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Project on :-transgenic animals : mechanism and its application

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Submitted by:- Submitted to:-

Saurabh Kumar Dr. Lokendra kr. Sharma
B.sc. LLb (Hons.) UGC- Asst. professor
6th semester Biotechnology programme
CUB1413115017 CUSB
CUSB (GAYA)

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 Acknowledgement

I hereby take the opportunity thank Dr. Lokendra sir, for his consent and the inspiration that he radiates.
His jovial behaviour and ease making attitude eased my tension and the initial doubts that I had about my
potentialities. I also want to thank my friends who helped me a lot in preparing this project. I have also
taken help from several books and websites for doing this.

Ultimately, I once again thank Dr. Lokendra Sir, who made indelible impact on me which shall go beyond
the pages of this project and reflect in all my endeavours of life.

Hoping Acceptance and Appreciation from you, I hereby submit this project.

-Saurabh kumar

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TABLE OF CONTENTS

 Project objective……………………………………………………………………. 04
 Introduction………………………………………………………………………… 04
 Definition………………………………………………………………………….04-05
Some imporntant terms and its definition ……………………………………….. 04-05
 Brief hstory about transgenic animals…………………………………………….05-06
 Mechanism to produce transgenic animals………………………………………. 06-08
Recombinant DNA ……………………………………………………………….06-07
Methods for inserting DNA into cell…………………………………………….. 07-08
 Some transgenic animals and its method of production ………………………… 08-10
 Some benefits of transgenic animals……………………………………………...10-11
 Some other example of transgenic animals……………………………………… 11
 Conclusion…………………………………………………………………………. 11

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Project objective:
The purpose of this project on transgenic animals is to discuss the methods, means, and morals of
this controversial new technology, to examine the effects of this technology on society. The
methods for creating genetically modified organisms are explained in detail. Then the applications
and directions of the science are reviewed. Moral and ethical issues are discussed regarding the
benefits to society versus the cost to the animal. Next, the laws regulating the development and
growth of the transgenic industry are investigated. The project aims to inform the transgenic
animals, mechanism and its application and it also includes different types of transgenic animals.

Introduction:
Transgenic animals contain a foreign gene which
has been experimentally inserted into the normal
genetic component. They have been used
experimentally to investigate gene function,
development and disease. Transgenic animals
have also proposed as a means of testing agents
for oncogenicity or virulence. In some instances
where very large amounts of material are required
for therapy the use of transgenic animals may be
one of the few viable production strategies.
However in some respects the products resemble
classical biological in that they derive from a
whole animal rather than from definable culture
systems. The considerations which apply are therefore a blend of those relevant to recombinant
DNA (rDNA) derived materials and materials from less defined sources.

Definition:
An organism whose genetic characteristics have been altered by the insertion of a modified gene
or a gene from another organism using the techniques of genetic engineering.1

Some important terms and its definition:

Forebears: the animals from which the egg and sperm used to create the genetic founder were
derived.

Host: the recipient mother in whom the embryonic genetic founder was implanted.

1
http://medical-dictionary.thefreedictionary.com/transgenic+animals

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Genetic founder: the transgenic animal resulting from the introduction of the foreign DNA into
the embryo or fertilized egg.

Production founder: a transgenic animal used as a source for the generation of production animal
herds.

Production animals: the immediate offspring of the production founder.

Brief history about transgenic animals:

In 1866, Gregor Mendel, a man know today as the “father of Genetics” published a paper called
“Experiments on Plant Hybridization”, where he studied inheritance and traits in peas. He
concluded that there were two laws governing the inheritance of plants:

 The law of segregation, and

 The law of independent assortment.

In 1869, Fredrick Meischer discovered and isolated DNA from a cell nuclei, although it was
unknown at the time that DNA was genetic material. Later, in 1928, Fredrick Griffith studied
transformations between virulent (disease causing) and non-virulent bacteria in mice. Griffith
discovered what he called the transformation principle, noticing that by injecting mice with a live
non-virulent stain of bacteria mixed with a dead virulent strain, the mouse would die because
something from the virulent strain was being absorbed by the non-virulent one to transform its
characteristics into a virulent strain. In 1944, Oswald Avery, Colin MacLeod, and Maclyn
McCarty, using the work Fredrick Griffith had started, made a major break through by concluding
the Griffith transforming substance was DNA, the inheritance molecule (discussed in Avery et al,
1979).

Then, in 1970, Hamilton Smith discovered restriction endonucleases (enzymes that cut DNA at
specific sequences), giving us the tools to begin to work with DNA. Paul Berg, in 1972, was the
first to join two strands of DNA from different sources into a single plasmid. Finally, in 1973,
Stanley Cohen and Herbert Boyer made the world’s first transgenic organism. Using recombinant
DNA techniques, Cohen and Boyer altered the genome of E. coli by adding a gene from another
type of bacteria. In 1974, Rudolf Jaenisch created the world’s first transgenic animal by inserting
foreign DNA (from an SV40 virus) into early stage mouse embryos; not only did the mice carry
the modified gene but the mice also transferred the gene to their progeny . Since those landmark
transgenic experiments were performed, transgenic technology has emerged into the forefront of
research, and along with it has appeared legislation for regulating it. In 1975, the Asilomar
conference, lead by Paul Berg, was held in Pacific Grove, California to discuss the potential
biohazards and regulations for recombinant DNA technology. The conference came to an

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agreement that strict guidelines should be set by the National Institutes of Health in the United
States, and by comparable organizations in other countries, for regulating its use .2

Mechanism to produce transgenic animals:

A number of methods are currently in use for the creation of transgenic animals. One favoured
method involves the inoculation of the DNA into the pronucleus of a fertilized ovum, followed by
implantation into pseudo pregnant females. This results in a proportion of animals carrying the
transgene in the germ line which may be high in some species (e.g. mouse 5-30%) or low in others
(e.g. cows and sheep, 1-5%). Depending on the time when the transgene is incorporated into the
cellular DNA, mosaic animals may develop in which certain cell lineages carry the transgene while
others do not.

Other methods of creating transgenic animals involving retroviral infection of the embryo at an
early cleavage stage in the blastocyst result in only a proportion of the cells carrying the transgene,
and therefore a high proportion of mosaic animals some of which may not have the transgene
incorporated in the germ line at all. Methods for predictable site specific integration of sequences
into the host genome would have advantages for both controlled expression and safety.

The basic theory or method to produce transgenic animals are discussed below:

Before discussing the different methods I would like to discuss recombinant DNA.

Recombinant DNA
DNA is made up of four different compounds called bases,
adenine, thymine, cytosine, and guanine. They come together to
form a double stranded chain (Figure-1). Adenine is normally
paired with thymine, and cytosine is normally paired with
guanine. The order of the bases (the base pair sequence) is what
determines the biochemical function of the DNA molecule. Each
organism has a specific DNA sequence that makes up their
genome, and the differences in their genome allows each
organism to express different characteristics.

Recombinant DNA (rDNA) is an unnatural arrangement of figure -1 (ds DNA)

2
https://web.wpi.edu>pubs>unrestricted

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 DNA, made by combining different DNA stands
(sequences) to form a new sequence that would not
normally occur in nature. rDNA is often called “chimeric”
DNA in reference to the mythological chimera, an
amalgamation of several animals. The DNA is usually cut
using various restcriction enzymes, mixed, annealed to
allow compatible sticky ends to adhere, then sealed using DNA ligase. rDNA is usually inserted
into a “cloning vehicle” or “vector” whose purpose is to help amplify the material. Four different
kinds of vector can be used: plasmid, virus, cosmid, or an artificial chromosome. Plasmids are
used most frequently due to their ease of use and large copy number (Above fig). Plasmids are
small pieces of circular DNA that replicate in bacteria and are separate from the bacterial
chromosome. The process of inserting foreign DNA into bacteria is termed transformation, since
once inside the cell the plasmid DNA becomes expressed to give the bacteria new properties.
Plasmids usually contain an origin of replication, a specific DNA sequence where DNA replication
is initiated. Also contained within the vector is a multiple cloning site (MCS) that contains many
restriction sites to allow flexibility in inserting various types of cut DNA. Lastly, plasmids contain
a selectable marker that allows cells containing it to be selected for.3

Methods for Inserting DNA Into Cells:

There are various ways for inserting foreign DNA into a cell, including microinjection, chemicals,
electroporation, viruses, or sperm manipulation. When a plasmid, virus, or bacteriophage is used
to transfer genetic materials it is called transduction. In higher-level organisms like eukaryotes
(plants and animals), the genetic incorporation of DNA is called transfection.

DNA Microinjection:

Microinjection is the most reliable method for introducing DNA into a cell. This technique was
commonly used to create most of the early transgenic animals (although this technique was not
used to create the very first transgenic animal which used virus delivery). Using a newly fertilized
egg created by in vitro fertilization, an egg is held in place by a micro-tube suction device, and a
separate glass needle approximately 0.1 μm in diameter is used to insert transgenic DNA into the
male pronucleus (discussed in more detail below).

DNA Chemical Methods:

Some bacteria have the ability to uptake extra-cellular DNA, but this process is rare in nature.
However, some bacteria can be made “competent” to take up DNA by using various salts,
especially calcium or rubidium. Chemical competency is a good method for transforming bacterial
cells. For eukaryotic cells, chemical methods include complexing the DNA with a positively
charged polymer, then binding that polymer to the cell membrane. The DNA complex is released

3
abid

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into the cell, where is can be taken it to the nucleus and expressed. Human and mouse cells can be
efficiently and easily transformed when exposed to the packaged DNA.

DNA Electroporation:

DNA is a negatively charged molecule due to the presence of phosphate groups in its backbone.
So when it is exposed to a current of electricity, DNA will migrate towards the positive anode.
Electroporation is performed by exposing a cell culture mixed with transgenic DNA to a pulse of
high voltage electricity. The DNA is pulled through the cell membranes by the electric current.
Once inside the cell the DNA moves through the cytoplasm and becomes incorporated with the
genetic material inside the cell.

DNA Viral Delivery:

One of the most sophisticated methods for introducing foreign DNA into cells uses viruses. DNA
viral delivery was used to create the worl’s first transgenic animal. Jaenisch’s group infected
mouse embryos with a virus containing SV40 sequences, creating the first transgenic animal.
Normally a virus would destroy the host organism but using genetic engineering it was altered and
allowed the viral DNA to incorporat into the genome. This method involves encasing the altered
viral DNA in a protein coat that will allow the virus to infect the target cells. Viral delivery is
efficient, and improves the chances of transgene expression. In some case, like the Adeno-
associated viruses (AAV), the viral DNA inserts at specific host DNA locations. The two biggest
limitations of viral delivery are the size of the DNA molecule that can fit inside the virus, and the
chance that a chimera will be made without the ability to pass on its transgene.

Sperm Manipulation:

Some scientists are trying to develop new ways to incorporate modified DNA into a cell by using
recombinant sperm. In 2004, Noriyoshi Sakai and Kayoko Kurita were able to create a transgenic
fish by modifying sperm from a zebrafish to perform in vitro fertilization. The transgenic fish grew
into a fully developed adult. This technique is being expanded to other animals.

Some transgenic animals and its method of production:

Transgenic mice:-

Transgenic mice is a mice which is made by the genetic engineering or we called by the alteration
in their gene.

Method to prouduce

The transgene containing the DNA sequences necessary for the expression of a functional protein
is injected into the male (larger) pronucleus of uncleaved fertilized eggs through a

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micropipette.The early embryos are then
transferred into the reproductive tract of a
mouse rendered “pseudopregnant” by
hormonal therapy. The resulting pups
(founders) are tested for incorporation of the
transgene by assaying genomic DNA from
their tails. Founder animals that have
incorporated the transgene are mated with
nontransgenic mice and their offspring are
mated with each other to confirm germ-line
integration and to establish a line of
homozygous transgenic mice. Several
transgenic lines that have incorporated
different numbers of transgenes at different
integration sites (and thus express various
amounts of the protein of interest) are usually
studied.

Transgenic cow:-

Transgenic cows are those cow which is different from the normal cows because of altered DNA
or recombinant DNA.

Method to produce:- First design the gene construct. It is a

unit of DNA which includes an antibiotic resistant gene, a
tissue specific promoter sequence, the desired gene and a
stop sequence. After it sourcing of DNA and make a gene
construct. Here gene construct means all the information
needed for transfection into a bovine cell and expression of
the desired gene in cow. After doing this there will be
transfection of bovine cells and select a positive transgene.
Then make a transgenic embryo using nuclear transfer and
then confirm that it is a transgenic cow. A short description
is tried to be shown in the figure.

Transgenic fish:-

Transgenic fish are those fish which is genetically modified and different than its normal species.

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Method of production:-

The sperm and egg cell of the fish is taken and it is altered according to the
desire. The whole methoid is tried to explain in the figure.

Transgenic goat:-

Goat which is production of the genetically modified genes is known as

transgenic goat.

Method of production:-

The methods to prouce goat is well shown in the below figure.

Some benefits of transgenic animals:

Drug producing goat:

This is the first approved example of transgenic animal. The drug produced by this is called Atryn
which is anti-thrombin protein purified from the genitically modified goats.

Silk producing goat:

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This goat is made by the transferring silk gene from Orb spiders into goat. Each goat produces
severls germs of silk protein in her milk, the silk is extracted, dried to a white powder, and spun
into fibres. The fibres are stronger and more flexible.

There is also many other benefits of transgenic animals, which can be used for research, medicine
and industrial purpose.

Some other example of transgenic animals:

 Tracy the sheep-

This was the first transgenic animal produced in the year 1997 to produce a recombinant
protein drug in her milk. Alpha -1-antitrypsin was found in the milk of this sheep which is
useful to treat emphysema and cystic fibrosis.
 Andi – This was the first transgenic primate born in January 2000. It was the only live
monkey carrying the GFP gene.
 Alba – The EGFP bunny created in the year 2000 as a transgenic atwork.
 Glofish – it was developed in Singapore as a way to monitor water pollution.

Conclusion:
Transgenic animals may have advantages over existing production methods with respect to the
quantity and quality of the source material, which may reduce production costs and simplify
downstream processing. Other than veterinary and environmental concerns, which are outside the
scope of this document, the issues they raise are principally those of using a whole organism in
production rather than a potentially more predictable cell culture or fermentation system based on
a seed lot. These include microbiological and virological concerns, possible difficulties in
purification and the consistency of the production and purification process.

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