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university

Concepts of
biotechnology
Term paper

Topic – transgenic
animals

Submitted by :
Submitted to :
Isha nayyar Mr.
Harsh
Btb – a
Roll no.19
1040070134
 INTRODUCTION
Transgenic animals are those whose genetic material has been altered using genetic
engineering techniques. These techniques are generally known as recombinant DNA
technology.

In other words , the nucleus of all cells in every living organism contains genes made up
of DNA. These genes store information that regulates how our bodies form and function.
Genes can be altered artificially, so that some characteristics of an animal are changed.
For example, an embryo can have an extra, functioning gene from another source
artificially introduced into it, or a gene introduced which can knock out the functioning of
another particular gene in the embryo. Animals that have their DNA manipulated in this
way are knows as transgenic animals.

With this technology, DNA molecules from different sources are combined into one
molecule to create a new set of genes. This DNA is then transferred into an organism,
giving it modified or novel traits
• The new gene is inherited by offspring in the same way as the organism's own
genes.
• The transgenic condition is achieved by injecting the foreign gene into the
fertilized egg or into embryonic cells.
• The injected gene becomes part of the host cell's deoxyribonucleic acid (DNA)
within the chromosome and is then inherited by all the cells produced during
embryonic development. It is thus present in all the cells of the resulting adult
organism and is inherited by all its descendants.
• This technique can be used to generate animals or plants that express genes
conferring, for example, improved meat yield or resistance to specific diseases.
• It can also be used to create organisms that function as biological factories
manufacturing hormones and other biological compounds used to treat human

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 diseases. For example, bacteria can produce the hormone insulin, which is used
to treat diabetes. Such uses of transgenic organisms are widely referred to as
genetic engineering.

HISTORICAL DEVELOPMENT

• Prior to the development of molecular genetics, the only way of studying the
regulation and function of mammalian genes was through the observation of
inherited characteristics or spontaneous mutations.
• Long before Mendel and any molecular genetic knowledge, selective breeding
was a common practice among farmers for the enhancement of chosen traits, e.g.,
increased milk production.
• During the 1970s, the first chimeric mice were produced.
• The cells of two different embryos of different strains were combined together at
an early stage of development (eight cells) to form a single embryo that
subsequently developed into a chimeric adult, exhibiting characteristics of each
strain.
• The mutual contributions of developmental biology and genetic engineering
permitted rapid development of the techniques for the creation of transgenic
animals.
• DNA microinjection, the first technique to prove successful in mammals, was first
applied to mice (Gordon and Ruddle, 1981) and then to various other species such
as rats, rabbits, sheep, pigs, birds, and fish.
• Two other main techniques were then developed: those of retrovirus-mediated
transgenesis(Jaenisch , 1976) and embryonic stem (ES) cell-mediated gene
transfer (Gossler et al., 1986).
• Since 1981, when the term transgenic was first used by J.W. Gordon and F.H.
Ruddle (1981), there has been rapid development in the use of genetically
engineered animals as investigators have found an increasing number of
applications for the technology.

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HOW ARE THEY CREATED ?
Materials required:

• Vectors generally used are

1. Fish vector (a plasmid) e.g - pRSV

2. P element vector (derived from drosophila and is a bacterial plasmid vector) e.g
.-pUC8.
3. BPV vectors (derived from bovine papilloma virus”transforming region”
+pBR32)
4. Mammalian virus vector :SV40
5. Retrovirus Vectors(derived from pBR32+retroviruses sequences)

• Promoters : a promoter sequence is the site to which RNA polymerase first binds
during the initiation of transcription;the affinity of RNA Polymerase to promoter
sequence in several order of magnitudes higher than that for other DNA
sequences. The presence of promoter sequence is very essential for the
transcription of DNA ;the promoter must be located at the end which has
initiation codon AUG .e.g –RSV(Rous sarcoma virys) ,beta lactoglobulin
promoter.
• Reporter or marker gene –produces a phenotype which is either easily or
specifically detected or that allow a differential multiplication of the cells ,the
former is called scorable vector while the latter is called selectable markers.e.g. of
scorable are CAT gene from E.coli transposan Tn9.

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The above mentioned , are the essential elements for the creation of
transgenic animals.

Process :

Firstly a suitable vector is chosen and the required gene is inserted into it i.e.
recombinant DNA is produced. Then,this recombinant DNA will be introduced in
the host cells (cultured cells) . selection of transformed cells and identification of the
cell containing the gene (using reporter or marker gene).multiplication of the
introduced gene in host.

Then it is transferred into an organism and that organism is called transgenic

organism .

Techniques used:

1. DNA MICROINJECTION - Direct microinjection of a chosen gene construct

from another member of the same species or from a different species, into the
pronucleus of a fertilized ovum.

2. EMBRYONIC STEM CELL MEDIATED GENE - Insertion of desired DNA

sequence into an in vitro culture of embryonic stem (ES) cells. Stem cells can give
rise to a complete organism. The cells are then incorporated into an embryo at the
blastocyst stage of development.

3.RETROVIRUS MEDIATED GENE TRANSFER - Retroviruses are used as

vectors which infects the host cells and the retrovirus integrate into the germ cells to
work.

DNA MICROINJECTION :

Method involves:

• Microinjection refers to the process of using a very fine needle to insert

substances at a microscopic or borderline macroscopic level into a single living
cell.
• It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers
in diameter penetrates the cell membrane and/or the nuclear envelope.
• The desired contents are then injected into the desired sub-cellular compartment
and the needle is removed.

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 • Microinjection is normally performed under a specialized optical microscope
setup called a micromanipulator.
• The process is frequently used as a vector in genetic engineering and transgenetics
to insert genetic material into a single cell.
• Microinjection can also be used in the cloning of organisms, and in the study of
cell biology and viruses.
• This method involves the direct microinjection of a chosen gene construct (a
single gene or a combination of genes) from another member of the same species
or from a different species, into the pronucleus of a fertilized ovum.
• It is one of the first methods that proved to be effective in mammals .
• The introduced DNA may lead to the over- or under-expression of certain genes
or to the expression of genes entirely new to the animal species. The insertion of
DNA is, however, a random process, and there is a high probability that the
introduced gene will not insert itself into a site on the host DNA that will permit
its expression.
• The manipulated fertilized ovum is transferred into the oviduct of a recipient
female, or foster mother that has been induced to act as a recipient by mating with
a vasectomized male.
• A major advantage of this method is its applicability to a wide variety of species

EMBRYONIC STEM CELL - MEDIATED GENE TRANSFER

This method involves:

• isolation of totipotent stem cells (stem cells that can develop into any type of
specialized cell) from embryos
• the desired gene is inserted into these cells
• cells containing the desired DNA are incorporated into the host’s embryo,
resulting in a chimeric animal
• Unlike the other methods, which require live transgenic offspring to test for the
presence of the desired transgene, this method allows testing for transgenes at the
cell stage.
• prior insertion of the desired DNA sequence by homologous recombination into
an in vitro culture of embryonic stem (ES) cells.
• Stem cells are undifferentiated cells that have the potential to differentiate into
any type of cell (somatic and germ cells) and therefore to give rise to a complete
organism.
• These cells are then incorporated into an embryo at the blastocyst stage of
development.The result is a chimeric animal.
• ES cell-mediated gene transfer is the method of choice for gene inactivation, the
so-called knock-out method.

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 • This technique is of particular importance for the study of the genetic control of
developmental processes.
• This technique works particularly well in mice.
• It has the advantage of allowing precise targeting of defined mutations in the
gene via homologous recombination

RETROVIRUS MEDIATED GENE TRANSFER

Method involves:

• A retrovirus is a virus that carries its genetic material in the form of RNA rather
than DNA .
• This method produces chimeras i.e. altered animals with mixed DNA.retroviruses
used as vectors to transfer genetic material into the host cell, resulting in a
chimera, an organism consisting of tissues or parts of diverse genetic constitution
• chimeras are inbred for as many as 20 generations until homozygous (carrying the
desired transgene in every cell) transgenic offspring are born At this stage
embryos carrying the transgene can be frozen and stored for subsequent
implantation
• The method was successfully used in 1974 when a simian virus was inserted into
mice embryos, resulting in mice carrying this DNA.
• To increase the probability of expression, gene transfer is mediated by means of a
carrier or vector, generally a virus or a plasmid and Retroviruses are commonly
used as vectors to transfer genetic material into the cell, taking advantage of their
ability to infect host cells in this way.
• Transmission of the transgene is possible only if the retrovirus integrates into
some of the germ cells.
• For any of these techniques the success rate in terms of live birth of animals
containing the transgene is extremely low.
• Providing that the genetic manipulation does not lead to abortion, the result is a
first generation (F1) of animals that need to be tested for the expression of the
transgene.
• Depending on the technique used, the F1 generation may result in chimeras.
• At this stage embryos carrying the transgene can be frozen and stored for
subsequent implantation

• At the end to conclude :.

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Gene transfer by microinjection is the predominant method used to produce transgenic
farm animals. Since the insertion of DNA results in a random process, transgenic animals
are mated to ensure that their offspring acquire the desired transgene. However, the
success rate of producing transgenic animals individually by these methods is very low
and it may be more efficient to use cloning techniques to increase their numbers. For
example, gene transfer studies revealed that only 0.6% of transgenic pigs were born with
a desired gene after 7,000 eggs were injected with a specific transgene.

PURPOSE OF TRANSGENIC ANIMALS :

• Contribution to human welfare

The benefits of these animals to human welfare can be grouped into areas:

1 . Agriculture

2 . Medicine

3 . Industry

The examples below are not intended to be complete but only to provide a sampling of
the benefits.

1. Agricultural Applications

• Transgenesis will allow larger herds with specific traits.

a).Breeding .

• Farmers have always used selective breeding to produce animals that exhibit
desired traits (e.g., increased milk production, high growth rate).

• Traditional breeding is a time-consuming, difficult task. When technology using

molecular biology was developed, it became possible to develop traits in animals
in a shorter time and with more precision. In addition, it offers the farmer an easy
way to increase yields. Scientists can improve the size of livestock genetically.

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 b) Quality :

• Transgenic cows exist that produce more milk or milk with less lactose or
cholesterol.

• pigs and cattle that have more meat on them and sheep that grow more wool.

• In the past, farmers used growth hormones to spur the development of animals
but this technique was problematic, especially since residue of the hormones
remained in the animal product.

c) Disease resistance :

• Scientists are attempting to produce disease-resistant animals, such as influenza-

resistant pigs, but a very limited number of genes are currently known to be
responsible for resistance to diseases in farm animals.

2. Medical Applications

a) Xeno-transplantation
Patients die every year for lack of a replacement heart, liver, or kidney. Transgenic pigs
may provide the transplant organs needed to alleviate the shortfall. Currently,
xenotransplantation is hampered by a pig protein that can cause donor rejection but
research is underway to remove the pig protein and replace it with a human protein.

b) Nutritional supplements and pharmaceuticals

• Products such as insulin, growth hormone, and blood anti-clotting factors may soon
be or have already been obtained from the milk of transgenic cows, sheep, or goats.

• Research is also underway to manufacture milk through transgenesis for treatment of

debilitating diseases such as phenylketonuria (PKU), hereditary emphysema, and
cystic fibrosis.

• In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk at 2.4
grams per litre. This transgenic milk is a more nutritionally balanced product than
natural bovine milk and could be given to babies or the elderly with special

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 nutritional or digestive needs.Rosie’s milk contains the human gene alpha-
lactalbumin.

c) Human gene therapy

• Human gene therapy involves adding a normal copy of a gene (transgene) to the
genome of a person carrying defective copies of the gene. The potential for treatments
for the 5,000 named genetic diseases is huge and transgenic animals could play a role.
For example, the A. I. Virtanen Institute in Finland produced a calf with a gene that
makes the substance that promotes the growth of red cells in humans.

3. Industrial Applications :

• In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into
the cells of lactating goats. The goats began to manufacture silk along with their milk
and secrete tiny silk strands from their body by the bucketful. By extracting polymer
strands from the milk and weaving them into thread, the scientists can create a light,
tough, flexible material that could be used in such applications as military
uniforms,medical microsutures, and tennis racket strings.

• Toxicity-sensitive transgenic animals have been produced for chemical safety testing.

• Microorganisms have been engineered to produce a wide variety of proteins, which in

turn can produce enzymes that can speed up industrial chemical reactions.

ETHICAL CONCERNS SURROUNDING TRANSGENESIS

This article focuses on the benefits of the technology; however, thoughtful ethical
decision-making cannot be ignored by the biotechnology industry, scientists, policy-
makers, and the public. These ethical issues are :

1. Use of animals in biotechnological research causes great suffering to the animals. But
most people seem to accept some animal suffering to serve the basic interest and welfare
of mankind , this attitude has been termed as interest-sensitive speciesism.

2. It is felt that by using animals for the production of pharmaceutical proteins we reduce
them to mere factories. This seems not to recognise that animals also are living beings
which feel pleasure and pain just as we do.

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3. Some people feel that animals should be regarded as equal to humans in that they have
the same basic rights as human beings. However, in most societies animals are relegated
to a position several steps below that of man.

4. An argument attempts to focus on integrity of species in that each biological species

has a right to exist as a separate identifiable entity. But biologists do not regard a species
as a fixed, water-tight entity; rather they are regarded as dynamic, constantly evolving
groups.

5. Finally, the introduction of human genes into animals, and vice versa, may be seen by
many as clouding the definition of "humanness". But most of the known human genes are
not unique, and comparable genes do occur in animals. In addition, many retroviruses
have integrated into the human genome without any recognisable devaluation of our
humanness.

6.It is unethical to modify an animal's genetic make-up for a specific purpose, without
knowing if there will be any side-effects that will cause suffering to the animal.

Conclusion:

Interestingly, the creation of transgenic animals has resulted in a shift in the use of
laboratory animals — from the use of higher-order species such as dogs to lower-order
species such as mice — and has decreased the number of animals used in such
experimentation, especially in the development of disease models. This is certainly a
good turn of events since transgenic technology holds great potential in many fields,
including agriculture, medicine, and industry.

RELIGIOUS VIEWS OF TRANSGENIC ANIMALS :

Against transgenic animals:

• God laid down the structure of creation and any tampering with it is sinful.
• Manipulating DNA is manipulating 'life itself' - and this is tampering with something
that God did not intend humanity to meddle with

In favour of transgenic animals:

• As human beings have been given 'dominion' over the animals, they are entitled to
tamper with them.
• Palaeontology shows that the structure of creation has changed over time as some
species became extinct and new ones came into being. They say that this shows that
there is nothing fixed about the structure of creation.

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TRANSGENIC ANIMAL AND RELIGIOUS FOOD LAWS:

• Transgenic animals pose problems for religions that restrict the foods that their
species believers can eat, since they may produce animals that appear to be one
species, but contain some elements of a forbidden.

SOME TRANSGENIC ANIMALS:

Gene transferred Objective Acheivments Remarks

Cattle ,goat,sheep and Gene expressed Experimental
swine – in mammary stages; alpha –
Gene farming tissues ; proteins trypsin recovered
Human gene alpha 1 secreted in milk from tracey’s (a
antitrypsin ,tissue in functional sheep)at the rate
plasminogen activator,blood form of 1.5kg per
clotting factor 9 ,and protein lactation.
C
Rabbits Gene expressed Experimental
in mammary stages
Human genes – gene farming tissues ; proteins
interlukin2 ,growth harvested from
harmone , tissue milk
plasminogen activator ,

Bovine gene – alpha

lactoglobulin
Fish

Salmon or rainbow trout Increased body Upto 60% Transgenes

growth harmone growth increase in size stably
inherited ,growth
Anti-freeze protein gene improved by
,alpha globin gene , chicken selection
del crystalline protein gene Variable Genes expressed .transgenes are
etc. in transgenic stably integrated.
individuals
Mice

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human haemoglobin and
specific circulating
immunoglobulins(antibodies) Proteins from Genes Experimental
blood serum for expressed stages
blood ;proteins released
transfusion and in blood serum
disease
diagnosis

SUMMARY
• Transgenic animals are those whose genetic material has been altered using
genetic engineering techniques. These techniques are generally known as
recombinant DNA technology.

• Process used

Firstly a suitable vector is chosen and the required gene is inserted into it i.e.
recombinant DNA is produced. Then,this recombinant DNA will be introduced in the
host cells (cultured cells) . selection of transformed ce

lls and identification of the cell containing the gene (using reporter or marker
gene).multiplication of the introduced gene in host.

Techniques used:

1. DNA microinjection
2. retrovirus mediated gene transfer
3. embryonic stem cell – mediated gene transfer

• their importance lies in all the fields – agricultural , industrial ,and in medicines

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• considering them the creations of god and keeping in mind the issue of extinction
of species there are many ethical issues surrounding transgenesis.
• There are number of transgenic animals produced in the past and many are in
experimental stages as the research work is going on.
• A very dangerous consequence of transgenesis is the possibility of creating a
destructive organism (chimera) ; kill of certain animals ; unintentional side
effects.

REFERENCE

1. Meyers , R.A. (ed. 1997) - Molecular biology and

biotechnology : a comprehensive reference.

2. Old , R.W. and Primrose , S.B.(ed.2001) – Principles of

gene manipulations : an introduction to genetic engineering.

Internet links :

• http://www.actionbioscience.org/biotech
• www.sciencedirect.com
• Google > wikipedia >transgenic animals
• www.users-rcn.com

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