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example, to study genes involved in executing the dopaminergic cell fate,
Doitsidou et al. (2008)
devised a worm-sorter-based screen using a transgenetic
strain with all dopaminergic neurons exclusively labeled by a cell type-specific GFP
reporter. As a failure to execute dopaminergic cell differentiation can result in fewer
GFP positive neurons, mutants with reduced GFP fluorescence were sought. To
control for the variability in fluorescence intensities among individual worms, they
introduced a broadly expressed red fluorescence protein (RFP) reporter into the
transgenic strain as an internal reference for GFP/RFP ratiometric measurements.
The worm sorter was accordingly set to detect a reduced GFP/RFP ratio. This
screening strategy was highly sensitive as it allowed the identification of mutants
lacking GFP expression in only one or two of the eight dopaminergic neurons. In
comparison with a manual screen performed in parallel, the worm-sorter-based
screen displayed a higher efficiency in isolating mutants, as
50,000 individual
worms per hour were screened - in contrast to
1000 manually screened with a
microscope. The automated screen identified 22 mutants over a few days, whereas
the manual screen isolated 10 mutants over a few months.
4. Enhancer Screens
It is estimated that only
30% of the approximately 20,000 genes encoded in the
C. elegans genome show a visible, lethal, or sterile phenotype after loss or reduction
in function (
Hodgkin and Herman, 1998; Johnsen and Baillie, 1991
). The majority of
genes are phenotypically silent upon loss of function under laboratory conditions.
Sometimes, it is because the function of these genes can be compensated either by
homologous genes with high structural and functional similarity or through buffer-
ing of regulatory networks via nonhomologous genes acting in related pathways
(
Hartman et al., 2001; Wagner, 2000
). These genes, which are thought to constitute a
large part of most genetic pathways, are unlikely to be recovered in direct simple
screens. Identifying these genes necessitates the loss of two or more genes simulta-
neously. One screening strategy to accomplish this task is the use of an enhancer
screen, which is usually conducted on a starting strain with a defined phenotypic
defect caused by a mutation in a single gene. Any gene whose functional disruption
can enhance the defects of the starting perturbation is referred to as an enhancer of
the starting mutant.
Two types of genetic outcomes are possible between enhancers and a starting
gene: (1) synergistic enhancement in which the combined severity is more than the
sum of both single mutant phenotypes; (2) additive enhancement in which the
severity of the combined defects equals to the sum of the individual defects.
The type of genetic enhancement can be informative for constructing genetic path-
ways (see also discussion in Part III).
To devise an enhancer screen, genetic nature, particularly dosage effects (loss of
function or reduction of function) of starting alleles used for enhancer screens should
be taken into careful consideration because the dosage nature of the starting alleles
affects the types of enhancer genes that can be identified.
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