International University – Vietnam National University

SCHOOL OF BIOTECHNOLOGY

GENETICS
Lab 5&6:

CULTURING AND MATING

DROSOPHILA MELANOGASTER

Instructor: MSc. Tong Thi Hang

Section: Wednesday morning
Group Members:
Trinh Le Hoang Minh – BTBTIU18152
Le Nha Tu – BTBTIU18264
Tran Ngoc Thanh Thu – BTBTIU18226
Nguyen Thi Trang – BTBTIU18248
Nguyen Le Uyen Vy – BTBTIU18284

SEMESTER II (2019-2020)
Ho Chi Minh City, July 31, 2020
 Contribution table

Full name Contribution Signature

percent
Trinh Le Hoang Minh

Le Nha Tu

Tran Ngoc Thanh Thu

Nguyen Thi Trang

Nguyen Le Uyen Vy
TABLE OF CONTENTS
I. INTRODUCTION………………………………………………………..........................2
a. Drosophila
melanogaster………………….……………………………………………………..2
b. Gal4/UAS
system………………………………………………………………………………..3
c. dUCH………………………………………………………………………….….4
II. OBJECTIVES………………..………………………………………………………….4
III. MATERIALS AND PROCEDURE………………………………………….………..5
a. Materials………………………...………………………………………………5
b. Procedure for handling flies……………………………………...………….....5
c. Procedure for observing mating result ………………………………..……...5
IV. RESULT AND DISCUSSION………………………………………..……………..…7
a. 3 crosses using GMR promoter………………………………………………..7
b. 3 crosses using GMR promoter………………………………………………..9
V. REFERENCES………………………………………………………………….………11

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 I. INTRODUCTION

a. Drosophila melanogaster (fruit fly): is an important model organism that can be

found near overripe fruits and rotten fruits. It has been used as a powerful genetic tool
in the study of genetics since it has many characteristics of a good experimental
model:
• Short life cycle
• The high reproductive rate
• Small chromosome number
• Small and easy to handle
• Easy to edit genome
• Independent gene expression
• Phenotypic markers
• High similarity to human genes

• Morphology

Most of fruit flies are small, about 2–4 mm long, but some are larger than a house fly.
Drosophila’s body has three main parts: head, thorax and abdomen. The major structures of the
head are 2 big compound eyes, 2 antennas and a mouth. Compound eye is one of the most
advanced forms of eye among insects. The unit structure of it is ommatidia. There are 760
ommatidia per compound eye, moreover, a cornea, eight photoreceptor cells (R1–8), many
pigment cells, and some support cells are also found in each ommatidium. Reddish pigment
cells are found in wild‐type Drosophila, excess blue light is absorbed by them; therefore,
ambient light is not made the fly blind. The thorax has 6 legs, 2 wings and 2 halters. Most of
fruit flies are typically pale yellow to reddish brown or black and transverse black rings across
the abdomen with brick red eyes. Many species have distinct black patterns on the wings with
plumose (feathery) and arista antennae, bristling on the head and thorax.

(Source: https://infovisual.info/en/biology-animal/morphology-of-a-fly-dorsal)

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 • Sexing flies
Sexing flies is critical when making crosses, so we need to be careful in identifying the
difference between the sexes
Males can be easily differentiated from females having differences in size and color.
The male is usually smaller than the female. Notice that males are generally smaller and
have a darker and more rounded abdomen. Females also tend to be larger. In addition, males
have tarsal sex combs on their first pair of legs. These are black and very distinctive but
can only be seen under relatively high magnification

• Life cycle:
Drosophila life cycle includes an egg, larval (worm-like) form, pupa and finally
emergence as a flying adult

(Source: https://www.cherrybiotech.com/scientific-note/drosophila-life-cycle-and-fly-anatomy)

Day 0: Female lays eggs

Day 1: Eggs hatch

Day 2: First instar (one day in length)

Day 3: Second instar (one day in length)

Day 5: Third and final instar (two days in length)

Day 7: Larvae begin roaming stage. Pupariation (pupal formation) occurs 120 hours after egg
laying

Day 11-12: Adults emerge from the pupa case. Females become sexually mature after 8-10
hours

b. GAL4/UAS system:

The GAL4-UAS system is a biochemical method used to study gene expression and
function in organisms such as the Drosophila melanogaster. GAL4/UAS system is used for
expressing dUCH protein in transgenic flies.

Gal4 expression can be regulated by any chosen promoter. In this lab, we will use GMR
promoter which express only in eyes and Act5C promoter which express in the whole cells

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 The wild-type (Canton S) are use as the control flies.

c. dUCH
Drosophila ubiquitin carboxyl-terminal hydrolase (dUCH) is the protein in Drosophila
melanogaster that is used for analyzing the role of the protein’s function in transgenic
Drosophila.
• dUCH gene overexpression

GAL4 is attached after the promoter. Upstream Activation Sequence region (UAS) is
attached to dUCH gene sequences (UAS-dUCH). GAL4 expression is controlled by a tissue-
specific promoter
(GMR driving promoter). The expressed Gal4 bound to UAS element on the upstream of
dUCH gene in transgenic Drosophila GAL4 expression is controlled by a tissue-specific
promoter
 caused the dUCH gene overexpression in F1

• dUCH gene knockdown

GAL4 is attached after the promoter. Upstream Activation Sequence region (UAS) is
attached to reverse dUCH gene sequences (UAS-dUCH-IR). When the GAL4 / UAS system
is activated, GAL4 bound to UAS element on the upstream of dUCH-IR gene in transgenic
Drosophila
 induces gene knockdown in F1

II. OBJECTIVES
Throughout this lab section, students should be able apply their knowledge into practical
experiment, including:

• Culturing fruit flies – one of the most popular genetics model

• Distinguish male and female fruit flies, which is important for mating step
• Identify various stage in life cycle of Drosophila Melanogaster
• Practicing using stereomicroscope
• Understand the mechanism of Gal4/UAS system as well as GMR and Act5C promoter
• Observing and guessing the effect of knockdown and overexpressed dUCH protein on
different part of fruit fly

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III. MATERIALS AND PROCEDURE
Since medium vials for culturing of the fruit fly were prepared by our TA, we carry out the
following lab work. Each group is responsible for one of 6 types of required crosses.

a. Materials:

Ether Culture tube

Small paintbrushes Cotton mesh

Stereo dissecting microscope

b. Procedure for handling flies

1. Strike lightly the base of the prepared culture tube containing flies for crossing on the
palm of the hand, so the flies will drop to the bottom. Avoid flies dropping strongly then
being stuck into the medium.

2. Remove the plug and quickly invert this tube over an empty tube. Strike lightly to convert
the flies from the prepared culture tube to the empty tube. Quickly cap these two tubes.

3. Expose the flies to 2 – 3 drops of Ether for them to stop moving in a few minutes (short
faint, not death). Be careful as overetherization will kill the flies.

4. Remove the cap and transfer etherized flies to a plate on the tables.

5. Observes the flies with the light source to determine male or female ones following the
cross requirement.

6. Open the plug of a new culture tube, keep the tube horizontally and gently transfer
selected flies (3 female and 3 male) by using a small paintbrush.

7. Quickly move the other non-selected flies into the initial medium.

8. Wait until the flies revive before turn the tube vertically otherwise the flies come into
contact with the moist medium, wetting their wings and unable to fly out of the medium.

In the next one week, we were provided with crossing results of our instructor and TAs
because our sample did not have enough time (about 10 days) to obtain complete results.

c. Procedure for observing mating result

• Testing for the ability of reacting to different light condition by the movement of fruit
flies)

We also observed to compare movement of among fruit flies in 3 different big tubes: wild
type, knockdown and overexpression. In this lab section, the TA performed for us as the
following steps:

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1. Strike lightly at the same time the base of 3 tubes containing 3 types of flies on the palm
of the hand, making the flies drop to the bottom.

2. Put these 3 tubes vertically. Observe the movement.

3. Repeat step 1. Put these 3 tubes horizontally. Observe the movement.

*Carry out the above steps in turn under white light, no light (dark), and UV light.

• Observing F1 phenotypes of 6 crosses

- For the 3 crosses using Act5C promoter, closely observe the phenotype of fruit flies
with the naked eye while they are put in culturing tube.

- For the 3 crosses using GMR promoter, closely observe the phenotype of fruit flies by
Stereo dissecting microscope
1. Strike lightly the culture tube containing, so the flies will drop to the bottom.
2. Remove the plug and quickly invert this tube over an empty tube. Strike lightly to
convert the flies from the prepared culture tube to the empty tube. Quickly cap these
two tubes.
3. Expose the flies to 2 – 3 drops of Ether for them to stop moving in a few minutes (short
faint, not death).
4. Remove the cap and transfer 3 etherized flies to a plate on the tables.
5. Place the plate on the stage plate of stereo microscope.
6. Switch on the light source(s) and adjust the eyepiece(s) so that you can look through
the microscope comfortably without straining your eyes.
7. While looking through the eyepiece(s), slowly turn the focus knob until the fly comes
into view. Once you can see the outline of the fly, turn even more slowly to focus as
sharply as possible. If you are not able to see anything, try moving the plate around
slightly on the stage plate to make sure it is directly below the objective lens and then
try focusing again.
8. Observer the eyes of the flies.

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 IV. RESUTL AND DISCUSSION
a. 3 crosses using GMR promoter

• F1 Genotype

♂ +; +; UAS_dUCH +; +; UAS_dUCH.IR
♀
+; +; +

GMRGal 4 UASdUCH GMRGal 4 UASdUCH .IR GMRGal 4

GMR_Gal4; +; + 100% ;+; 100% ;+;
+ + + + 100%
+
;+; +

• F1 Phenotype
dUCH dUCH
Control
Overexpression Knockdown
#23-4 V26468
CannonS
+; +; UAS_dUCH +; +;
+; +; +
UAS_dUCH.IR

♀ Eyes GMR_Gal4; +; +
Fused ommatidia Long bristles and Wild type eye:
and loss of mildy rough eye, brick-red eye
pigmentation, deep orange color
wrinkled surface,
brown color

When crossing the female fruit fly carrying the GMR-GAL4 gene complex with male
one carrying the UAS-dUCH gene complex, the promoter GMR will attach on the Gal4 and
causes the overexpression of dUCH in eye imaginal discs. As a result, the rough eye
phenotype is observed in adults. Besides, this abnormal phenotype also is resulted from the

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induction of caspase-dependent apoptosis followed by compensatory proliferation. This death
cell process is also caused by the overexpression of dUCH.

The knockdown dUCH also influences to the abnormal development of eye tissue, and
then affect the eye phenotype of fruit flies. When dUCH is knocked down in cells line, it
leads to a mistake in the eye structure in fruit fly - rough eye pattern. The apoptosis
phenomenon also has been seen. That why we see rough eye phenotype in adult flies in the
KD GMR Petri.

• Movement Results
White light No light UV light
(horizontal)
Vertical Horizontal Vertical Horizontal
Overexpression They Most of Most of them Most of All of them have
move them did not move slowly them have no movement
slowly move (a few flies no towards UV light
move faster) movement (avoid UV light)
Knockdown They A few flies They move All of them A few flies move
move move to with faster did not towards UV light
faster another end speed move to the
slowly (most empty end
of them keep
moving at
the bottom)
Wild type The The fastest The fastest The fastest Most of them
fastest movement movement movement move fast
movement

Why Mutant Drosophila move slowly in the vertical and horizontal tube?
Changes in the eye structure of fruit flies affect on their ability to detecting and
receiving light because of causing the damage of photoreceptors cells. It is the reason why
wild type fruit flies move faster than the mutant one. For the knockdown dUCH Drosophila,
the level of tyrosine hydroxylase (TH) decreased significantly, this leads the poor ability
climb in fruit flies. There are many studies that have reported that the direct relationship
between TH and dopamine in the brain Drosophila. This decrease is the factors causing slow
movement in mutated fruit flies

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Why does mutant Drosophila (especially duch overexpressed flies) move slowly and
cannot move to UV Light?

The compound eye of Drosophila melanogaster contains an array of some 800

ommatidia or simple eyes. Each ommatidium comprises 8 photoreceptor neurons (R cells,
designated R1–R8) and a set of accessory cells, including lens-secreting cone cells and pigment
cells. Among those 8 photoreceptor neurons, R7 is the primary receptor for UV light. Since the
dUCH overexpression caused the impairment of R7 cell differentiation, flies that contain
overexpressed dUCH may lose the ability to convert light into signals that are sent to brain.
Therefore, mutant Drosophila cannot recognizer UV Light and they tend to move slowly and
stay away from UV Light.

b. 3 crosses using Act5C promoter

dUCH dUCH
Control
Overexpression Knockdown
#23-4 V26468
CannonS
+; +; UAS_dUCH +; +;
+; +; +
UAS_dUCH.IR

Curly wings, a Curly wings, a few ▪ Wild type,

All few pupas not pupas not straight wings, a
♂ +; Act5C_GAL4; + developed
tissues developed few pupas not
developed

▪ Occurring a
special
individual:
curly wings,
length in body
and yellow
color

The target protein, UCH-L1, is a member of ubiquitin-proteasome system, so it is thought to

participate in intracellular protein degradation and turnover process. Therefore, any change
about UCH-L1 must cause abnormal phenotypes in adult fruit flies, or even cause lethal death

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Actin5C is ubiquitously expressed, including in the male germline, and the protein has
been found as a component of the sperm proteome. In this experiment, the Act5C
promoter has been used to drive “ubiquitous” high-level expression of Gal4.

It is proved that knockdown dUCH resulted in the abnormal development of tissue, and
then affect the survivability of fruit flies. When dUCH is knocked down in all cells line,
this will cause pupal lethal effect. That is the reason why we have seen many pupae can
not develop in tube 4.

On the other hand, there are many experiments shows that there is a relationship
between the overexpression of dUCH and the downregulate of mitogen-activated
protein kinase (MAPK) pathway. As we all know, the MAPK pathway plays a role in
the regulation of gene expression, cellular growth, and survival, so abnormal MAPK
signaling may be the reason leading to the pupal lethal effect at tube 3.

Those Drosophila that are alive in tube 3 and 4, must caring the Cyo (“Curly O”) -
balancer chromosome which is dominant for curly wings and also has a recessive lethal
mutation. Therefore, those alive Drosophila must have curved wings. However, as we
observed in the lab, a few alive fruit flies have straight wings. The reason for this
problem maybe from mutation during meiosis.

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 V. REFERENCES
Farzana Khan Perveen (February 28th 2018). Introduction to Drosophila, Drosophila
melanogaster - Model for Recent Advances in Genetics and Therapeutics, Farzana Khan
Perveen, IntechOpen, DOI: 10.5772/67731. Available from:
https://www.intechopen.com/books/drosophila-melanogaster-model-for-recent-advances-in-
genetics-and-therapeutics/introduction-to-drosophila

An introduction to fruit flies. (2017, July 11). Retrieved July 26, 2020, from:
https://depts.washington.edu/cberglab/wordpress/outreach/an-introduction-to-fruit-flies/

Home Science Tools. How to use a stereo microscope. Available at:

https://learning-center.homesciencetools.com/article/using-a-stereo-microscope-science-
lesson/

Dang Thi Phuong Thao ( February 28th, 2018). Drosophila Model in the Study Role of UCH-
L1. Available at:
https://www.intechopen.com/books/drosophila-melanogaster-model-for-recent-advances-in-
genetics-and-therapeutics/drosophila-model-in-the-study-role-of-uch-l1

Dang Thi Phuong Thao, An N.T.Phan, Masamitsu Yamaguchi, Tran Linh Thuoc (2012 April).
Overexpression of ubiquitin carboxyl terminal hydrolase impairs multiple pathways during eye
development in Drosophila melanogaster. Available at:
https://www.researchgate.net/publication/224819543_Overexpression_of_ubiquitin_carboxyl
_terminal_hydrolase_impairs_multiple_pathways_during_eye_development_in_Drosophila_
melanogaster

Explore MAPK. Understanding the MAPK pathway as it relates to oncology. Retrieved from:
https://www.genentechoncology.com/pathways/cancer-tumor-targets/mapk.html

Cure FFI (October 27, 2014). Introduction to Drosophila: genotypes, recombination and
balancer chromosomes. Available at: https://www.cureffi.org/2014/10/27/genetics-16/

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