Worm Breeder's Gazette 15(5): 19 (February 1, 1999)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England
For a variety of purposes, it may be desirable to generate samples
of C. elegans which are predominantly or completely composed
of males, either wild-type XO males or sexually transformed XX
males. Several strains have been constructed for this purpose,
and have been deposited at the CGC Stock Center.
1. CB4951: spe-12(hc76) I; him-8(e1489) IV. Strains carrying
him mutations, which lead to meiotic loss of X chromosomes
at a high rate, have been a traditionally convenient source of
XO males. The strongest of these is him-8(e1489), which
has a male frequency of about 37%. Combining this with spe-12,a mutation which prevents sperm activation in hermaphrodites
but not in males, creates an obligate male/female strain, for
which the XO male frequency in an exponential culture is 64%.
If desired, further enrichment of adult males can be achieved
by filtration through appropriate nylon mesh (see Lewis &
Fleming, 1995). However, selective filtration of males only works
with synchronized adult populations.
2. CB4017: fem-1(hc17ts) him-8(e1489) IV; unc-1(e1598dm)
X . This strain can be used for generating samples of pure
larval or adult XO males (Zarkower & Hodgkin, 1992). The strain
is temperature-sensitive, growing well at 15C. To make pure males,
synchronize a culture by bleaching and shift to 25C. All XX animals
will mature into Unc females. Add wild-type males to the culture,
wait 24 hours for extensive mating to occur, then collect eggs
by standard bleaching procedure. Spread the eggs in a band across
the diameter of one or more unseeded 9cm NGM plates, and place
a small drop (100 microlitres) of glacial acetic acid at the edge
of the plate, orthogonal to the band of eggs. This creates a steep
pH gradient. As the eggs hatch, larvae that can move well (patroclinous
XO males, genotype fem-1 him-8/+ +; +/O) are able to swim
away from the repellent acid, but all other progeny carry the
dominant unc-1 mutation, and are too uncoordinated to move
away from the band of eggs. The males can be collected by washing
off the half-plate distal to the acid drop, and are arrested as
starved L1 larvae, so they will commence synchronous larval growth
when provided with food. This procedure gives 99 - 100% pure larval
XO males, but it is distinctly inconvenient, and also hard to
scale up.
3. CB4689: dpy-28(y1ts) III; him-8(e1489) IV. The
y1 mutation is a temperature-sensitive allele of the dosage
compensation gene dpy-28 (Plenefisch et al., 1989), so
the strain can be grown at 15C. At restrictive temperature, most
XX animals die as embryos or young larvae, whereas the XO males
are unaffected. However, even at 25C there are some viable XX
hermaphrodites, as well as many dying XX animals, so the percentage
of males is less than 100%.
4. CB5362: tra-2(ar221ts) II ; xol-1(y9) X. The ar221 mutation is a tra-2 allele isolated by Jane Hubbard, which turns out to be an excellent temperature-sensitive mutation, much easier to work with than the classic tra-2(b202ts). The xol-1(y9) mutation enhances the masculinization of tra-2 XX males (Miller et al., 1988). At 15C, most of the population in a culture of CB5362 consists of Egl hermaphrodites, so the strain can be grown in bulk. At 25C, all animals are good phenotypic XX males, with mating behavior. At least half are fertile, capable of siring numerous progeny. The disadvantage of this strain is that the males are all XX rather than XO, although for some purposes this may be a convenient property.