Arranzado Mirah Mae Transgenic Flies FINALS

Bicol University MA Bio Ed 211
GRADUATE SCHOOL Advanced Genetics

Legazpi City 1st Semester 2021-2022
MIRAH MAE D. ARRANZADO Dr. Maria Eden A. Ante

2nd year, MABioEd Student Professor
TRANSGENIC FLY VIRTUAL LAB WORKSHEET

INTRODUCTION
Go to http://www.hhmi.org/biointeractive/transgenic-fly-virtual-lab. Start the Virtual Lab and maximize
the screen if you wish. Answer the following questions in the spaces provided.
TRANSGENIC FLY LAB INTRODUCTION
Read the Transgenic Fly Lab Introduction. Answer the following questions in the spaces provided.
1. What is the purpose of this virtual lab? What are transgenic organisms used for in research?
This virtual lab provides a virtual interactive lab space where you can explore the techniques used
to make transgenic flies and to be able to describe how RNA is used to produce transgenic
organisms, explain how these organisms are used to execute the biological processes like the
circadian rhythms and its response under different conditions. The transgenic organism used is a
Drosophila fruit fly
2. Click on "more on experimental design." What gene will you be studying as a graduate student in this
lab? The period gene that controls the circadian rhythm in fruit flies.
3. What is the role of a promoter (regulatory region) of a gene?
The promoter is located upstream of the gene which is the site of transcription.
4. What occurs when the appropriate signals turn on the promoter?

When appropriate signals turn on the promoter, then transcription is initiated converting DNA to
mRNA.
5. What is the role of a reporter gene?

The reporter gene is indicator and will report the activity of the gene under study to which they are
linked.
6. Why is the luciferase gene such a popularly used reporter gene in experiments?
Luciferase has a half-life of 2-4 hours, convenient enough to be used as a reporter gene to study
biological processes in a day or in a short period of time.
7. Won't it be interesting to turn an ordinary fruit fly into a fly that glows? However, the light produced
has no practical function for your fly. What does the light from this transgenic organism demonstrate to
you?
The light from these transgenic organisms will monitor and will serve as an indicator of the activity
of the period gene on the molecular clock of the flies.
8. In summary, transgenic per luc fruit flies will produce light in a pattern that reflects the
transcriptional activity of the period gene.
9. List the basic steps and substeps of the lab procedure:

1. Preparing a transgenic fly
 Prepare DNA that will be incorporated in the fly genome.
 Prepare fly embryos
 Inject fly embryos with DNA
 Breed flies
 Select transgenic progeny
 Examine light output from transgenic adults.
2. Use of transgenic fly to study circadian rhythm and genetics.
 Measure per luc expression under light-dark conditions.
 Examine different body parts for per luc expression.
10. What does the period gene normally control for the fly?
Period gene controls the molecular clock or the circadian rhythm of the fruit fly.
11. What can a mutation in the period gene alter?

Mutation on period gene can alter the normal oscillation period of 24 hours.
12. In this lab, the period gene is linked to the luciferase gene. Why? What does this elegant model allow
us to do?
The luciferase gene will serve as the indicator in monitoring the changes of molecular clock of he
flies, so when period gene is “on” light is produced on the cells where period gene transcription is
occurring.
Click to Enter the Lab. (Click the window on the left-hand side of the screen to enter the lab.) Follow the
instructions in the lab (left-hand window) and answer the following questions pertaining to each part from
the information Notebook window on the right.
PART 1: PREPARE DNA THAT WILL BE INCORPORATED INTO THE FLY GENOME
1. What is "construct DNA"?
Construct DNA is a process of preparing a DNA that is to be inserted to the fruit fly genome. It
includes incorporating target segments of a particular trait.
Click on "learn more about the technical details of DNA preparation."

2. Where is the promoter of the period gene?
The promoter of the period gene is located adjacent to the DNA sequence coding for luciferase
gene.
3. If the construct DNA's promoter is activated, what will occur and what will be produced?
The activation of the promoter in the promoter sequence at the right time will result in the
transcription and translation of luciferase protein to produce luciferase enzyme.
4. How are DNA sequences "put together" in the laboratory?

DNA sequence are put together in the laboratory using restriction enzymes and ligases as cutting
and pasting tools.
5. The construct DNA contains a gene that confers the red eye trait. What is the significance of this
"clever device" built into the construct DNA? What does it enable us to check for?
The red eye will indicate the success of incorporating the construct gene to the the fly genome.
Normal fruit flies has white eyes, so if the new fruit fly has red eyes it means the construct gene was
successfully incorporated in its genome.
6. The embryos normally develop white eyes. If the embryos incorporate the new DNA, they will pass the
gene on to their progeny. What color eyes will the progeny have?
Since the segment of the construct DNA confers red eyes, so the progeny will express the red color
for the eye.
At this point, if you wish to perform a BLAST sequence analysis of the construct DNA, click on the
appropriate link. (This is not a required portion of the virtual lab.)
Move through the steps in the virtual lab window on the left. Be sure to watch the virtual lab animation
and follow the instructions.
7. After being filled, what does the glass needle contain?

The glass needle contains the construct DNA and the vector that carries the transposase enzyme.
8. What is the purpose of the pump to which the glass needle is attached?
The pump regulates the amount of DNA solution injected to the embryo, to make sure the only the
right amount of DNA can be embedded and incorporated to the fruit fly.
Be sure to answer all virtual quiz questions as they appear on your screen. Submit them for grading. Look
over your results, and review the questions that were answered incorrectly.
PART 2: PREPARE EMBRYOS FOR INJECTION
1. Why is it important to use embryos that are less than 30 minutes old?
Embryos to be used should be less than 30 minutes old to make sure that the cells that will become
the germ cell are not yet differentiated and can still incorporate new DNA.
2. What is a germ cell? Where are germ cells found in fly embryos?
Germ cells are the future egg and sperms found on the posterior portion of the fly.
3. If the DNA integrates properly into the germ cells, the adult fly will contain the germ cells that contain
the construct DNA. After mating, these flies can then produce progeny that will contain the construct
DNA.
Click and review the lab equipment used in this experiment. Be sure to proceed through the virtual lab in
the window on the left.
Answer all virtual quiz questions as they appear on your screen. Submit them for grading. Look over your
results, and review the questions that were answered incorrectly.
PART 3: INJECT FLY EMBRYOS WITH DNA

1. The DNA is injected into which end of the embryo? Posterior end of the embryos.
2. Explain why embryo survival rates are so low in this type of experiment. (Include all reasons.)
This part of the experiment requires skill and patience because these embryos may dry or die out if
the procedure takes too long. If injection is not handled properly it may cause rupture to the
embryonic membrane and cytoplasm can leak out, fluid loss can damage the embryos. Injecting too
little DNA solution means small chance of having desired progeny, while too much may cause
embryos to explode. At the end of the experiment only 10% of the initial numbers of embryo
survives, since mature embryos are destroyed.
3. What are "transformant progeny"?

Transformant progeny are those offspring which successfully incorporated the construct DNA
thereby expressing desired traits.
Be sure to proceed through the virtual lab in the window on the left.
4. How much time does it take for the embryos to reach adulthood?
It takes at least 7 days for the flies to reach adulthood.
Be sure to answer all virtual quiz questions as they appear on your screen. Submit them for
grading. Look over your results, and review the questions that were answered incorrectly
PART 4: BREED FLIES
1. The adult flies are white eyed. Does this mean there are no transgenic flies?
No, adult have white eyes it is their germ cells that contains the transgenic gene, so it is their
offspring which will express the desired trait of the gene,
2. Your task now is to mate these adults with noninjected white-eyed flies. What do you expect from this
mating?
Some but not all of the offspring will contain and express the per luc DNA. So some may have red
or white eyes.
3. What do you expect all of their cells to contain? What color will their eyes be?
Since the are the progeny containing the construct DNA expressing the gens that codes for the red
eyes, so their eyes are colored red.
4. What occurs in the vial during the first two to three days?
The 1st 2 to 3 days is the mating of the injected flies with noninjected flies, and settling of embryos
on the apgar ready for collection.
PART 5: SELECT TRANSGENIC PROGENY

1. When looking at the progeny, what is the visible marker?
Red eyes are the visible marker of transgenic flies.
2. What does the CO2 do to the flies?
Carbon dioxide is used to anesthetize the flies, to render them unconscious whule undergoing
observation under the microscope.
3. Did your experiment work? YES If you answered "No,” answer question #4 and 5 and then proceed to
Part 6.
4. What are four reasons why the experiment might have failed?
a. ________________________________________________________________________________
b. ________________________________________________________________________________
c. ________________________________________________________________________________
d. ________________________________________________________________________________
5. None of your flies had red eyes. The ability to troubleshoot an experiment is a vital skill in research.
What are a few possible explanations of what went wrong?
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
_____________________________________________________________________________________
PART 6: EXAMINE LIGHT OUTPUT FROM TRANSGENIC ADULTS
1. What was your original hypothesis at the beginning of the experiment?
The presence of pre luc gene in the construct DNA in the cells of the fly results in light a emission
which reflects the transcriptional activity of the period gene.
2. What is luciferin? What is its function?

Luciferin is a substrate that produces the enzyme luciferase which emits light.
3. How often are light emissions measured in this experiment?

Light emissions are measures every hour, for 5 days.
4. Analyze the graph before clicking on it. What initial conclusions can be drawn from it?
During the 5 day period, light emission decreases periodical from day 1 to day 5. Having its peak
during lights off condition.
Be sure to answer all virtual quiz questions as they appear on your screen. Submit them for
grading. Look over your results, and review the questions that were answered incorrectly
USE TRANSGENIC FLIES

You should now be in to the next section, Use Transgenic Flies. To redirect there yourself, choose the
bottom right tab. Note that there are three experiments in this section.
Experiment Selection
• In the virtual lab window to the left, select Experiment 1 to begin this portion of the virtual lab. Once
you have selected an experiment, click to select the hypothesis that you think is the correct one for that
experiment.
• Proceed through the following questions pertaining to the experiment that you are completing.
• You must complete all three experiments in order, along with the accompanying questions that follow.
Experiment 1: Measure per-luc Gene Expression While Keeping Lights Off

1. What is your hypothesis?
Daily light emission will be detected from flies kept in constant darkness.
2. According to the Methods, what is the variable you are manipulating in this experiment?
The effect of external light-dark conditions to the light emission that reflects the period gene.
3. Look at the graph before clicking on it. What initial conclusions can be drawn from it?
External factors cannot stop light emissions from the flies, circadian rhythm has its own internal
control of gene expression independent from external factors.
4. What is the purpose of days 1-5?

This will be the duration wherein the flies will be exposed to 12 hour lights on and 12 hour lights
off.
5. Is your hypothesis correct or incorrect?

YES, it is correct.
6. Explain the conclusion of Experiment 1 in your own words. (Scroll to bottom of screen.)
The light-dark conditions did not stop the light emissions, because circadian rhythm has its internal
gene that control its expression and not on necessarily on external light cues. These shows that
organisms have constant sleep and wake cycles due to the intrincic biological clock that controls
this regulation.
Experiment 2: Measure per-luc Gene Expression While Changing Light On/Off Times
Changing the light-dark conditions cycles that flies are kept in resets the flies’ internal clock,
resulting in a shift of the peaks and troughs in the daily light emission oscillations.
2. According to the Methods, what is the variable you are manipulating in this experiment?
The change in the light-dark condition cycle and its effect on the period gene activity.
3. Look at the graph before clicking on it. What initial conclusions can be drawn from it?
At the time the lights are on and off were reversed, the peaks and troughs of the graph also shifted.
The peak of biolumiscence are now during “lights on” when normally it should be during “lights
off”.
Click on the graph and complete the Graph Analysis for Experiment 2. Print the data window if you wish.
4. Is your hypothesis correct or incorrect? YES, it is correct.
Click on the graph and complete the Graph Analysis for Experiment 1. Print the data window if
you wish.
6. Explain the conclusion of Experiment 2 in your own words. (Scroll to bottom of screen)
When the timing of the external light sources were changed, the oscillation of the gene
expression also changed.
Experiment 3: Examine Different Fly Body Parts for per-luc Gene Expression
Oscillating per gene expression is throughout the Drosophila’s body
2. Analyze the graphs before clicking on them. What initial conclusions can be drawn from each?
a.Head: head exhibits the light emissions
b.Leg: has the highest light emissions
c.Wing:
3. What is the purpose of the following: a. Days 1-2
For observing the flies under the 12 hour light and 12 hour dark condition.
b. Days 3-7
observing the flies in constant darkness.
c. Days 8-10
exposure in 12 hour light-12 hour dark condition but different from the firs, daytime is noon to
midnight.
Click on the wing graph and complete the Graph Analysis for Experiment 3. Print the data window if you
wish.
4. Is your hypothesis correct or incorrect? YES , it is correct.
5. Explain the conclusion of Experiment 3 in your own words. (Scroll to bottom of screen.)
Gene expression was seen throughout the flies’ body, having the highest emission peak on the legs.
This experiment allows us to observe the gene activity all over the flies body.
After completing all three experiments, proceed to the next section.

TRANSGENIC FLY VIRTUAL LAB: CONCLUSION
1. What are the three main conclusions that can be drawn from the three different experiments?
Experiment 1:
The light-dark conditions did not stop the light emissions, because circadian rhythm has its internal
gene that control its expression and not on necessarily on external light cues. These show that
organisms have constant sleep and wake cycles due to the intrinsic biological clock that controls this
regulation.
Experiment 2:
When the timing of the external light sources was changed, the oscillation of the gene
expression also changed. That is why the peak and troughs of the graph also shifted,
showing the gene responding to the shift of the light-dark cycle.
Experiment 3:
Gene expression was seen throughout the flies’ body, having the highest emission peak on the legs.
This experiment allows us to observe the gene activity all over the flies’ body.
Click on "View lab status and quiz summaries." Print the "Lab Status and Quiz Summaries," write your
name on it, and turn it in to your instructor. (Staple the data analysis sheets to this packet if you printed
them as you completed the three experiments.)

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Bicol University MA Bio Ed 211

GRADUATE SCHOOL Advanced Genetics

MIRAH MAE D. ARRANZADO Dr. Maria Eden A. Ante

TRANSGENIC FLY VIRTUAL LAB WORKSHEET

4. What occurs when the appropriate signals turn on the promoter?

5. What is the role of a reporter gene?

9. List the basic steps and substeps of the lab procedure:

11. What can a mutation in the period gene alter?

Click on "learn more about the technical details of DNA preparation."

4. How are DNA sequences "put together" in the laboratory?

7. After being filled, what does the glass needle contain?

PART 3: INJECT FLY EMBRYOS WITH DNA

3. What are "transformant progeny"?

PART 4: BREED FLIES

PART 5: SELECT TRANSGENIC PROGENY

2. What is luciferin? What is its function?

3. How often are light emissions measured in this experiment?

USE TRANSGENIC FLIES

Experiment 1: Measure per-luc Gene Expression While Keeping Lights Off

4. What is the purpose of days 1-5?

5. Is your hypothesis correct or incorrect?

4. Is your hypothesis correct or incorrect? YES, it is correct.

4. Is your hypothesis correct or incorrect? YES , it is correct.

After completing all three experiments, proceed to the next section.

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