BISC 219/F10: Gene Linkage

Genetic Manipulations

Matings:
To start a mating, place 4 to 5 young adult (or L4) males on a small plate with 2 to 3 young adult (or L4) hermaphrodites. We will use plates seeded with a very small drop of OP50 bacteria for our crosses. Using only a small central area of the plate helps our worms find each other quickly and mate. The self-cross progeny produced by the hermaphrodite must be distinguished from outcross progeny. Generally, the hermaphrodite is homozygous for a visible marker and the progeny from self-crossing will show the same phenotype as the hermaphrodite, while progeny from a cross with a wild type male (outcross) will be heterozygous for the maternal marker and thus appear wild type if the mutation is recessive. When no other way is available to distinguish selfcross from outcross progeny, only males are scored. It is the outcross progeny of a mating that are of interest; essentially, all male progeny are crossprogeny. For this reason it is critical than the male transfer not be contaminated with any eggs or hermaphrodites, as they will be confused with crossprogeny.

When the male progeny from a cross are scored, remember that the parental males remain on the plate (usually 3 or 4 animals). These males will be the oldest and largest males and should not be scored among progeny phenotypes. Generally, the parental males are no longer fertile by the time the cross is scored.

In cases where male progeny are to be used for a subsequent cross, care must be taken to use the young adult males and to avoid the parental males, since only the young males will carry the marker of interest.

In cases where outcrossed hermaphrodite progeny are required, only L4 hermaphrodites are picked since adult hermaphrodite progeny will not be virgin: adults will have mated with sibling males.

Scoring Phenotypic Ratios
Since worms crawl constantly around the plate, the only reliable method of counting animals is to remove them from the plate as they are scored. Individuals can be picked up with the wire pick and incinerated in the flame. Many investigators prefer to count one phenotypic class at a time. Unless you have a very good memory it is probably best to jot down sub-totals several times while scoring a plate. In general, populations are scored after four days of incubation at 20°C. At this time virtually all F1 progeny are adults, while small larvae on the plate represent second generation progeny and are not scored.

Lab 1: Becoming a "Worm Wrangler"

Today we will:

1. Observe the video that shows examples of some mutant strains as well as showing wild-type males and larvae.
2. Learn to recognize: males, hermaphrodite larval stages, mutant phenotypes.
3. Practice moving worms from one plate to another.
4. Set up your first set of crosses to examine the inheritance differences between autosomal and X-linked mutations.