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.

Strains for making enriched or pure male samples

Jonathan Hodgkin

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.