The Genetics and Cell Biology of Fertilization - Caenorhabditis Elegans: Molecular Genetics and Development - page 346

screens that focus specifically on this phenotype can identify many new fertility
genes (G. Singaravelu, D. Shakes, and A. Singson, unpublished).
The ts alleles isolated in conditional screens can be used to determine the temporal
requirements for a given protein ' s function or synthesis, and their availability can
greatly simplify genetic manipulations ( L ' Hernault et al., 1988; Putiri et al., 2004 ).
However, although their phenotypes may represent a wide range of severities, ts
alleles are rarely amorphic (null) and investigators must therefore be cautious when
relying on them to make inferences about gene functions/interactions. Null pheno-
types are best defined using molecularly verified amorphic mutants.
Reverse genetic methods may also be used to study the function of fertility genes
( Geldziler et al.,2004 ). Microarray analysis in C. elegans has helped identify numer-
ous candidates for reverse genetic approaches; 1343 spermatogenesis-enriched and
1652 oogenesis-enriched genes were initially identified ( Reinke et al.,2004 ), and
subsequent studies have built on to this original list ( Reinke and Cutter, 2009 ). Many
of these genes may function in the events of fertilization.
RNAi is often used as a quick, loss-of-function, reverse genetic approach ( Kamath
and Ahringer, 2003; Maeda et al., 2001 ) (also see chapter by Cipriani and Piano).
Injecting, feeding, or soaking worms to introduce dsRNA can systemically trigger
gene-specific silencing in both the treated animal and its F 1 progeny ( Fire et al.,
1998; Kamath and Ahringer, 2003; Timmons and Fire, 1998; Timmons et al., 2001 ).
RNAi has been successfully used to study C. elegans genes with early sperm or
oocyte development functions ( Sumiyoshi et al., 2002 ), but most C. elegans sperm
genes have proven refractory to this method for unknown reasons ( Geldziler et al.,
2004 ). Consequently, RNAi has not been a productive method for studying gene
candidates with fertilization functions, and systematic RNAi screens have undoubt-
edly failed to identify many key molecules required for spermatogenesis and sperm
function. Nevertheless, injection and soaking-based RNAi approaches may be useful
in identifying at least some egg-sterile mutants ( Kadandale et al., 2005b; Maeda
et al., 2001 ).
RNAi and similar knockdown methods are relatively easy ways to assess gene
function, but RNAi-induced phenotypes are not genetic mutations and therefore
require careful interpretation. Genes differ greatly in the extent to which they can be
functionally reduced by RNAi; RNAi phenotypes range from no effect to total loss of
function. RNAi phenotypes are also highly sensitive to experimental subtleties such
as genetic background and exposure method, time, and temperature. For instance,
the effective RNAi knockdown of the egg-1 and egg-2 genes requires injection or
soaking treatment at 25 C to see a complete fertilization defect ( Kadandale et al.,
2005b; Lee and Schedl, 2001; Maeda et al., 2001 ). Feeding RNAi yields only
incomplete knockdown phenotypes at best. Such variability can confound analyses
and makes proper controls critically important.
Since RNAi methods do not provide actual germ line mutations, other methods are
required for further genetic studies/manipulations ( Liu et al., 1999 ). Deletion library
screening is one technique used extensively in C. elegans as a powerful large-scale
method for deriving true knockout mutations in target genes known only via their