Lab 2: Gene Linkage Finale
Complete the Lab Entry Survey
Please click on the following link and complete the lab entry survey, preferably BEFORE you come to Lab 1. The password is found in the welcome message in your 219 First Class LAB conference.
Lab Entry Survey
Your participation in this 10 minute survey will help us make adjustments to the course over the next year. Thank you!
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 team's 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 don't kill the worms when you pick the next ones after sterilizing because your pick is too hot!
To DO Today: Worm Wrangling
Each of you needs to become facile worm wranglers. The more you do today to acquire the skills needed to quickly and successfully recognize and manipulate the appropriate stage and class of worm, the more successful your experiments will be. Recognize that there is a significant learning curve to acquiring the skills you need and, please, don't be frustrated if you are slow or aren't 100% successful today at the tasks we have described below. By the end of the semester, you will be astonished at how fast and facile you are at these manipulations! The more you practice, the better your crosses will be in the next step of your work today. So take your time with this part of today's work. Getting confident and comfortable with recognizing worm stages (particularly L3 and L4's), differentiating males from hermaphrodites, and knowing which are wild type worms and which are single or double mutants are crucial. You can do it!
After you have made your worm pick, find a plate of N2 (wild type) worms for you and your partner to share.
- Identify all the stages of worm development: egg, L1, L2, L3, L4, young adult, mature adult. Pay particular attention to the L3 and L4 stages and BE SURE that you can differentiate those from more immature and from adult worms. Use the pictures of worm stages in the Worm Info section of the wiki
- In the adult and L4 worm population, identify males and differentiate them from hermaphrodites. Use the pictures of them in the Worm Info section of the wiki. BE SURE you can pick out males and L3 and L4 worms from a mixed population before you move on.
- Get a new plate of media seeded with OP50 bacteria (worm food). Practice picking worms from one plate to this new plate. It is very important not to gouge the media as the worms will burrow into it and not be easy to find later for scoring or using for other crosses. The best way to do this is to flame your worm pick, touch the bottom of the shovel end lightly to the bacterial food (it acts as glue), and then touch your pick to a worm you want to select. Move the pick over to the new media and touch lightly in the center of the new plate to remove the worm. FLAME your worm pick! Observe to see if the worm is on the new plate and moving.
- Acquire a plate of unc and dpy mutant worms for you and your partner to share. Differentiate unc & dpy single and double mutants from wild type in the adult and the late larval stages.
Now you are ready to set up some crosses and start your first investigation!
Autosomal vs. X-linked Genes
In Series 1, a brief investigation that you will start today and finish in Lab2, 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?
Series 1 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).
Background Information on Genetic Manipulations
General Instructions for Setting up and Scoring Matings:
To start a mating, you will 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. These seeded plates have been prepared for you by our hard working lab specialists. If you were working as an investigator in a worm lab, you would be growing the bacteria to use as "worm food", pouring the agar into sterile plates, and seeding the plates with the bacteria yourself. Yes, you should appreciate that this has been done for you! When setting up a mating, it is important to use only a small central area of the plate. Doing so helps our worms find each other quickly and helps insure a successful mating of the parentals. In the F1 progeny, any self-cross progeny produced by the parental 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.
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 to the cross plate.(These worms should still have the "clearing" at the center where the vulva is developing.) Because these mutant strains of worms do not move well, they tend to form piles of worms. GENTLY spread the piles out with your worm pick to find L4 worms to transfer.
- 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 if one or both are X-linked. (You will make another copy of these cross diagrams to submit as homework.)
- 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 mutant worms that are significant, if found, will be smaller than the others and look like sticks.
Take a few photos of representative worms of each strain. Make sure you have at least one good picture to use as evidence that one of the three strains has a mutation that is X-linked. Directions for using the NikonSM1500 microscope and camera can be found in the Resources section of the wiki or downloadable as a .doc file: BISC219/F10: How to Use the NikonSM1500
- When you have formed your conclusion about which strain has an X-linked mutation and whether it is the dumpy or the uncoordinated phenotype that comes from an X-linked gene, make note of your findings in your notebook and email your lab instructor with your preliminary id of the X-linked mutation (dpy or unc)and its strain name (MB1, MB2, or MB3).
- Transfer two wild-type L3 or L4 hermaphrodites from each of the plates of the strains that you have determined are NOT sex-linked (meaning the dpy and unc mutations are autosomal). We know that two of the three strains lack a sex-linked mutation and in those strains, one has linked autosomal mutations (dpy unc/+ + ) and the other has unlinked autosomal mutations: dpy/+; unc/+.
Once you have identified which of the strains has the sex-linked mutation, you need not carry it further to the F2 generation, but if you are worried that you may not have identified the sex-linked strain correctly, it is fine to transfer a couple of wild type L3 or L4 hermaphrodites from that strain to a new plate after you set up the other two strain transfers. You can always discard the x-linked strain progeny, if you don't need them when we score the automosomal strains.
- 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.
Always Check the Weekly Calendar and, most importantly, the Assignment Section of the wiki to find out what you must submit accomplish outside of lab and for Graded Homework each week. There are complete descriptions of your homework and grading rubrics in the Assignments section of the wiki that should help you.
Links to Labs& Project Info
Lab 11: RT PCR reactions
Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start
Lab 2: Sex-Linked or Autosomal Finale
Background: Classical Forward Genetics and Gene Mapping
Lab 2: Mutant Hunt
Lab 3: Linkage Test Part 1
Lab 4: Linkage Test Part 2, Mapping and Complementation
Lab 5: Mapping Part 2
Lab 6: Score & DNA sequencing analysis
Schedule of Reverse Genetics Project
RNAi General Information
Lab 5: Picking your gene to RNAi
Lab 6: Cloning your gene of interest
Lab 7: Picking your transformant
Lab 8: Plasmid purification and transformation
Lab 9: Induction of bacteria for RNAi
Lab 10: Scoring your worms and RNA purification