Characterization of Mutants

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CHARACTERIZATION OF MUTANTS

Behavior Testing

The common mutant that all of the groups will be working with has a behavioral phenotype involving the ability to fly. During Laboratory Session 1 you will flight test 4-5 lines of flies to identify the flight-impaired line. As a homework assignment you will use the a computer tutorial to genetically map this common mutant to a location on one of the Drosophila chromosomes.

The mutants for your independent projects all have a recessive lethal phenotype and the chromosome containing the P-element insertion is paired with a sister chromosome known as a balancer chromosome. Balancer chromosomes contain large inversion mutations that effectively block recombination. The balancer chromosomes also have recessive lethal alleles with a visible dominant mutant phenotype. Recessive lethal mutations in alleles on the 2^nd chromosome are balanced over CyO, a balancer with a curly wing phenotype. 3^rd chromosome mutations are balanced over MKRS, a balancer with a stubble bristle phenotype or TM3, a balancer that carries an altered haltere¹. You may flight test your unknown mutant also but remember that the balancer phenotype probably affects flight ability. In fact, the common mutant that we are using is also recessive lethal and is usually balanced over CyO. Several weeks prior to this laboratory, flies in this mutant line were crossed with wild-type flies to remove the balancer chromosome so that accurate measures of flight ability could be obtained.

PROTOCOL FOR FLIGHT TESTING

• Place the flight-testing apparatus² upright on a table with a light source at the top.

• There are 10 flies in each vial. Hold the vial of flies to be tested in the apparatus and remove the plug.

• Flies that fly normally will emerge from the vial and fly up toward the light source, give these flies a score of 3.

• Some flies will fly but will not be able to gain altitude and will therefore fly horizontally, give these flies a score of 2.

• Some flies will attempt to fly but will not be able to maintain altitude, they will fly downward at an angle, give these flies a score of 1.

• Some flies will drop straight down when they attempt to fly, these flies are flight-less, give them a score of 0,

• Sometimes flies that cannot fly will not attempt to fly, they will emerge from the vial and walk around on the outside of the vial, you may gently shake these flies off, they will probably be flightless.

• Generate an average weighted score for each line tested. The flight-impaired mutant line should be significantly different from the other lines.

Example of Data from Flight Testing

Score	Number	Weighted score
3	3	9
2	5	10
1	2	2
0	0	0

Average weighted score = 9+10+2/10 = 2.1

• Record you data in your laboratory notebook, show your calculation. What type of statistical method could you use to determine whether one line was significantly different from the others? Two of the lines of flies that you tested have a P-element insertion. Could all of the flies in lines other than those you determined to be the common mutant fly normally (did all of these flies obtain a flight score of 3)? If not, list possible reasons for their inability to fly. One of the lines that you tested was a red-eyed wild type strain called Canton S. At the end of your testing your instructor will tell you which line this was. Could all of the flies in this group fly normally? Give answers to these questions in your laboratory notebook.

Genetic Mapping of the Common Mutant

See tutorial.

Gene Expression Pattern: lacZ Staining

During Laboratory Sessions 2 and 3 you will determine the reporter gene expression pattern in embryos, larvae and adult flies for both of your mutant lines. The X-gal staining can be done on whole mounts of embryos and larvae after the chorion or epidermal sheath has been made permeable. You will be provided with a sample of embryos and larvae³. The embryos will range in age from 4 to 24 hours AEL (after egg-laying). After 24 hours embryos should hatch from the egg chamber and emerge as first instar larvae. You should find some larvae mixed with the eggs.

In order to visualize lac Z expression in adult tissues, the fly must be sectioned. Since the heating involved in most embedding protocols inactivates proteins, the flies are frozen in a polyethylene glycol/polyvinyl-based embedding material and sectioned on a cryostat. Transverse and longitudinal sections of flies will be provided.

You will follow the protocols below to determine the lac Z expression pattern in your mutant lines. Answer the following questions as a part of your notes for this laboratory.

• Many of the mutants that you are working with have recessive lethal mutantions that are maintained over a balancer chromosome. Consequently, all of the adults will be heterozygous for the P-element. What are the possible genotypes of the embryos? Some of the embryos are expected to have no lac Z expression. Why? What is the genotype of these flies? Would you also expect any of the adults to have no lac Z expression? Why?

lac Z Staining Procedures

Embryos

You will receive an agar plate onto which adult flies from your mutant line(s) were allowed to lay eggs for 24 hours. The adult flies were then removed and the fertilized eggs were allowed to develop for an additional 4 hours. The embryos are now 4-28 hours old.

1) Collect the embryos and larvae from the agar plate by first removing the clump of yeast that is on the plate and then washing the plate with 50% bleach. Collect the embryos and larvae floating in bleach with a wide mouth plastic pipette and place them in a specimen bottle.

2) Add a couple more ml of bleach and allow the embryos to sit for 5 -10 min. This process will dechorionate the embryos.

3) Remove all but about 1 ml of the bleach using a small bore pipette. Transfer the remaining bleach, along with the embryos, to a 1.5 ml plastic tube. Centrifuge at low speed (2000-3000 RPM) to pellet the embryos and then remove the remaining bleach. To avoid sucking up the embryos use a drawn-out glass pipette to remove the final volume. Wash the embryos 3 X with dH₂O. Be careful not to remove the embryos during the removal of liquid. Pelleting the embryos helps cut down on lose during these washes.

4) Add 1 ml of n-heptane that has been saturated with formaldehyde to the specimen bottle and let the embryos fix for 1-2 hours. The embryos should turn light yellow. Whitish embryos are not well fixed and will not stain well.

5) Remove as much heptane as possible by pelleting and pipeting.

6) Wash the embryos with PBS-Triton-X 100 (PBS-T) 4-5 times. On the last wash be sure to remove all of the PBS-T.

7) Add 100 µl of X-gal staining solution (see below). Stain 1-5 days.

8) Remove the staining solution using a drawn out pipette. Rinse the embryos 3 times in PBS with Triton-X100. Remove PBS and add about 200 µl 10% glycerol. Place the embryos with glycerol on a glass slide; place a cover slip over the embryos and seal the coverslip to the slide with nail polish.

9) View the embryos under phase contrast optics. Describe what you see. Take photos of representative embryos.

Recipe for staining solution:

100 µl 50mM ferricyanide
100µl 50 mM ferrocyanide
780 µl 1X PBS

• heat the above ingredients to 55^oC

• add 2 mg X-Gal dissolved in 20µl DMSO

• keep at 55 ^oC until needed

ADULTS

In order to study the lacZ expression pattern in adults, it is necessary to prepare frozen section of adult flies and to fix and stain the tissue within a short period of time. Because even experienced researchers cannot produce usable sections on demand with 100% assurance, it will not be practical for you to prepare these materials yourselves. Therefore, you will be provided with sections of adult flies for the common mutant and for your unknown. Follow the procedure below for lac Z staining.

1) Fix the tissue on the slide in 4% paraformaldehyde in PBS for 15-20 minutes. Several staining dishes filled with fixative will be available in the front of the room. Each dish holds 10 slides - groups can share these dishes.

2) Wash the slides twice in 1X PBS for 5 minutes. Fresh staining dishes are available for these washes.

3) Prepare a humidifier chamber consisting of a plastic container lined with wet paper towel.

4) After the last wash, remove the excess PBS with paper towel, place the slide on a flat surface and apply 100µl of pre-warmed staining solution + X-gal (see above) to each slide. Place a cover slip on the slide. Place the slide in the humidifier chamber. The slides will stain until the next laboratory session.

5) At the end of the staining process, float the cover slips off of the slides by immersing them in 1X PBS for 2-5 minutes.

6) Remove the excess PBS with a paper towel. Place a drop of 70% glycerol on the slide and cover with a cover slip. Seal the cover slip to the slide with nail polish.

7) View the embryos under phase contrast optics. Describe what you see. Take photos of representative sections.

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¹There are several balancers for each chromosome. Information about other Balancer chromosomes can be found in the Abberations section of Flybase.

²This is merely a large plastic box with an arm hole cut in one side. I usually attach a plastic bag to the arm hole to prevent flies from escaping.

³To obtain eggs and larvae, place flies in a clean bottle without food and cover the bottle with a 35 mm petri plate filled with grape or apple agar. It also helps to place a small amount of baker's yeast on the plate. Turn the bottle upside down an leave overnight. Females will lay their eggs on the agar plate. Remove the plate and collect embryos. If you leave the plate for 24 hours, some of the embryos will have emerged as 1^st instar larvae.