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sequence (
Jansen et al., 1997
). Pools of mutagenized worms are split for long-term
viable freezing and DNA extraction. The DNA pools are then screened for deletions
in the gene of interest via PCR and populations containing such deletions are
sequentially subdivided to isolate a pure strain homozygous for the deletion. Such
a strategy was used to demonstrate an essential role for various genes in fertilization
(
Kadandale et al., 2005b; Parry et al., 2009; Xu and Sternberg, 2003
). New methods
for gene knockout based on DNA transposition will also be important for generating
fertility gene mutations (see chapter by Robert and Bessereau).
III. Maintaining Sterile Mutants
Once sterility mutations have been generated and identified it becomes necessary to
maintain them. Nonconditional recessive spe hermaphrodites cannot be maintained as
self-fertile homozygotes, but the mutant chromosome can be propagated by crossing
spe hermaphrodites with wild-type males. These crosses are referred to in the literature
variously as ''rescue,'' propagation, complementation, or maintenance crosses (
Fig. 5
).
Since these maintenance crosses are nothing more than standard genetic backcrosses
(
Fig. 5
B), many spe mutant strains have been cleared of extraneous mutations.
Nonconditional egg-sterile mutants cannot be complemented by mating, however,
and must be maintained using labor-intensive ''sib selection'' (
Fig. 5D
) prior to genetic
balancing (see below); the mutation of interest is recovered from a heterozygous
sibling of the homozygous sterile mutant. Since sibling heterozygotes are often
phenotypically wild-type, worms must be individually plated and followed for the
segregation of sterile progeny to assure the correct genotype and maintain the strain.
This must be done with each generation.
Because fertile males are required to maintain sperm-sterile mutants as described
above, a ready supply is essential. In C. elegans, males naturally arise at too low a
frequency (1 in 1000, due to nondisjunction of the X chromosome) to be useful for
this purpose (although heat shocking hermaphrodites at 25-30
C for
>
6 h will
increase this frequency) (
Hodgkin, 1999
). Consequently, many researchers keep
daily matings of wild-type worms (which generate 50% male progeny) to ensure a
sufficient quantity (
Fig. 5A
). Alternatively, specific him (high incidence of males)
strains may be used (
Hodgkin, 1997
). These mutants show increased X chromosome
loss and therefore generate many males. him-3, him-5, and him-8 do not affect
spermatogenesis and are routinely used for this purpose (
Nelson and Ward, 1980;
Zannoni et al., 2003
). They do affect chromosome number and ploidy, however, so
their use must be monitored to ensure they are not affecting the original function/
process-of-interest. Although these are the most widely used methods for obtaining
males, other methods such as heat-shock, ethanol treatment, and RNAi against him
genes can generate males for crossing (
Fay, 2006; Hodgkin, 1999
).
Maintaining sterile strains as heterozygotes is tedious and requires manual selec-
tion, making it impractical for large-scale operations. Fortunately, a variety of
genetic balancers allow mutants of interest to be maintained and propagated without
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