TRANSGENIC FLY

The Transgenic Fly Virtual Laboratory

SUBMITTED BY:
Aquino, Charlyn C.
De Jesus, John Thronn A.
BSED – III

SUBMITTED TO:
Mrs. Jamaica L. Delos Reyes
SSED13 Teacher
I. Rationale
The activity aims to achieve the following:
1. Describe how recombinant DNA technology is used to produce transgenic
organisms; and
2. Explain how transgenic organisms can be used to explore biological
processes.

II. Answers to Questions

TRANSGENIC FLY LAB INTRODUCTION

1. What is the purpose of this virtual lab? What are transgenic organisms
used for in research?
To create transgenic flies in order to study on how to use transgenic
organisms to study biological processes. Transgenic organisms contain
DNA that is inserted experimentally and are used to study many biological
processes. We will be using Drosophila (fruit fly) as our transgenic
organism.

2. Click on "more on experimental design." What gene will you be studying

as a graduate student in this lab?
The period gene.

3. What is the role of a promoter (regulatory region) of a gene?

The promoter lies before the coding region of the gene. RNA polymerase
and accessory proteins (transcription factors) bind to the promoter to
initiate production of an mRNA transcript, thus the DNA is transcribed
into mRNA.

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

The DNA is transcribed into mRNA.

5. What is the role of a reporter gene?

They "report" on the activity of the promoter to which they are linked.
Expression of the reporter is easily monitored and permits the function or
whereabouts of the `target' sequence to be tracked.

6. Why is the luciferase gene such a popularly used reporter gene in

experiments?
It glows and is therefore easily recognizable. Luciferase has a half-life of
2-4 hours, short enough to show fluctuations within a day. It is convenient,
relatively inexpensive, and gives quantitative measurements
instantaneously.
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?
Light production is an external marker of internal molecular events. The
light from the transgenic organism demonstrates the expression of the
gene. The light produced is the window into the molecular clock in the fly
that regulates its daily rhythms.

8. In summary, transgenic per-luc flies will produce light in a pattern that

reflects the transcriptional activity of the period gene.

9. List the basic steps and sub steps of the lab procedure:
Make transgenic flies.

- Prepare DNA that will be incorporated into the fly genome.

- Prepare fly embryos.
- Inject fly embryos with DNA.
- Breed flies.
- Select transgenic progeny.
- Examine light output from transgenic adults

Use transgenic flies to study circadian rhythms and genetics.

- Measure per-luc gene expression under different light-dark conditions.

- Examine different fly body parts for per-luc expression.

10. What does the period gene normally control for the fly?
The molecular clock.

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

The mutation in the period gene will alter the circadian rhythms of
Drosophila or ‘an altered period (altered clock molecules)’.

12. In this lab, the period gene is linked to the luciferase gene. Why? What
does this elegant model allow us to do?
When the period gene is expressed (by attaching the period gene with the
luciferase gene), light is produced in the cells where period gene
transcription is occurring. This elegant model allows us to look at changes
in genes simply by looking at the glow of these transgenic flies.

PART 1: PREPARE DNA THAT WILL BE INCORPORATED INTO

THE FLY GENOME

1. What is "construct DNA"?

 A construct DNA is a type of DNA which consists small segments of
DNA, each with a critical function, then spliced together. It contains the
period promoter, luciferase coding region, mini-white promoter and
coding region and the P element.

2. Where is the promoter of the period gene?

All the experimental DNA to be integrated into the fly's genome is
contained within a circularized DNA vector. The promoter sequence of the
period gene is adjacent to the DNA sequence coding for the luciferase
gene.

3. If the construct DNA's promoter is activated, what will occur and what
will be produced?
The activation of the promoter sequence will result in the transcription and
translation of the luciferase protein, and the active enzyme luciferase will
be produced.

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

DNA sequences are put together using restriction enzymes and ligases
which are the molecular biologist's principal 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?
We will know whether the DNA has been inserted into the fly genome.
We can check eye color to determine whether the new gene has been
incorporated.

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?
Red eyes.

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

Construct and transposase DNA solution.

8. What is the purpose of the pump to which the glass needle is attached?
It regulates the amount of force ejected into the needle and into the tubing
and allows the careful regulation of the volume of the DNA solution to be
injected in the embryo.

PART 2: PREPARE EMBRYOS FOR INJECTION

1. Why is it important to use embryos that are less than 30 minutes old?
Cells that are actually germ cells are not yet differentiated and can
incorporate new DNA.
2. What is a germ cell? Where are germ cells found in fly embryos?
A germ cell is a type of cell which is basically destined to become a sperm
or an egg. They are found in the posterior region of the fly embryo.
3. If the DNA integrates properly into the germ cells, the adult fly will
contain germ cells that contain construct DNA. After mating, these flies
can then produce progeny that will contain the construct DNA in all
cells.

PART 3: INJECT FLY EMBRYOS WITH DNA

1. The DNA is injected into which end of the embryo?

The posterior part of the fly embryo.

2. Explain why embryo survival rates are so low in this type of experiment.
(Include all reasons.)
Although when the needle enters the right place in the embryo,
considerable damage to the embryo occurs. The outer embryonic
membrane is broken and cytoplasm may leak out. Survival of the embryo
depends on the repair of the membrane and compensation for fluid loss.
Too little DNA and there is little chance for DNA to be incorporated; too
much solution and the embryo may explode. Furthermore, some cells may
have matured, and DNA will not be incorporated. Following the injection,
if the injected embryos are too mature, they are destroyed.

3. What are "transformant progeny"?

These are the 10% of the injected flies which have offspring that contain
the DNA.

4. How much time does it take for the embryos to reach adulthood?
Few days after placed in the nursery flask.

PART 4: BREED FLIES

1. The adult flies are white eyed. Does this mean there are no transgenic
flies?
No, some of them have germ cells that contain the transgene.

2. Your task now is to mate these adults with noninjected white-eyed flies.
What do you expect from this mating?
That some of the resulting progeny will be transgenics.

3. What do you expect all of their cells to contain? What color will their
eyes be?
All cells should contain the per-luc DNA and their eyes will be red.
4. What occurs in the vial during the first two to three days?
In the vial during the first two to three days the flies mate, breed, and
grow. The embryos begin to grow on the agar plate.

PART 5: SELECT TRANSGENIC PROGENY

1. When looking at the progeny, what is the visible marker?

Red eyes.

2. What does the CO2 do to the flies?

CO2 anesthetize the flies.

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 original hypothesis of mine at beginning of the experiment was- the
presence of the per-luc construct DNA in the cells of the fly would result
in light emissions that reflect the transcriptional activity of the period
gene.

2. What is luciferin? What is its function?

Luciferin is a gene that is contains fluorescent protein. its function is to
produce light when then gene of interest is expressed. After a fly eats this
special food, all of its cells will contain luciferin. Only in those cells in
which the period promoter is activated will the enzyme luciferase be
produce and light is produced.

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

It is observed every hour for five (5) days.

4. Analyze the graph before clicking on it. What initial conclusions can be
drawn from it?
 From this, we can conclude that flies that expressed light have expressed
the transcriptional activity of the period gene. The bioluminescence
increases at dark and decreases with light each day. However, the amount
of bioluminescence decreases overall throughout the 5 day period.
5. Conclusions: During what time of day did light emissions peak? And
trough?
Peak was just before 6AM and trough was just before 6 PM each day.

III. Conclusion
IV. References

https://media.hhmi.org/biointeractive/vlabs/transgenic_fly/index.html