RNAi Methods and Screening: RNAi Based High-Throughput Genetic Interaction Screening - Caenorhabditis Elegans: Molecular Genetics and Development - page 90

suppression of temperature-sensitive mutations. This chapter covers our entire set
of protocols, from setting up the experiment and screening schedule, to scoring
the results. The rapid acquisition of high-quality images of each experiment
allows the management of a large number of samples per screening cycle and
opens up new possibilities for quantitative scoring, computerized image analysis,
and the ability to review results independent of the time constraints that are
associated with large-scale screening.
I. Introduction: Large-Scale RNAi Screening in C. elegans
A powerful way to help decipher a gene ' s function is to disturb it and analyze the
effect that is produced. The result provides a clue about the processes in which
the gene product is required, or more generally, the response of the system to the
perturbation. Extending this idea to genome-wide analyses has given new insights
into the molecular mechanisms underlying basic cell biological processes ( Mohr
et al., 2010; Perrimon and Mathey-Prevot, 2007 ). C. elegans in particular has been
used successfully as a model animal for large-scale genetic, RNAi, and other types of
screening - including chemical genetic screens to link genes, proteins, or small
molecules with biological roles (e.g., Burns et al., 2006; Kwok et al., 2006; Min
et al., 2007 ).The reasons that C. elegans is such a good in vivo model system both are
biological and technical. C. elegans displays many developmental programs and
behaviors that are conserved across metazoans, yet is unique in that its development
is completely mapped out to single-cell resolution, so that its entire lineage of cell
fates is defined ( Sulston and Horvitz, 1977 ). On a practical, experimental level, a
number of functional genomic tools are available (i.e., described in Wormbook:
http://www.Wormbook.org ) . C. elegans can growwell within wells of a 96-well plate
( van Haaften et al., 2004 ), and it is small enough to be handled through microfluidic
devices like liquid-handling robots or Fluorescence Activated Cell Sorting machines
without damage (e.g., Ben-Yakar and Bourgeois, 2009; Ben-Yakar et al., 2009;
Doitsidou et al., 2008; Fernandez et al., 2010; Stoeckius et al., 2009 ). Combining
these features makes C. elegans a premiere animal model for high-throughput in vivo
biology.
In large-scale screening, we are now moving toward ever higher throughput. A
systematic approach that explores condition-specific genetic analysis is potentially
infinite. The simple case of reducing the function of two genes simultaneously under
laboratory conditions explores an experimental space of over 200,000,000 possible
interactions among the 20,000 genes in the C. elegans genome. Adding complexities
derived from specific alleles, from diverse genetic background, and from the possi-
bility of cell-specific interactions can expand the systematic search space dramatically.
A solution to this problem is to perform even-more complex screens that take advan-
tage of strategies that look for multiple genetic effects on phenotypes ( Rockman and
Kruglyak, 2009 ). Other approaches include exploring systematically genetic interac-
tions, as has been successfully done in yeast ( Boone et al., 2007; Costanzo et al.,2010;