Transgenesis in Transgenic Animals and Its Applications

Middle East Journal of Applied Science & Technology (MEJAST)
(Peer Reviewed & Quarterly International Journal) Volume 2, Issue 2, Pages 21-28, April-June 2019
21 | P a g e Website: www.mejast.com
Transgenesis in Transgenic Animals and Its Applications
Muhammad Saeed1
, Muhammad Mohsin Aslam1
, Sidra Batool1
, Ayesha Ghazanfar1
& Rana Khalid Iqbal*1
*1
Institute of Molecular Biology & Biotechnology Bahauddin Zakariya University, Multan-60800, Pakistan.
Corresponding Author Email: khalid.iqbal@bzu.edu.pk
Article Received: 12 December 2018 Article Accepted: 17 March 2019 Article Published: 03 June 2019
1. INTRODUCTION
From a long the breeders have the struggle to improve the qualities of the animals such as to increase the production
of the animals, to increase the milk yield and also work to improve the feeding efficiency.in the earlier though the
phenotypic breeding was the best way to increase the desired qualities in the farm animals, but the selection based
on the genetic improvement has greatly increased the growth of the animals and the other desired characteristics
(Houdebine 2002; Mohammed et al. 2016). Before the genetic marker development, the selection was done on the
base of inherited characteristics. And earlier before Mendel and any molecular level study and discovery the
selective breeding was the main practice done by the farmers to increase the desired traits (Kessler et al. 2004; Ye et
al. 2011).
Transgenic animals are the animals that contain a foreign gene integrated into their genomes. The Transgenesis is
used to insert the gene of the required trait in the animals to take the expression of that gene in the animals and then
in its offspring by using the recombinant DNA technology. When the foreign gene is inserted in the animals it is
inserted from the germ line because all the cells should have the same expression of that gene. If we alter the germ
line then the modification will be transferred to the next generation also and we get the animals of our desired
traits.so the limitation in the Transgenesis is the efficiency and the control of the expression of the gene in the
production of the transgenic animals. Transgenesis include the alteration of the whole organism instead of the
animal cell and there may be the alteration of the body functions in vivo. The first experiment of Transgenesis in the
mammals was done on the mice(Gordon et al. 1980; Mohammed et al. 2016) then rabbits then pigs, sheep, and the
cattle (Hammer et al.; Murray et al. 2010).
The transgenic animals are very important to humans in many ways they are important in medicine. They are used
as the main source of biologically active proteins, as a source of organ transplantation (xenotransplantation) and
also used for the research purposes in cell level and also in gene therapy. In the agricultural level, they are also
important such as resistance to diseases, decrease in environmental impact, and enhance the carcass composition
and wool production and locational performances also (Tozaki et al. 2019).
ABSTRACT
Animal breeding is a process that is being used from many years ago. In the earlier days, the breeders use to increase the qualities in the animals by
using breeding and other methods. But by the invention of the molecular techniques, it is become easier to manipulate any animal and to enhance the
traits or different qualities in the animals. In this regard, different methods have used these methods are discussed in this article. The animal
transgenesis is used for different purposes such as in research purposes, like organ transplant source for humans, as protein extraction sources, as drug
extraction process, in hormones production, and in many other purposes.
Keywords: Transgene, Transgenic animal, Microinjection, Transposons.

Transgenesis help in the enhancement of the nutrients from the animals which constitute the quality of the whole
food and specific nutritional composition of the product. By the Transgenesis we can insert nutritional benefit traits
in the animals. For example, when we increase the omega 3 fatty acid amount in the fish which is being consumed
by the humans in the result there was a decrease in the heart diseases (Lai et al. 2006; Kumar et al. 2015). The
reason for carcass alteration is to lower the amount of fat that Is present in the carcass. So by altering the
metabolism or the uptake of the cholesterol the amount of fat will be decreased in the meat, egg, and cheese of the
animals. Or we can also insert the beneficial fats like omega 3 fatty acids which can get from the fishes (Lai et al.
2006; Kumar et al. 2015).
The main development in the methods which are used to transfer the genes in the animals is DNA microinjection,
retrovirus-mediated gene transfer, embryonic stem cell, and the somatic cell nuclear transfer methods to increase
the efficiency of the transferred gene. Each technique has different limitations and also has long term effects on
animals production (Zhang and Yu 2008; Pons et al. 2014).
2. TRANSGENIC ANIMALS
The animals whose genome has been altered by inserting the DNA by using human interventions are known as
transgenic animals. For the formation of transgenic animals, the DNA from outside is to be inserted in the genome
of the animals by the help of recombinant DNA technology, then the construct is stabled and maintained then it is
transferred in the animal to show expression and then passed into the offspring.
The first transgenic animal produced was the mice in1980(Sosa et al. 2010; Lomax and DeWitt 2013). In the earlier
days, the DNA was transferred by the microinjection in the fertilized egg of the mouse. So now it is not possible to
handle the integration of foreign genes by using this technique. The mice generated by using this technique are
called “overexpressors”.
The transgenic animals are created for many purposes such as to study the gene functions it is seen that about 95%
of the transgenic animals that are used for research purposes are the mice because 80% genes of the mouse works
similar to the humans genes, they have the short reproductive cycle and we can easily alter the embryo of the mice.
The transgenic animals are also used to build model organisms which are used for the study of different genetic
diseases. They are also used to improve the traits in the animals and for the production of the new animal products
(Melo et al. 2007).
Table 1: Names of transgenic species
Family Transgenic Species
Mammals mice rat rabbit sheep
Birds chickens Japanese quail
Fishes zebrafish goldfish
(Sosa et al. 2010)

3. FORMATION OF THE TRANSGENE
Before cloning it is necessary to form its multiple copies either using Polymerase chain reaction or by using vector.
In this process first extract the DNA cut it and then transfer it in small pieces then ligate into the vector by DNA
ligase and we get multiple copies of the DNA. (Kerr and Wellnitz 2003; Donovan et al. 2005; Maksimenko et al.
2013; Mohammed et al. 2016). The transgene formation requires the few main parts assembly. First is the
promoter, it is the regulatory sequence which tells that where and when the transgene is active, an exon, a protein
coding sequence which is obtained from the cDNA, stop codon, intron, poly A and enhancer sequences. (Zhang and
Yu 2008)
The promoter is obtained from an upstream portion of the mammalian genes which has the transcriptional starting
portion and regulatory transcriptional sequences. The protein is the cDNA sequence which is extracted from the
RNA of our desired gene. This contains a start codon(AUG) and also contain the stop codon(UGA, UAG, UAA).
This also contains thee Kozak sequence due to that the ribosome can identify the proper translation start and stop
codon sites present on the mRNA(Murray et al.). The introns cause a greater expression of the gene. The intron has
important effects on the stability of the mRNA and used as the translocator from the nucleus to the cytoplasm. The
stop codons, Poly A sequence, terminator sequences present in the transgene and the enhancer sequences control
the proper pattern of gene expression.
4. METHODS USED TO TRANSFER THE TRANSGENE
The formation of the transgenic animals indicates that the transgene is present in the gametes or in the germ cells so
that it may transfer to the progeny. For this purpose, the DNA is to be inserted the organisms by using different
methods(WARD et al. 2016). The main methods used to transfer the transgene are microinjection, embryonic stem
cell-mediated method, by use of the retrovirus-mediated method, sperm-mediated, by using transposons and
nuclear transfer method.
4.1. DNA microinjection
The DNA microinjection into the nucleus of the embryos was the first successful techniques in mammals. This
technique was firstly used in 1980 in which the gene was transferred into the mouse by using DNA microinjection.
In this technique, the transgene construct is transferred into the fertilized oocyte by the help of a needle. In this
method, the target cell is placed under the microscope and the other two micromanipulators, one holds the pipette
and other one hold the needle whose diameter is of about 0.5-5 micrometer are used for penetration in the cell
membrane (Maksimenko et al. 2013). This method based on the random insertion of the transgene through the
cellular DNA repairing pathway and the efficiency of integration is 1-4% (Melo et al. 2007).
The protocol of the method is the female is given an injection of gonadotropin of the pregnant female which cause
her to release more eggs three to four times. These eggs are then fertilized in glass or in the vivo(Mohammed et al.
2016). After in vivo fertilization the eggs are taken are the oviduct is removed. At this stage, the two pronuclei have
not fused till yet. Now we take the constructed gene of interest and is injected with the use of microinjection
(Brundige et al. 2018). The pronucleus of the male is chosen for integration because of its large size. When the

pronuclei fuse produces the zygote then the embryo is implanted in the surrogate mother. Then this mother is made
pregnant by giving her the hormones or mating with the male that made her false pregnant and made the uterus
capable of receiving the egg. The success rate of microinjection is very low. This method is efficient in mammals
but not efficient in fishes, Xenopus, and chicken. The integration of the transgene is injected in the gonad
syncytium in the insects and worms. Its main advantage is its wide application to different varieties of species.
(Pfeifer 2006)
4.2. Retrovirus-mediated gene transfer
Retrovirus is the type of virus containing RNA as genetic material. These viruses are used as the vector to transfer
the gene in the cell. This virus when an attack on the cell the RNA cannot integrate into the cell so the enzyme
reverse transcriptase is present which convert the RNA into the cDNA then it will convert it into the DNA which
will then integrate into the host genome. We remove the gag-pol and enveloped proteins which cause the infection
and then they are replaced with the gene of interest and these two things are added in another compliment cell and
then allowed to attack the embryo and then the embryo allowed to grow at blastocyst level and then it will be
transplanted in the surrogate mother this method gives efficient transfer in many species of mammals(Pfeifer 2006)
4.3. Gene transfer by use of transposons
The transposons are short DNA sequences which are naturally present in the genome and they have the capacity to
replicate by themselves and can integrate into multiple sites. It is seen that when we insert the foreign DNA
especially in non-mammals embryo cytoplasm it is not integrated into the genome. So to increase the efficiency of
integration the scientists use the transposons. These transposons contain minimum one gene that codes for
transposase that can initiate the integration of the foreign DNA. The protocol is we firstly insert the gene of interest
in the transposons. Then these hybrid transposons are microinjected in the embryo which is of about one day. The
gene of interest then will integrate into the embryo. This method is used for the production of transgenic insects,
fishes, silkworms, chicken, and mammals(Mohammed et al. 2016)
4.4. Transgene transfer by using embryonic stem cells
The embryonic stem cell is the cells that are taken from the embryo blastula stage and are capable of differentiating
into any other type of the cell. These stem cells are pluripotent means can differentiate into any type of cell, so these
have major use in the research field, medicine field, and agricultural research. The ES cells are taken from the
blastula stage and are grown in vitro. Then these grown ES cells are transfected with the gene of interest. Then these
cells are selected by using any type of marker(Mohammed et al. 2016). These cells then transferred in the blastocyst
of the animal and allow transferring in somatic and the germ line tissues. Then they are transferred to further
generations. The stable transgenic lines are then obtained from those animals which have the stem cells in their
germ cells. Then these embryos are-cultured and are injected with the ES cells that are transfected and then they are
transferred into the blastula of the surrogate mother. There it integrates and forms the chimera animal, which
contains two types of cells containing foreign DNA and others are non-containing. The complete homozygous
animal can be developed by using selective breeding (Ittner and Götz 2007).

4.5. Transgene transfer by sperm mediated method
The sperm is used as a vector not only to transfer their own DNA but act as a vehicle for the foreign DNA also. This
method is difficult to use because of the degradation of DNA(Zhang et al. 2008). By using this method transgenic
mice and rabbits were formed by mixing the sperm with the DNA and in the presence of the dimethyl sulfoxide, by
the help of fertilization. The DNA, when exposed to the sperm cells, are joined with the sperm head by
protein-protein interactions. It is seen that the two main steps are involved in this method.in the first, the DNA join
with the head of sperm cell by protein interactions. The second is the delivery of the sperm cell to the oocyte for the
fertilization.
The sperm cell is taken and its plasma membrane is damaged by freezing or thawing, then the sperm cell is
transferred in the cytoplasm of the oocyte where it integrates by the fertilization process. This method is best for
mice and also in pigs this technique is successfully done in the cattle(Mohammed et al. 2016).
Many researches have been done to increase the efficiency of the DNA to bind with the head of the sperm cell one
method is to attach the DNA with the sperm cell by using the antibody. This protein will function as the recognizers
of the binding sites for the DNA that are common in sperm sites of cattle, pigs sheep and humans.
5. APPLICATIONS OF THE TRANSGENIC ANIMALS
5.1. Applications in human health
The most important application of the transgenic animals is the productions of recombinant proteins in the milk of
the animals produced by the mammary glands and use it for the benefit of mankind. This process is known as Gene
Pharming. The mammary glands are the best site for the production of the proteins because we can collect it easily
without giving any harm to the animals.
5.2. Production of the recombinant proteins
Many novel proteins are being got from the transgenic animal's different types of proteins are being extracted from
the mammary glands of the transgenic sheep and the goats such as Antithrombin III, the antitrypsin and the tissue
plasminogen activator. Another protein is taken from the rabbit's milk is glycosidase.
Table II: proteins obtained from the transgenic animals. (Zhang and Yu 2008)
Name of protein Source of the protein Treatment
Antithrombin III Goat Thrombosis
Tissue plasminogen activator Sheep, Pig thrombosis
Human serum albumin Cow, sheep Maintenance of blood volume
Monoclonal antibodies Chicken, cow, goat Vaccine production
Fibrinogen Cow, sheep Wound healing

5.3. Source of blood substitutes
The transgenic swine are developed which has the capability of production of hemoglobin that works as same as the
human hemoglobin has the capacity of binding the oxygen with it and can be extracted from the blood. (5r150)
5.4. Model for human genetic diseases
Many genetic diseases study are done on animals such as rabbits, mice, goats, and pigs. They are best used for the
experiments of diseases such as cystic fibrosis, cancer and also for the treatment and study of different
neurodegenerative diseases.
5.5. Disease resistance
The application of Transgenesis is the alteration of the genes that controlled the immunity which is the major
histocompatibility complex (MHC) they also increase the resistance capacity of the animals to the diseases.in this
way transgenic mice have been developed which show resistance against the transmissible gastroenteritis by the
production of antibodies in their milk.
The transgenic cows have been developed which produce lysostaphin by their mammary glands which act as a
resistance against the Staphylococcus aureus bacteria which cause mastitis disease in the cow.
5.6. Tissue repair source
The induced pluripotent stem cells are directly inserted in the damaged retina of the mice where then the stem cell
is grafted and repair the nerve cells that are damaged.
5.7. Source of organ transplantation (xenotransplantation)
The limited availability of organ donor in humans they move to the production of the organs in the transgenic
animals. The transgenic pigs, goats are used as the source of organ transplantations such as heart, kidneys, liver,
and lungs.
6. CONCLUSION
Transgenesis is the technique in which a foreign gene is inserted in the host DNA to increase the quality of the
desired trait in the host. There are different methods to manipulate the DNA that are discussed in the article, these
are DNA microinjection, and Retrovirus-mediated gene transfer, Gene transfer by use of transposons. The
manipulation of DNA of animals is very useful in many ways. By increasing the qualities or different traits in
animals are helping the humans in many ways they are acting as drug sources, as for testing animals, as therapeutic
protein sources, like an organ transplant source. The animal transgenesis technology has a major advantage that by
increasing population the animal usage increased and their population so by using this technique the animals are
grown fastly and increased the qualities and traits.
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