonus at the synapse, such as the serotonin reuptake transporter. The second
category includes those genes that influence indirectly the process of interest.
Taking our example, mutations that constitutively switch on a signal to feed
would stimulate drinking and they could be members of the dopaminergic