The Asian Journal of Animal Science (June to November, 2009), Vol. 4 Issue 1 : (126-129)
A CASE STUDY
Genetically engineered zebra fish-fluorescent beauties with practical applications
PRIYA NAGARE, B.A. AGLAVE AND M.O. LOKHANDE
Accepted : March, 2009
G
See end of the article for
authors’ affiliations
Correspondence to :
B.A. AGLAVE
Department of Biotechnology,
Institute of Life Sciences, H.P.T.
Arts and R.Y.K. Science College,
NASHIK (M.P.) INDIA
Key words : Zebrafish,
Glowfish, Zebra danios,
Brachydanio rerio
low fish or fluorescent zebra fish are
beautiful and unique creations of
science. Glofish is the name given to
genetically modified zebra da nios
(Brachydanio rerio). Their name is
correctly spelled as glofish, but is
sometimes incorrectly spelled as glowfish.
This is a common mistake because the
fish do seem to glow when they encounter
environmental toxins. The now infamous
luminescent zebrafish, sold as glofish, the
only transgenic fish commercially
available, as of 2004, was originally
developed in a laboratory in Singapore to
be a living indicator of water pollution. The
difference between regular zebra danios
and glofish are that researchers in
Singapore added a fluorescence gene
from a sea coral to zebra danio eggs to
produce glofish. This gene causes the
genetically modified fish to fluoresce or
light up in the presence of environmental
toxins. Other than this, they are identical
to regular zebra danios, with the exception
that the genetically modified glofish have
neon colours. Regular zebra danios are
gold and blue striped or gold and silver
striped fish. Both glofish and regular zebra
danios are peaceful fish that do well in a
community tank. Originally created to
detect environmental toxins, glofish have
been for sale in pet stores since 2003 for
the general public to buy and place in their
aquariums. There is some concern about
what might happen if these genetically
altered fish find their way into natural
rivers and streams. The creators of glofish
state that they don’t pose an ecological
threat because glowfish, like regular zebra
danios, won’t survive in the wild because
they are tropical fish and need a water
temperature of about 24-26 °C (75-79 °F).
It has been five years now and so far
there doesn’t seem to be a problem.
Although some people do question the
ethics of selling genetically altered fish to
the public just because they are prettier
than the original fish. Glofish come in a
variety of colours, including red, green, and
orange. They can be purchased for about
$6-7 each. Glofish are reported to be as
healthy as regular zebra danios and their
care is identical. They reach an adult size
of approximately 2 inches (5 cm). They
are omnivores that eat a variety of
aquarium fare.
Development of transgenic fluorescent
zebra fish:
Mechanism:
The process, illustrated in this chart,
begins by adding a fluorescence gene to
the fish before it hatches from its egg.
Once the gene integrates into the genome
(i.e., genetic code) of the embryo, the
developing fish will be able to pass the
fluorescence gene along to its offspring
upon maturity. Because of this, the gene
only needs to be added to one embryo;
from that point forward, all subsequent
fluorescent fish are the result of traditional
breeding.
The gloFish was produced by
injecting zebra fish eggs with the gene of
a sea anemone which makes it red
coloured. The researchers have also
produced green fish by injecting the eggs
with a fluorescent marker gene from
jellyfish, which is commonly used in
experiments.
To enable the fishes’ use as pollution
detectors, the scientists have pinpointed
gene promoters which act as on/off
switches in the presence of certain
triggers. One type of switch is activated
by the sex hormone oestrogen, which can
contaminate water. Other glow switches
can be stress-induced and will respond to
the presence of damaging chemicals like
heavy metals.
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PRIYA NAGARE, B.A. AGLAVE
AND
M.O. LOKHANDE
127
gene. Later, a team of Taiwanese researchers at the
National University of Taiwan, headed by Professor HuaiJen Tsai succeeded in creating a medaka (rice fish) with
a fluorescent green colour.
The scientists from NUS and businessmen Alan
Blake and Richard Crockett from Yorktown Technologies,
a company in Austin, Texas, met and a deal was signed
whereby Yorktown obtained the worldwide rights to
market the gloFish. At around the same time, a separate
deal was made between Taikong, the largest aquarium
fish producer in Taiwan and the Taiwanese researchers
to market the green medaka in Taiwan under the name
TK-1. In spring of 2003, Taiwan became the first to
authorize sales of a genetically modified organism as a
pet. One hundred thousand fish were reported sold in
less than a month at US$18.60 a piece. It should be
clarified that the fluorescent medaka are not gloFish, as
they are not marketed by Yorktown Technologies, but
instead by Taikong Corp under a different brand name.
Early development:
The original zebrafish (Danio rerio) from which the
gloFish was developed is a native of rivers in India and
Bangladesh. It measures three centimetres long and has
gold and dark blue stripes, and over 200 million have been
sold in the last 50 years in the United States ornamental
fish market. Despite the number of zebrafish sold, they
have never established any reproducing populations in the
United States, primarily because they are tropical fish,
unable to survive in the temperate North American
climate.
In 1999, Dr. Zhiyuan Gong and his colleagues at the
National University of Singapore extracted the green
fluorescent protein (GFP) gene from a jellyfish that
naturally produced bright green bioluminescence. They
inserted the gene into the zebrafish genome, causing the
fish to glow brightly under both natural white light and
ultraviolet light. Their goal was to develop a fish that could
detect pollution by selectively fluorescing in the presence
of environmental toxins. The development of the always
fluorescing fish was the first step in this process. Shortly
thereafter, his team developed a line of red fluorescent
zebra fish by adding a gene from a sea coral and yellow
fluorescent zebra fish, by adding a variant of the jellyfish
[Asian. J. Animal Sci., June to Nov., 2009, Vol. 4 (1) ]
Introduction to the U.S. market:
The gloFish was introduced to the United States
market in February 3, 2002 by Yorktown Technologies of
Austin, Texas, after more than two years of extensive
environmental research and consultation with various
Federal and State agencies, as well as leading experts in
the field of risk assessment. The definitive environmental
risk assessment was made by the U.S. Food and Drug
Administration (FDA), which has jurisdiction over all
genetically modified animals, including fluorescent zebra
fish, since they consider the inserted gene to be a drug.
Their official statement, made on 9 December 2003, was
as follows:
“Because tropical aquarium fish are not used for
food purposes, they pose no threat to the food supply.
There is no evidence that these genetically engineered
zebra danio fish pose any more threat to the environment
than their unmodified counterparts which have long been
widely sold in the United States. In the absence of a clear
risk to the public health, the FDA finds no reason to
regulate these particular fish.”
Similar findings were reached by the State of
California Department of Fish and Game and the State
of Florida Transgenic Aquatic Task Force.
Marketing of the fish was met by protests from a
non-governmental organization called the Center for Food
Safety. They were concerned that approval of the GloFish
based only on a Food and Drug Administration risk
assessment would create a precedent of inadequate
scrutiny of biotech. animals in general.
To prevent this, the group, along with one of their
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GENETICALLY ENGINEERED ZEBRA FISH-FLUORESCENT BEAUTIES WITH PRACTICAL APPLICATIONS
sister organizations, filed a lawsuit in US Federal District
Court to block the sale of the gloFish. The lawsuit sought
a court order stating that the sale of transgenic fish is
subject to federal regulation beyond the FDA’s charter,
and as such should not be sold without more extensive
approvals. In the opinion of Joseph Mendelson, the Center
for Food Safety’s legal director:
It’s clear this sets a precedent for genetically
engineered animals. It opens the dams to a whole host of
nonfood genetically engineered organisms. That’s
unacceptable to us and runs counter to things the National
Academy of Sciences and other scientific review boards
have said, particularly when it comes to mobile GM
organisms like fish and insects.
The Center for Food Safety’s suit was dismissed on
March 30, 2005.
Developments during 2006-2007:
GloFish have continued to be successfully marketed
throughout the United States. After more than three years
of availability, there are no reports of any ecological
concerns associated with their sale.
In addition to the red fluorescent zebrafish,
trademarked as “Starfire Red”, Yorktown Technologies
released a green fluorescent zebrafish and an orange
fluorescent zebrafish in mid-2006. The new lines of fish
are trademarked as “Electric Green” and “Sunburst
Orange”, and incorporate genes from sea coral. Despite
the speculation of aquarium enthusiasts that the eggs are
pressure treated to make them infertile, it has been found
some gloFish are indeed fertile and will reproduce in a
captive environment.
As of January 2007, sale or possession of gloFish is
illegal in California due to a regulation that restricts all
genetically modified fish. The regulation was implemented
before the marketing of gloFish, largely due to concern
about a fast-growing biotech. Salmon. Although the Fish
and Game Commission declined to grant an exception
(solely on ethical grounds) in December 2003, it later
reversed course and decided to move forward with the
process of exempting GloFish from the regulation.
However, due to the State’s interpretation of the California
Environmental Quality Act, Yorktown Technologies was
informed by State attorneys that it would first need to
complete an extremely expensive study, which could cost
hundreds of thousands of dollars and take years to
complete. According to the company’s web site, they have
thus far declined to undertake this study.
As of January 2007, Canada also prohibits import or
sale of the fish, due to what they report is a lack of
sufficient information to make a decision with regard to
[Asian. J. Animal Sci., June to Nov., 2009, Vol. 4 (1) ]
safety.
The import, sale and possession of these fish is not
permitted within the European Union. On November 9,
2006, however, the Netherlands’ Ministry of Housing,
Spatial Planning and the Environment (VROM) found
1,400 fluorescent fish, which were sold in various
aquarium shops.
Applications:
Transgenic zebra fish as pollution indicators:
The transgenic zebra fish can detect water pollution
by changing colour. Scientists have developed
commercially viable zebra fish that can be used as
pollution indicators making them a simple alternative to a
complicated pollution testing system.
The glowing zebra fish can be used to identify
pollutants in drinking water. The fish light up when exposed
to polychlorinated biphenyls (PCBs), which are known to
cause cancer in human beings. These fish are much more
sensitive than water current testing systems and can detect
low concentrations of PCBs. Testing water using
fluorescent zebra fish takes less time and cost than testing
with conventional equipment.
Normal zebra fish are usually black and silver in
colour while transgenic zebra fish are capable of producing
green or red fluorescent colours. The fluorescence in fish
is under the control of genes. In order to trigger off the
genes in the fish to be of any use, inducible gene promoters
are used to act as control switches to activate different
tissues of the fish. At present, scientists have succeeded
in isolating two types of gene promoters in the zebra fish:
an estrogen-inducible promoter and a stess-responsive
promoter.
The promoters have been used to drive the fluorescent
colour genes in transgenic zebra fish. Such fluorescent
coloured transgenic fish will be able to respond through
the estrogenic promoter when exposed to toxicants. The
fish will display the fluorescent colour that has been
assigned to detect the particular contaminants. Scientists
have developed as many as five colours indicating a
particular pollutant. The detection of pollutants by
transgenic fish is very quick and economical too.
Furthermore, these fish are also biodegradable. All these
factors make them suitable pollutant indicator.
Fluorescent fish as experimental organisms:
The fluorescent zebra fish is an alternative to
labor atory mice for testing new medicines. The
fluorescent fish can be used to show the impacts of
experimental drugs on cancerous tumours. The zebra fish
are a better option because their physiology resembles
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PRIYA NAGARE, B.A. AGLAVE
that of humans and they react similarly to cancerous
tissue. The fluorescent zebra fish can test experimental
drugs for cancers in less time and at less cost then
conventional laboratory mice. Liver cancer can be
implanted in zebra fish in only six weeks. It takes about
68 weeks to implant liver cancer in mice. Furthermore, a
zebra fish can lay hundreds of eggs in a week, compared
to a dozen or two by a mouse – producing a larger test
sample in a shorter span of time. Real time images enable
scientists to monitor how the internal organs grow, are
affected by tumours and heal after treatment.
The gene that makes jelly fish fluoresce is
transplanted into the liver of zebra fish that are later
implanted with cancer cells for testing. This allows
scientists to monitor cancer development in the fish’s liver
through a special microscope and evaluate the impact of
drugs.
Fluorescent zebra fish have been particularly helpful
in understanding cellular disease and development, as well
as cancer and gene therapy.
Transgenic zebra fish and environmental risk:
Some believe that transgenic fluorescent zebra fish
may adversely affect wild populations of zebra fish once
it escapes from the aquaria to the natural environment.
They believe that the transgenic fish may cross breed
with the wild zebra fish and produce fluorescent offspring.
However, research shows that the viability of fluorescent
zebra fish in nature is comparatively less than that of wild
zebra fish. The fluorescent fish is also more susceptible
to diseases, giving a higher mortality rate. The wild type
fish generally grows to a bigger size than the transgenic
fish, due to the additional burden of expressing the
fluorescent protein in glow fish. The fecundity of
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M.O. LOKHANDE
transgenic female fish is 50% less than that of wild type
females and also the fertility of transgenic males is less
than 90% that of wild type males.
Beca use fluorescence carries additional
bioenergetics cost of biosynthesis, energy distribution, and
predator avoidance, fluorescent fish are likely to have
reduced fitness. According to Darwin’s theory of
evolution, nature selects only those organisms which are
more adaptable to the environment. The risk of predation
is also more in case of fluorescent fish; therefore, it is
likely that in spite of escapes of fluorescent fish, nature
will select only wild zebra fish. Fluorescent zebra fish
may not establish their population in a natural environment.
Some people are worried about potential toxicity or
allergen issues associated with fluorescent transgenic
zebra fish. But fluorescent proteins occur naturally and
are wildely dispersed in marine ecosystems with no known
harmful effects through the food chain. Fluorescent
proteins have been widely used in many cell types and
organisms and no adverse health affects have been
reported. The fluorescent proteins also don’t share any
similarity in amino acid sequence to known allergens.
Therefore, it is unlikely that they may cause environmental
problems.
Authors’ affiliations
PRIYANKA NAGARE AND M.O. LOKHANDE,
Department of Biotechnology, H.P.T. Arts and R.Y.K.
Science College, NASHIK (M.S.) INDIA
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