Chapter 10:
Production of transgenic fish: Creation of a gene construct, methods of gene transfer. Production of Transgenic Fish
Application of genetic engineering technique to produce fish with enhanced growth, disease
resistance and cold tolerance. The production of transgenic fish involves the introduction of
foreign DNA into the genome of a fish species to confer new traits
or characteristics that are not present naturally. This process is a
key area of genetic engineering and has potential applications in
aquaculture, environmental research, and biomedical studies.
Transgenic fish can be designed to grow faster, resist disease,
tolerate environmental stresses, or possess other beneficial traits,
thus offering significant advantages for fish farming and
environmental management.
Dr. Sharmin Suraiya
B. Sc in Fisheries (Hons.) & [Link]. in Biotechnology (BAU, Bangladesh)
PhD in Biotechnology (KGSP PhD Scholar-2014, PKNU, S. Korea)
Assistant Professor
Department of Fisheries and Marine Bioscience
Jashore University of Science and Technology, Jashore.
Mobile: +8801790054236
The most common way to create Steps Involved in the Production of Transgenic Fish
fluorescent lines in zebrafish is to use the
Tol2 transposon-based system, which
contains the gene of interest associated 1. Selection of the Target Fish Species
with a fluorescent protein, such as eGFP.
After injection of the construct, different •The first step in producing transgenic fish is to select the species in which genetic
embryos will have integrated a different modification is desired. The most commonly used species in transgenic fish research and
number of repetitions, which results in
different levels of fluorescence, so the
aquaculture are salmon, tilapia, carp, and zebrafish. The choice of species depends on factors
line will need to be outcrossed with WT like their economic importance, growth rate, and ease of genetic manipulation.
fo r s eve ra l ge n e rat i o n s , to o b ta i n 2. Gene Selection
individuals with only one repetition and
stable fluorescence. •The next step is to choose the gene(s) to be introduced into the fish genome. The chosen
genes could come from a variety of sources:
◦Other species: Genes that confer beneficial traits like disease resistance, enhanced
growth rate, or stress tolerance.
◦Human genes: In some cases, transgenic fish are engineered to produce proteins or
substances used in medicine or biotechnology (e.g., insulin, growth hormone).
◦Plants or microorganisms: For traits like enhanced nutrient uptake or disease resistance.
Commonly used genes in transgenic fish include:
•Growth hormone genes (e.g., the Atlantic salmon growth hormone gene).
•Antibiotic resistance genes or disease resistance genes.
•Bioluminescent proteins (e.g., green fluorescent protein (GFP) from jellyfish) for tracking and
monitoring.
3. Gene Cloning and Vector Construction
•Gene Cloning: Once the gene of interest is selected, it needs to be cloned into a
vector. The gene is inserted into a plasmid or viral vector, which is a piece of DNA
that can be introduced into the fish genome. The vector usually includes:
◦A promoter: A DNA sequence that ensures the gene is expressed in the fish.
◦Selectable markers: To help identify cells that have successfully incorporated
the gene (e.g., antibiotic resistance markers).
◦The gene of interest.
•Constructing the Vector: The foreign gene is then inserted into the vector using
techniques like restriction enzyme digestion and ligase to ensure the DNA is
properly inserted.
Figure . Tol2-mediated transgenesis in zebrafish. (a) Stable transgenesis. Transposase mRNA synthesized by in vitro transcription and a
plasmid DNA harboring the Tol2 cis regulatory elements (yellow) are co-injected into zebrafish fertilized eggs. The transposase synthesized
from mRNA catalyzes the integration of the excised transgenic sequences placed between the Tol2 short terminal repeats into the genome.
The injected embryos are raised and crossed with wild-type fish. The integrated transgene is transmitted to the F1 generation showing non-
mosaic transgene expression. (b) Mosaic F0 transgenic zebrafish can be created either through the microinjection into fertilized eggs or
through electroporation in adults (TEAZ) of the Tol2 transposase and the construct containing the cis regulatory elements. A fluorescent
marker gene (e.g., GFP) is often present in the construct to identify the transgenics.
4. Gene Transfer into Fish Cells
There are several methods used for transferring the foreign gene into the fish genome:
[Link]:
◦This is one of the most commonly used techniques. The vector carrying the gene of interest is
microinjected directly into the fertilized egg (embryo) of the fish. This method involves:
•Using a fine needle to inject the DNA into the fertilized egg or one-cell-stage embryo.
•The foreign DNA is integrated into the genome of the embryo, and if successful, the
transgene is passed to the offspring.
◦Limitations: Microinjection is technically demanding and requires specialized equipment.
[Link]:
◦In this method, embryos or cells are exposed to an electric field that makes their membranes
temporarily permeable, allowing foreign DNA to enter the cells. It is often used for larger-scale
production and in species that are difficult to modify with microinjection.
◦Limitations: Not as widely applicable as microinjection, and may cause cell damage.
[Link] Vectors:
◦Another technique involves using viruses as vectors to deliver the foreign gene. Modified viruses
are used to infect fish embryos, and the viral genome integrates into the fish genome, carrying the
foreign gene with it. This method is often more efficient than microinjection or electroporation in
some species.
◦Limitations: Safety concerns with viral contamination.
[Link]-Mediated Gene Transfer:
◦This method involves mixing sperm with the DNA construct and then fertilizing eggs. The DNA is
incorporated into the genome through the fertilization process.
◦This method is still under development and is not as commonly used as other methods.
5. Selection of Transgenic Fish
Applications of Transgenic Fish
After the foreign DNA is introduced into the fish, not all embryos will successfully incorporate the gene.
Thus, a selection process is necessary to identify those that have incorporated the transgene: [Link] (Fish Farming):
•Antibiotic Selection: If an antibiotic resistance gene was used as a marker, fish that have incorporated ◦The most prominent application of transgenic fish is in aquaculture. By introducing genes
the foreign DNA can be grown in an environment with an antibiotic to select for those that have that promote faster growth, disease resistance, and improved feed conversion, transgenic
successfully taken up the DNA. fish can help increase production efficiency and reduce costs.
•Fluorescent Markers: In some cases, the foreign gene may include a marker (e.g., green fluorescent ◦For example, AquAdvantage Salmon, which contains a growth hormone gene from the
protein (GFP)), making it easier to identify transgenic fish under UV light. Pacific chinook salmon, grows faster than non-transgenic salmon, allowing for more efficient
•PCR and Southern Blotting: Molecular techniques such as PCR (Polymerase Chain Reaction) and fish farming.
Southern blotting can be used to confirm the presence of the transgene in the genome of the fish.
6. Breeding and Propagation [Link] Resistance:
◦Genetic modification can provide fish with enhanced resistance to viral and bacterial
•Once transgenic fish are identified, they are bred to produce offspring that carry the foreign gene. Over diseases. For example, fish engineered with genes that confer resistance to viral diseases like
successive generations, the transgene is propagated, and the trait of interest (e.g., faster growth, disease Viral Hemorrhagic Septicemia (VHS) or Infectious Salmon Anemia (ISA) can reduce the need
resistance) becomes established in the population. for antibiotics and other chemical treatments in aquaculture.
•Careful breeding and selection are required to ensure the transgene is inherited correctly and expressed
effectively in the offspring. [Link] Stress Tolerance:
◦Transgenic fish can be modified to tolerate adverse environmental conditions such as
temperature extremes, low oxygen levels, or high salinity. This can help fish farming
operations in regions with fluctuating environmental conditions.
[Link] and Ecological Research:
•Transgenic fish are valuable tools in environmental research, where they can
be used to study the effects of pollutants, toxins, or endocrine-disrupting
chemicals on aquatic life.
•Bioremediation: Transgenic fish could be used in efforts to clean up
environmental contaminants.
[Link] Research:
•Transgenic fish, especially zebrafish, are widely used in biomedical research
due to their transparent embryos, rapid development, and genetic similarity
to humans. They are often used to study genetic diseases, developmental
biology, and the effects of drugs or chemicals.
Ethical and Regulatory Considerations
What is gene transfer?
The production and use of transgenic fish are surrounded by various ethical, ecological, and Definition:
regulatory concerns:
A genetic engineering technique to transfer genes using vectors
[Link] Risks:
◦There is concern that transgenic fish might escape into wild populations, potentially leading to from one organism to another or from one cell to another in
genetic contamination and disruption of local ecosystems. As such, transgenic fish are often order to treat disease, construct GMO and economically
engineered to be sterile to prevent breeding with wild populations.
important organisms are known as gene transfer.
[Link] Safety: A short history:
◦Concerns about the safety of consuming genetically modified fish have led to regulatory
scrutiny. Rigorous testing is needed to ensure that transgenic fish are safe for human The first evidence of gene transfer was reported in bacteria by
consumption and do not pose health risks.
Frederick Griffith in 1928. He had named it a transformation. In
[Link] Welfare: 1944, Avery had demonstrated that the material transfer
◦The welfare of transgenic fish during breeding and farming, including potential issues of
abnormal development or suffering, is a concern for animal rights organizations. between bacteria during transformation is the nucleic acid (DNA)
which was later validated by Hershey and Chase in 1952.
[Link] Oversight:
◦In many countries, regulatory bodies like the U.S. Food and Drug Administration (FDA) and
Environmental Protection Agency (EPA), or similar organizations in other countries, oversee the Although the exogenous gene transfer in a eukaryotic cell (in
approval and regulation of genetically modified fish. vitro) was demonstrated by Rogers in 1970.
Types of gene transfer
Usually, the gene transfer techniques used in various applications can be categorized
into two broader categories.
•Natural methods of gene transfer
•Artificial methods of gene transfer
Classification of gene transfer techniques.
The natural mode of gene transfer: In this technique natural agents like live cells,
bacteria, other DNA molecules and viruses are used to transmit genes. Some of them
are:
[Link] transformation
[Link]
[Link] transduction
[Link] transduction
[Link]
[Link] tumefaciens mediated gene transfer
Another classification of gene transfer is based on which organism it affects. It can be categorized into
The artificial method of gene transfer: In this technique, an exogenous gene is transmitted horizontal gene transfer and vertical gene transfer
to another cell or organism using artificial means either physical or chemical agents. Some of Horizontal gene transfer vs vertical gene transfer:
the common artificial techniques are:
When gene transfer occurs between two unrelated individuals or organisms, this type of gene transfer
Physical methods is known as horizontal gene transfer. It is also called the lateral gene transfer method.
•Electroporation
Usually, it is commonly observed between prokaryotic and eukaryotic cells. The origin of antibiotic
•Biolistics transformation resistance in bacteria is the classic example of the horizontal gene transfer by natural means.
•Gene gun
On the other side in the vertical method of gene transfer, the genes are transmitted from parents to
•Protoplast fusion their offspring.
•Microinjection
This means the transfer of genetic material or genes from one cell to another or one organism to
another related organism is known as the vertical mode of gene transfer.
Chemical methods
•Liposome mediated gene transfer Several examples of horizontal gene transfer are:
•Calcium phosphate mediated gene transfer
Transformation: Alteration in a gene by uptaking genetic material from unrelated organisms is known
•DEAE- Dextran mediated gene transfer as transformation. It is used in transformation experiments to transmit genes in bacteria to infect a
•Gene transfer using polyethylene glycol host.
Transduction: Transfer of genetic material from one bacterium to another by the virus or phase is
known as transduction.
Conjugation: The gene transfer of one cell to another using the bacterial plasmid from donor to
recipient cell is known as conjugation.
Transposable elements: The transposons or transposable elements are the mobile genetic sequences
that can carry a gene or DNA and can transfer it to other unrelated cells.
Cervical cancer caused by HPV is an example of horizontal gene transfer in which the infected cell
transmits the genetic material to another cell and spreads cancer.
On the other side, Transient vs stable gene transfer or transfection:
In the transient type of gene transfer, the foreign genetic material is never incorporated into the host
The gene transfer from one related cell to another or parental organisms to their genome and thus can’t be inherited into offspring.
progenies is known as a vertical mode of gene transfer. On the other side, in the stable gene transfer, the gene or foreign DNA inserts into the host genome
and transmits into the offspring.
The process of reproduction or the sexual mode of reproduction is a type of
The transient gene transfer is performed to produce a temporary high level of gene expression and
vertical mode of gene transfer. thus it is limited to somatic cells.
Crossbreeding in plants is the most common example of vertical gene transfer While in the stable gene transfer, once the gene is incorporated into the host genome it may express
normally, transmit to offspring and express normally in them too.
used by scientists for a long time. These two plants are sexually reproduced or
It is usually performed on germ cells.
crossed to produce a superior quality plant species.
Further to this, the meiosis and mitosis cell division process is a way of vertical
gene transfer in which genetic material is inherited from one identical cell to
another.
Infection of HIV from mother to child is an example of it. Here the infected patient
or cell inherited the genetic material of HIV along with their own to their offspring.
Resultantly, the progeny of the baby got infected by HIV.
Artificial Gene transfer techniques: Biolistic transformation
The biolistic transformation is also known as a biolistic particle delivery system.
Physical Gene transfer methods: In this method, a gene of interest coated with heavy metal ions is inserted
into the target cell by mechanical force.
Electroporation, biolistics transformation,
gene gun, protoplast fusion and Gene gun is a common name used for it. Molecules like DNA, RNA or protein
microinjection are some common physical can be inserted into a cell directly.
gene transfer techniques used often in
genetic engineering experiments. We are
discussing every method comprehensively
here.
Electroporation:
The electroporation technique is one of the
commonest and successful methods used so
far by scientists. In this method, the gene of
interest or the DNA is transferred into the
target cell under the influence of an
electrical pulse.
Under the pulse electric current, pores are
cre a t e d i n t h e c e l l w a l l o r t h e p l a s m a
membrane of a cell which allows DNA
intake.
The overview of the
Once the electrical current is discharged, electroporator machine.
the pores are closed. The electroporation
method is used for both stable and transient
gene expression.
Protoplast fusion: Microinjection:
A cell without a cell wall or cell membrane is known as a protoplast. The The microinjection method facilitates direct nuclear delivery of
protoplast fusion method is also used in the tissue culture methods in exogenous DNA. Using a fine needle, or injection, a DNA or gene is
which two protoplasts of somatic cells are fused to get a hybrid directly injected into the target cell nucleus without disrupting the
protoplast of two different cells. It is commonly practiced in plant endogenous pathway.
research to produce genetically altered plant species through their
The microinjection gene transfer technique is practiced to inject genes
protoplast fusion.
directly into oocytes, eggs and embryos.
Chemical methods of gene transfer: Liposome mediated gene transfer:
Liposome-mediated gene transfer, calcium phosphate mediated gene transfer,
Among all chemical methods, liposome-mediated gene transfer or
DEAE-Dextran mediated gene transfer and polyethylene glycol mediated gene
transfer are common chemical methods used in transformation experiments. lipofection is most effective and used widely. The liposome is a
phospholipid vesicle that inserts DNA once it interacts with the target
Before discussing various chemical methods to transfer a gene, we should have cell.
to understand how chemical-mediated gene transfer techniques actually work!
Because the whole mechanism is different. It is a hydrophobic vesicle that surrounds the naked DNA and interacts
with the cell wall or membrane. It inserts DNA into the cell cytoplasm.
Here the naked DNA is used to fuse the complex with a chemical agent (and
Although the method of making the liposome and incorporating DNA
therefore it is also known as naked-DNA mediated gene transfer). The
chemical and DNA complex either interact with the cell wall or membrane or
into it is tedious, time-consuming and labor-intensive
is processed through endocytosis. Once it enters into the cell cytoplasm, the
DNA is released from the chemical agent and incorporated into the genome.
In comparison with physical gene transfer techniques, the chemical method is
slightly less effective, though.
.
Calcium phosphate transfection:
Transfection with DEAE-dextran:
Yet another reliable chemical method of gene transfer is the calcium
The DEAE-dextran is the polycationic carbohydrate soluble complex that makes phosphate technique in which the DNA eluted in phosphate is mixed with
aggregate or complex with the DNA. The idea is to make a net charge of the the solution of calcium chloride and allows it to co-precipitate.
complex positive-charged so that it can interact with the negative charge of the
cell wall. When mixed with the target cell population, some complex DNA and
It is often employed before the discovery of electroporation. calcium phosphate precipitate when uptaken by the cells through the
process of endocytosis and our DNA is inserted in the target cell.
The technique is simple, cost-effective, reliable than other methods and
can transfect cells growing into the monolayer.
Note that it was the first method used to transfect the animal cells.
Virus mediated gene transfer:
The virus is the carrier of genetic material whose effort can help to transfer
DNA in a target cell. Viruses are one of the most effective genes transfer vehicles.
Adenovirus, Adeno-associated virus, HIV, lentivirus and other retroviruses can
be used for gene transfer. Although it can infect the host organism.
Process of gene transfer:
The steps in the process of gene transfer are similar to other genetic engineering
experiments. Common steps of the gene transfer method are:
•Isolation of gene of interest
•Selection of plasmid
•Restriction digestion of DNA
•Insertion of DNA into plasmid
•Selection of gene transfer technique
•Insertion of rRNA or altered plasmid into the target cell
•Cell culture
•Injection to the target organism
Let us briefly understand the process, The graphical illustration of the process of
cDNA synthesis and library preparation.
In the very first step, the organism and the gene of interest we wish to study and
insert into the recipient organism are selected and studied.
Soon after, the appropriate vector or plasmid is selected which will insert our
DNA. The plasmid must have promoter, enhancer, antibiotic resistance and
restriction digestion gene sequences in it.
After that, the DNA is digested using the restriction endonuclease and ligated into
the plasmid.
The method of gene transfer is selected and the altered plasmid, now known as
the recombinant DNA is transferred into the host cell.
The altered cells are grown into the culture media, under aseptic conditions.
Importance of gene transfer:
Why are we doing gene transfer? The answer is to create a variation or to produce
a new phenotype that is useful, beneficial and important for us.
We have been using gene transfer techniques for so many years. In ancient times,
crossing two different plants was common.
Inbreeding and crossbreeding were used to make various abiotic and biotic resistant
plant species.
Even, bacteria follow the natural mechanism of gene transfer to surviving in harsh
conditions. Gene transfer was not new to use, perhaps but a substantial
improvement in techniques was done in the mid 19s.
In the present time, the gene transfer techniques that we have are efficient enough
and accurate.
We are using gene transfer techniques to modify organisms’ genomes, especially the
plant genome. To create various genetically modified plant species.
GM plant species are created to fight against biotic and abiotic stress, increase the
nutrient values, decrease the cost and make it available for all.
Furthermore, genetically modified organisms are constructed to produce proteins,
therapeutics, drugs and other important biomolecules.
The gene transfer technique is further practiced to cure disease and treat patients.