Lab 2: Gene Linkage Finale
To start a mating, place 4 to 5 young adult (or L4) males on a plate with 4 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.
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Lab 1: Becoming a "Worm Wrangler"
Today we will:
- Observe the video that shows examples of some mutant strains as well as showing wild-type males and larvae.
- Learn to recognize: males, hermaphrodite larval stages, mutant phenotypes.
- Make your own worm pick
- Practice moving worms from one plate to another.
- Set up your first set of crosses to examine the inheritance differences between autosomal and X-linked mutations.
Making a Worm Pick
You will buy from your lab instructor (cost $10) a small piece of ridiculously expensive platinum wire to make the worm pick you will use this semester. You must keep track of your worm pick and mend it yourself if it breaks. If you return the wire to us at the end of the semester, you will be refunded your $10.
Directions for Making and Repairing a Worm Pick
Making/repairing a worm pick is a fairly easy task. It involves the expensive piece of platinum wire, a very cheap glass pipette and heat (flame). The key here is NOT to burn yourself making the pick - the glass and the wire become VERY HOT - you melt the glass to the platinum.
- Pay your $10 and receive your wire and glass pipette.
- Using tweezers insert about 3-4 millimeters of wire into the small hole at the end of the pipette.
- Hold the region where the wire and pipette meet over the flame until the glass melts to the wire and seals the open end completely. This should take less than a minute. WARNING - the wire and glass are VERY HOT at this point.
- After a minute or so gently tug the wire with tweezers to be sure it is attached to the pipette.
- Wrap a piece of your teams tape around the top end of your new worm pick.
- Write your name and lab day on the tape.
- With a diamond pencil (see instructor) flatten the tip of your worm pick wire into a "shovel" shape. Ask for help if you need it.
- Flame the end of your pick and happy worm farming!
If your wire happens to fall out of the pick or become loose during the semester, which is likely, just repeat the above steps with a new glass pipette.
Why does the wire have to be platinum? Platinum heats up and cools off very quickly which means you sterilize the tip easily between picking and you dont kill the worms when you pick the next ones after sterilizing because your pick is too hot!
Autosomal vs. X-linked Genes
In this experiment you will perform a number of crosses to illustrate important genetic concepts. Recessive X-linked mutations are expressed differently in males (C. elegans males are XO) versus females or hermaphrodites (XX). WHY? Think about hemizygosity. In the case of C. elegans, when a hermaphrodite is mated with wild type males, the male progeny receive their single X chromosome from the maternal parent. If the X chromosome carries a mutation, all male progeny will express the maternal mutant phenotype, while the crossprogeny hermaphrodites will be phenotypically wild type. Recessive autosomal mutations, on the other hand, are not expressed in any of the F1 progeny. Why not?
This exercise will also show that linked genes segregate differently than unlinked genes; that is, unlinked genes should give us expected ratios for dihybrid inheritance (such as 9:3:3:1 or testcross 1:1:1:1), whereas linked genes will produce ratios that depart from those of dihybrid inheritance.
To test these concepts, you will cross wild-type males with three different dumpy and uncoordinated strains (Dpy Unc). The Dpy and Unc mutations are autosomal and linked (on the same chromosome) in one strain, autosomal and unlinked (on different chromosomes) in another strain, and in the third strain, one of the mutations is autosomal and the other is X-linked. Your task is to sort these strains out, which will be accomplished by making a series of double heterozygotes and examining the progeny from the heterozygotes (self-progeny).
To Do in Lab Today (per group):
- Examine the four plates: MB1, MB2, MB3 and wild type males. In your notebook record phenotypic information about these worms. Drawings may help!
- Transfer 3-4 L4 or young adult males onto each of 3 cross plates.
- Label the cross plate using your GREEN Sharpie. Make sure the labeling on each plate is on the bottom or the side (NOT THE LID) with the cross scheme: wild type ♂ X (strain name) hermaphrodite (H)
- Add 4 or 5 L4 mutant hermaphrodites (these worms should still have the "clearing" at the center where the vulva is developing) to the cross plate. These strains of worms do not move well so they tend to form piles of worms. GENTLY spread the piles out with your worm pick to find the L4 worms.
- Repeat for each of the other 2 mutant strains.
- Put an elastic around the three plates.
- Put the plates in your group's plastic box. Make sure the box is labeled with your team color, your names, and your lab section.
- Put the box in the incubator on your lab section's designated shelf.
- Incubate your crosses at 23°C for 3 days.
You will end up with three crosses of wild type males with different mutants (refer to the introductory comments on crossing) - remember that it is essential that the ONLY wild type animals on the cross plates are males.
To Do Outside of Lab on the 3rd day
- Before examining your cross progeny, in your lab notebook create a diagram of each cross with the phenotype expected for the F1 progeny (both hermaphrodites and males) if your mutations are autosomal or X-linked.
- Examine the F1 progeny of each cross. Do you have cross progeny (phenotypically wild type) that signifies your cross was successful? If so, look for the presence of males. The hallmark of a successful cross is 30-50% male progeny. These should be wild type unless the Dpy or Unc mutation is X-linked. HINT the worms that you are looking for seem to be smaller than the others and look like sticks.
- When you have made your conclusion about which strain is X-linked and which gene it is make note of it in your notebook.
- Transfer two wild-type L3 or L4 hermaphrodites each from of the three plates containing autosomal Dpy Unc’s (remember that the mutations are unlinked in two cases; i.e., dpy/+; unc/+; and linked; i.e., dpy unc/+ + in one).
- To guarantee that all progeny are self progeny, make certain that you have transferred only L4 hermaphrodite animals - after you transfer, make certain that there are no other animals on the plate.
- Incubate at 23°C until your next class period.