RNAi Methods and Screening: RNAi Based High-Throughput Genetic Interaction Screening - Caenorhabditis Elegans: Molecular Genetics and Development - page 92

II. Methods
A. Large-Scale RNAi Screening
1. Selection of Worm Strains and Gene Targets
Depending on the focus of the research and the phenotype under scrutiny, the
screening can be done in the standard laboratory C. elegans strain N2, in RNAi
hypersensitive strains such as rrf-3(pk1426) or eri-1(mg369), lin-15(n744),orin
other mutant strains. The protocol that we present in this chapter is tailored for use
with ts strains, but could be adapted to use with transgenic reporter strains to evaluate
the presence or absence of fluorescence, or to screen for genetic modifiers of the
effects of small molecules dissolved in the liquid media.
For the evaluation of genetic interactions that affect embryonic development, we
can easily differentiate wild-type (WT), embryonic lethality, and sterility in the
images that we produce. Many other phenotypes can be readily observed such as
ruptured vulva, protruding vulva, dumpy, patchy coloration, larva lethal, and larval
arrest ( Fig. 1 ).
Mutants and Temperature-Sensitive Strains
Searching for modifiers of a gene of interest is possible by comparing the results
of an RNAi screen in a mutant strain with the results in the control strain N2. Using
hypomorphic or conditional alleles, it is possible to evaluate a gene ' s effect on
lethality through the RNAi knock-down of a second gene. Our lab focuses on
screening modifiers of genes that play a role in early embryonic development. We
use ts strains that allow us to do two types of screens at the same time: one looking for
an increase in lethality (enhancement) when the screen is performed at a semi-
permissive temperature, and one looking for a reduction in lethality (suppression)
when the screen is performed at a semi-restrictive temperature.
Temperature Sensitivity Curves
Each ts mutant allele shows specific rates of lethality at varying temperatures. The
strain(s) of interest must be tested at a minimum of four different temperatures to
determine the best allele to use for the screen (if more than one is available) and to
calculate the semi-permissive (less than 20% lethality) and semi-restrictive (more
than 80% lethality) temperatures ( Fig. 2 A). In our lab we begin by testing 15, 20,
22.5, and 25 C, because even different alleles of the same gene could demonstrate
different temperature sensitivities ( Fig. 2 B).
The procedure for determining temperature sensitivity is as follows:
1. Pick and plate L4s. For each mutant strain, collect at least 20 L4s on a medium
(60 mm 15 mm) NGM plate seeded with OP50. From this plate, transfer five
worms, 1 worm per well (labeled A1-A5), to each of four 12-well NGM plates
seeded with 20 m L OP50 bacteria per well.