PROJECT REPORT ON :-

ANIMAL CLOANING & APPLICATION & ETHICAL ISSUES

ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

UNIVERSITY OF CALCUTTA

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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

PREFACE

Biotechnology is a diverse field which involves either working with living cells or
using molecules derived from them for applications oriented toward human welfare
using varied types of tools and technologies. This report has been prepared as my
project for fulfillment of the practical syllabus of B.SC Zoology course DSE2:
Animal Biotechnology under CBCS semester 6 examination under University Of
Calcutta.
This project report is prepared with the view to include all the details regarding the
project “ANIMAL CLONING & APPLICATION & ETHICAL ISSUES” that I
carried out. In the course of the project I mainly studied how animals are cloned ,
techniques of cloning, methods of cloning , future of cloning, application and ethics of
cloning. we have taken an extensive study on every detailing of cloning and other
perspectives. In the above mentioned project report we were accompanied by our
Faculties Dr. Malabika Bhattacharjee.

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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

ACKNOWLEDGEMENT
I would like to express my special thank of gratitude to Dr.
Malabika Bhattacharjee who gave me the golden
opportunity to do this wonderful project on the topic of
‘Animal Cloning & Application & Ethical issues’ , which
also helped me in doing a lot research and I come to know
about so many new things. I’m really thankful to her
constant support, guidance and encouragement.
Secondly I would also liked to thank my friends , who
directly and indirectly helped me a lot in finishing this
project report within the limited.
I also acknowledge with deep sense of reverence, my
gratitude towards my parents and members of my family,
who has always supported morally as well as
economically.
It helped me to increase my knowledge and skills.
THANK YOU ALL FOR YOUR KIND SUPPORT AND
COURAGE.

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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

INDEX
SL. NO. TOPIC PAGE
1. INTRODUCTION OF CLONING 6
2. HISTORY OF CLONING 6
3. TYPES OF CLONING 6
A. DNA CLONING OR GENE CLONING 6
B. REPRODUCTIVE CLONING 7
C. THERAPEUTIC CLONING 8
4. IMPORTANCE OF CLONING 9
5. BRIEF IMPORTANCE OF CLONING 10
6. DOLLY THE SHEEP 11
7. BACKGROUND OF DOLLY 11
8. HOW DOLLY WAS FORMED 12
9. DOLLY’S LIFE AND DEATH 13
10. DRAWBACKS 14
11. ADVANTAGES OF CLONING 14
12. DISADVANTAGES OF CLONING 15
13. APPLICATION 16
14. ETHICAL ISSUES 17
15. CONCLUSION 19
16. REFERENCE 20

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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

Cloning is a process of generating a genetically identical copy of biological unit (

eg- DNA sequence, cell or organism). “Clone” comes from the Greek word “klon,”
meaning twin. Cloning happens often in nature—for example, when a cell
replicates itself asexually without any genetic alteration or recombination.
Prokaryotic organisms (organisms lacking a cell nucleus) such as bacteria
create genetically identical duplicates of themselves using binary fission or
budding. In eukaryotic organisms (organisms possessing a cell nucleus) such as
humans, all the cells that undergo mitosis, such as skin cells and cells lining the
gastrointestinal tract, are clones; the only exceptions are gametes (eggs and sperm),
which undergo meiosis and genetic recombination.

The first study of cloning took place in 1885, when German scientist Hans Adolf
Eduard Driesch began researching reproduction. In 1902, he was able to create a set
of twin salamanders by dividing an embryo into two separate, viable embryos,
according to the Genetic Science Learning Center.
In 1952 by American scientists Robert W. Briggs and Thomas J. King, who used
DNA from embryonic cells of the frog Rana pipiens to generate cloned tadpoles. In
1958 British biologist John Bertrand Gurdon successfully carried out nuclear
transfer using DNA from adult intestinal cells of African clawed frogs ( Xenopus
laevis).
In 1985 , British biologist John Gurdon cloned frogs from the skin of adult frogs. On
July 5, 1996, a female sheep gave birth to the now famous Dolly, a Finn Dorset
lamb- the first mammal to be cloned from cells of an adult animal at Roslin Institute
in Scotland.

There are three different types of artificial cloning-

1. Gene cloning or DNA cloning

2. Reproductive cloning

3. Therapeutic cloning

DNA cloning technique used to create many copies of DNA fragments. This
technique can be used to clone random fragments of DNA, specific sequence of
DNA or can be used to clone entire gene sequence.
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DNA cloning process comprises many steps – first the interested DNA fragment is
extracted from the genome , by the help of restriction enzyme or can be synthesized
chemically . Polymerase Chain Reaction or cloning vectors are used to produce
multiple copies of DNA fragments.

( Fig1: Gene Cloning or DNA cloning)

Fig1 :- History of cloning

Cloning which is used for producing the exact copy of an existing organisms are
called reproductive cloning. Reproductive cloning is performed using a technique
known as somatic cell nuclear transfer. During the procedure nucleus is extracted
from somatic cell . Somatic cell is any cell from the body other than germ cells.
Then the nucleus of the egg is also removed, the extracted nucleus is then
transferred into enucleated eggs. Eggs start to divide after receiving the external
stimuli. Eggs will divide and grow and finally divide into embryo. The embryo is
then implanted to gestational surrogate mother.
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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

Reproductive cloning have some issues like – cloning which are produced have
shorter lifespan compared with parental species.. Reproductive cloning also
imposes the risk of losing genetic diversity of the nature.

( Fig2: Reproductive Cloning )

Therapeutic cloning is used for medical treatment. This cloning is mainly used to
grow organs, which can be used to replace the non functional organs of the patients.
It can also be used to generate skin to treat burn victim, can be used to create nerve
to treat patients suffering from nerve degenerative disease. This cloning is also
done by using somatic cell nuclear transfer technique. The main difference is that
the dividing egg is used to extract stem cells.
Therapeutic cloning plays major roll in medical treatment. Doctors with the help of
therapeutic cloning technique can grow replacement organs for damaged body
parts of the patients. Therefore it reduces the usage of immunosuppressant drugs
and also risk of organ rejection by the body.
SCNT in the context of therapeutic cloning holds a huge potential for research and
clinical applications including the use of SCNT product as a vector for gene
delivery, the creation of animal models of human diseases, and cell replacement
therapy in regenerative medicine. The combination of therapeutic cloning and gene
therapy offers a great potential for patient- specific rescue of a genetic mutation of
the loss-of-function type, resulting in lowered or eliminated activity of a particular
protein. Therapeutic cloning used in cell replacement therapy has the potential to
create various types of tissues such as osteoblasts to counteract osteoporosis, and
spinal cord regeneration following trauma.
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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

( Fig 3 : Therapeutic cloning )

1) DNA cloning can be used to make proteins such as insulin with biomedical techniques.
2) It is used to develop recombinant versions of the non-functional gene to
understand the functioning of the normal gene. This is applied in gene therapies
also.
3) Control sequence of DNA can be identified and analyzed.
4) Therapeutic cloning technique help to from organ, and these organs are
genetically and immunologically same as patient’s own organ.
5) Animal cloning can help in preserving endangered species and to maintain biodiversity.
6) These cloned animals can be utilized in biomedical research and also used as model
for treating human diseases.
7) Cloned animals utilized as a commercial endeavor and help in producing large
quantity of food product.

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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

Reproductive cloning is a method used to make a clone or an identical copy of an

entire multicellular organism. Most multicellular organisms undergo reproduction
by sexual means, which involves the contribution of DNA from two individuals
(parents), making it impossible to generate an identical copy or a clone of either
parent.
The origins of nuclear transfer remount discoveries with amphibians by Spemann
(1938), who demonstrated that nuclei of newt salamanders are pluripotent up to
eight-cell stage, leading intensive studies with nuclear transfer in Rana pipiens and
Xenopus laevis, attempting to understand the nuclei participation of differentiated
cells in reprogramming. Studies by Brings and King (1952) showed that amphibian
oocytes receiving blastula nuclei could be reared to maturity.
In mammals the first nuclear transfer studies were done in mice, in which illmense
and Hoppe (1981) reported that this technique could be used to produce mice clones
from embryo cells. In domestic animals, Willadsen (1986) published the first report
with lamb clones production. This accomplish was confirmed after with bovines,
rabbits, swine, and others. And, at 1996 Dolly brought the accomplishment of
mammal nuclear transfer form adult cells.

( Fig 4 : Steps of Animal Cloning )

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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

Dolly the sheep may have been the world's most famous clone, but she was not the
first. Since 1996, when Dolly was born, other sheep have been cloned from adult
cells, as have mice, rabbits, horses and donkeys, pigs, goats and cattle. In 2004 a
mouse was cloned using a nucleus from an olfactory neuron, showing that the
donor nucleus can come from a tissue of the body that does not normally divide.
Dolly the Sheep, born in 1996, is by far the most famous cloned animal, but she was
nowhere near the first. Producing an animal clone from an adult cell is obviously
much more complex and difficult than growing a plant from a cutting. So when
scientists working at the Roslin Institute in Scotland produced Dolly, the only lamb
born from 277 attempts, it was a major news story around the world.

(Fig 5: Dr. Ian Wilmut, a British

scientist at the Roslin Institute in
Scotland who led the team that cloned
Dolly the sheep from an adult
cell)

BACKGROUND OF DOLLY

Finn Dorsets are a Common Scottish beep of sheep that are all white. Another
popular beat is the Scottish blackface , which is very similar to a Finn dorset,
expect that it is larger and , of course, has a black face . Both breds figured
prominently in the experiment that led to Dolly. A Finn Dorset has a life span
of about 12 years.
Dolly remained alive and well long after her birth, with a functional heart,
liver, brain, and other organs, all derived genetically from the nuclear DNA of
an adult mammary gland cell. The technique used to produce her later became
known as somatic cell nuclear transfer (SCNT).

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1. At first scientists collected an ovine mammary gland epithelial (OME) cell from
Finn Dorset and isolated the cell, grown in culture, and then starved in order to
arrest them in G 0 of the cell cycle.

2. They also collect egg from Scottish Blackface, the egg donor and from Scottish
blackface’s egg they they took out the nucleus with the help of a pipette and the
eggs become enucleated ,so there is no genetic material from the Scottish
blackface.

3. The karyoplast (the cell donating the nucleus) is injected into the space between
the zona pellucida and egg cell. The karyoplast and the cytoplast (the enucleated
egg) are then fused together with an electric current. This is a much gentler
procedure, compared to injecting the nucleus into the egg by poking another hole in
the egg’s membrane (the first hole was made when the egg’s chromosomes were
removed).

4. Hybrid cell is formed and the genetic material is totally similar to Finn
Dorset and the cytoplasmic material would be a mix up of Finn Dorset and
Scottish Blackface.

5. Once development begins, the embryo is encased in agar and incubated

temporarily in the oviduct of a blackface after which when the embryo reaches the
blastocyst it is implanted to the oviduct of the final surrogate mother, also a
blackface, which carries it to term.

6. The Scottish blackface gave birth to Dolly When the progeny was born the
progeny was exactly similar to the Finn Dorset.

(Fig 6: Light micrograph of a sheep egg being injected with an embryonic cell during sheep
cloning)
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(Fig 7: To clone a mammal such as a sheep, cells from the udder are isolated, grown in culture, and then
starved in order to arrest them in G 0 of the cell cycle. Unfertilized egg cells from another sheep are also
harvested and the nucleus is removed from them. An electrical stimulus fuses the G 0 udder cell with the
enucleated egg, thus placing a somatic cell nucleus into an undifferentiated cytoplasm The eggs that
result are put back into a foster mother, and the offspring are screened for DNA identical to the donor
sheep.)

By all accounts, Dolly was a healthy sheep right up to her death, at the age of six, on
February 14, 2003. The reason for euthanasia was a virus infection that gave rise to
lung cancer. Dolly’s breed of sheep, the Finn Dorset often reaches 11 or 12 years.
Like most cloned animals Dolly died somewhat prematurely. Dolly had telomeres
that were 20% shorter than normal for a sheep her age, which might have
contributed to premature aging. Dolly gave birth to six healthy lambs (one in 1998,
two in 1999, and triplets in 2000). When she was born many scientists feared she
might develop a variety of medical disorders because all her cells have abnormally
short telomeres. Telomere length decreases as cells divide; the cell used to clone
Dolly came from a six year old sheep and had been kept in cell culture for many
weeks.
At birth, Dolly’s genome was already middle-aged .Wilmut’s team analyzed
Dolly’s telomeres in 2000 and showed that they were indeed about 20 percent
shorter than is normal for a Finn
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Dorset of Dolly’s age. But the damaging effects of shortened telomeres on the
health of a cell and the physiology of the organism are still highly speculative. In
2001, Dolly was reported to have developed arthritis in her left knee and hip joint.
Arthritis is a common ailment in sheep, although it usually affects other joints.
There is no way to know if the arthritis was related to the length of her telomeres or
to the fact that she was a clone, simply because there are too few clones available
for the information to be meaningful.

One of the main drawbacks of cloning is that if the original organism has genetic
defects, these transfer to the clone as a copy of the original. The first clone, Dolly
the sheep, born to a surrogate in 1996, was a genetic copy of a six-year old sheep.
Dolly only lived to six years old herself, the bottom end of a sheep's average life
expectancy. At the age of five she developed arthritis, and the researchers put her to
sleep at age six because of tumors in her lungs, which may have been in the genome
of the original.
It may be normal that clones have a different sensitivity to stress compared to
conservative animals due to it they are more prone to pathogens which can also
increase the death rate of clones. To prevail over the low efficiency of cloning,
various different method have been tried such as using different types of donor cell
lines, a variety of culture systems, different fusion methods, and chemicals.35 Some
of these research efforts have led to minor upgrading in the quality of cloned
embryos, which is closely related with embryo development and offspring
productivity.

1. One big advantage of cloning is that it allows infertile couples to reproduce, and the
child would be genetically modified to fit what the parents want to appeal to other
people. During the process, somatic cells are acquired from the male’s sperm and
are injected into the female’s egg for fertilization. By the time the embryo has
developed, it is then implanted to a surrogate mother, who will carry it for 9 months
until birth. This means cloning can potentially ease fertility problems. The children
produced will have the DNAs and qualities of both parents, instead of just one of
them.

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2. Genetic cloning allows scientists to take small amounts of cells from a certain organ
and use them to generate and harvest new organs that are entirely functioning.
Considering the huge number of people on organ donation waiting lists, this is
extremely beneficial.
3. Starting with the same procedure as adult DNA cloning, therapeutic cloning allows
a resultant embryo to grow for days, where the stem cells would then be extracted
and encouraged to grow into human tissue or complete human organs that will be
used for transplants or treatments of certain diseases. The end result would not be a
human being, but rather a piece of nerve tissue, replacement organ or quantity of
skin.

1. There is still a lot of repercussions and effects of cloning that remain unknown to
date. After all, it is a new world of science that is still continually being discovered,
and there is no convincing way to tell what the mental, social and medical
consequences may be endured due to it. Also, the research and testing that are
needed before embarking on something as insane as cloning humans still does not
exist. Considering the problems that already exist with animal cloning, we can
safely presume that the procedure will be a very large obstacle to begin with.
2. One of the real possibilities of cloning is cell mutation, which is known to result
in new and more aggressive genetic diseases to begin within humans, creating a lot
of problems. In fact, many people believe that such an event will be the demise of
civilization.
3. As stated above, cloning can cause cell mutation, which is still highly possible
even if the technique uses the cells of the recipient’s organ. This can result to a
substantial difference in the cell make-up between the original and replicated organ.
4. The ability of humans to live greatly relies on the diversity of genes, which
comes from parents who have different sets of genes. One big disadvantage of
identical genes is that they will weaken our adaptations and power, which can make
us subjected to certain diseases easily. Moreover, we should remember that the
beauty of humanity lies in the differences among individuals, and cloning ruins this.
Not to mention that it would eliminate surprise and predict expectancy.
5. One of the most unfavorable consequences of cloning is in-breeding, where
everyone will be having the same genotypes, which can keep reproducing among
themselves. This means this procedure would lead us to extinction. As what Richard
Nicholson of the British Bulletin of Medical Ethics said, research on cloning may
well be “sowing the seeds of our own destruction.

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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

Aside from showing that whole animals can be cloned, there are practical reasons.
For millennia, humans have bred farm animals in attempts to improve them.
Genetic duplication allows an improved animal to be widely distributed relatively
quickly. In addition, a flock or herd of genetically identical animals will give wool,
milk, eggs, or meat of a more standardized quality. Conversely, genetically identical
animals will all be susceptible to the same infections, and epidemics will spread
faster and further.
The reason why Dolly was cloned using a mammary gland cell should now be
apparent. If a foreign protein is expressed in this tissue, it will be secreted into the
milk and be easy to harvest commercially. Once a good transgenic cell line has been
established in culture, nuclear transplantation can be used to generate several
genetically identical animals for production purposes. Such transgenic cells can be
stored over the long term by freezing in liquid nitrogen and have been referred to as
protoanimals.
The most important use of animal cloning is in combination with transgenesis.
Previously created transgenic animals may be cloned for speedier distribution of the
product. However, it is also possible to introduce transgenic DNA during the
cloning process. The Roslin Institute, where Dolly was born, has since cloned sheep
carrying the gene for human factor IX . The transgene was inserted into the nuclear
donor cells while in culture. A variety of other transgenic animals carrying
pharmaceutically important proteins have also been cloned.
Cloned mammalian animals can be made by replacing the chromosomes of an egg
cell with a nucleus from the individual to be cloned, followed by stimulation of cell
division and implantation of the resulting embryo. Cloned individuals, whether born
at the same or different times, will not be physically or behaviorally identical with
each other at comparable ages. Stem cells are cells that have an extensive ability to
self-renew and differentiate, and they are therefore important as a potential source
of cells for therapeutic transplantation. Embryonic stem cells derived through
nuclear transplantation into eggs are a potential source of pluripotent (embryonic)
stem cell lines that are immunologically similar to a patient’s cells. Research with
such cells has the goal of producing cells and tissues for therapeutic transplantation .

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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

ETHICAL ISSUES
Ethics identifies a code of values for our actions, especially toward other humans. In
simple terms, ethics could be considered a guide to separate right from wrong and
good from evil. The area of ethics that deals with the implications of biological
research and biotechnological applications, especially regarding medicine, is called
bioethics . It considers social and moral aspects and potential outcomes of the use of
biological and medical technologies.
Animal cloning may give rise to two different types of moral problems –
1. It may create some negative impacts on animals, human beings and the environment

2. It may disobey various important moral principles. The negative impacts that
occur to animals can be understood both narrowly and broadly. While the broadly
understood negative impact on animals includes the adverse effect of cloning on
other animals or threatened species.
Risk to clone animals
From the literature, the term "risk" is commonly used and has many different
meanings. Risk is defined as a "chance or possibility of danger, loss, injury, etc.” In
relation to animal cloning, risk can be defined as a possibility to cause potential
effects to cloned animals, surrogate dams and the environment. In one study it was
found that 5 out of 10 cloned pigs died between 3-130 days of age from sickness like
chronic diarrhoea, congestive heart failure and decreased growth rate which results
in a 50% mortality rate. In another study also the case of early death of cloned mice
has been reported.
Altering Gene Pool
If cloning becomes widespread, the genetic diversity of humans will go down. This
would result in the decrease in immunity of humans against diseases. Thus making
humans susceptible to epidemics and unknown diseases. Some advocate human
cloning as ethically unacceptable because it is seen as a threat to the entire human
evolution.
Philosophical Views
Several debates have held over a period of time to discuss the validity of animal
cloning. It was concluded to ban the use of products from cloned animals and to
prevent the use of animal cloning. For example, the U.S. Humane society which is
an animal protection organization has requested to ban on products coming from
cloned animals and their offspring. The vice president of this organization, Michael
Appleby stated that the animal welfare problems that already exist will increase
with the new biotechnological practices e.g., disease vulnerability.
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ANIMAL CLONING & APPLICATION & ETHICAL ISSUES DOLLY THE SHEEP

Religious views
Many religions have various guidelines or principles on doing things in the right
way, on how should we live. Various philosophers proposed many ethical traditions
or principles which can be classified into two categories 1) secular 2) spiritual.
Some ethical or moral theories are included in the secular division or western
division, where as religion comes under the spiritual division. According to the
Islamic religion research, one has to ultimately find the truth and to observe the
signs of Allah’s glory in order to understand natural phenomena and the hidden
story behind the universe.
Cloning goes against the basic belief of certain religions that only God has created
life and its various forms in nature. Humans cannot act as God. Ethically, it is
wrong for any human to have control over the genetic makeup of any other
individual. More so, the cloned individual would be generated for specific purposes.
This in essence is wrong wherein the purpose of an individual's life should be more
than just satisfying someone else's needs.
Use of cloned animals for food and health
The impact of food production from cloned livestock arose many questions. One
part of the question is ethics and the other part is science. The next question arises
about the safety of such food to eat. Finally, in 2008 after gathering the safety data
from USDA the US FDA approved the use of cloned animals for their food and
milk products.
The pain, sufferings, and difficulty that animals feel due to cloning procedures can
be justified by their noble uses like curing humans and animal diseases or
protecting threatened species. It should be permissible for this reasons because it is
helpful in solving the pain of human being and other animals but it should not be
permissible for the reasons like making animals more attractive for art purposes, for
example, the deer’s are cloned to get large antler and to make them more attractive
to hunters or production of chimera to keep them as an art object.
Illegal Cloning and Clones
Cloning could have legal implications as well. A cloned child having multiple
donors might complicate parental right issues as well as inheritance and marital
eligibility issues. Another view held by many experts, suggests that there is a
possibility of clones being developed without the concerned individual's consent.

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Advancement in cloning technology promise new possibilities but many

ethical challenges have emerged with it. . Decreasing the disease
susceptibility will be benefit animal welfare and agricultural productivity
so for this more research is needed for the understanding of the process
involving failures in pre and postnatal development.
In applied research, cloning using nucleus transfer opens up new approaches
to creating transgenic animals. Some proteins with therapeutic effect can be
cheaply produced in this way. Obtaining autologous replacement tissue
seems particularly promising in both medical and ethical terms, and the
associated research is accordingly particularly deserving of promotion. It is
not clear whether it will be possible to create better test models for human
diseases in livestock, but the considerable medical importance justifies
increased effort and support in this area as well.
Overall there appear to be a relatively high potential benefits for research
and medicine from cloning using nucleus transfer. In agriculture the
production of clones of breeding stock promises to improve animal
performance and quality while simultaneously reducing production costs. It
is likely that cloning techniques will add further weight to existing trends in
optimizing the performance potential of livestock, i.e. high-performance
animals.

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REFERENCE

1) Introduction to Biotechnology (William J. Thieman Michael A. Palladino )

2) ANIMAL CLONING- The Science of Nuclear Transfer ( Joseph Panno)
3) GENE CLONING AND DNA ANALYSIS- An Introduction (T.A. BROWN)
4) Animal Transgenesis and Cloning (Louis-Marie Houdebine )
5) Carter DB, Lai L, Park KW, Samuel M, Lattimer JC, Jordan KR, Estes DM, Besch-
Williford C, Prather RS. Phenotyping of transgenic cloned piglets. Cloning Stem
Cells 2002;4(2):131-45.
6) Ogonuki N, Inoue K, Yamamoto Y, Noguchi Y, Tanemura K, Suzuki O, Nakayama
H, Doi K, Ohtomo Y, Satoh M, Nishida A. Early death of mice cloned from somatic
cells. Nat Genet 2002;30(3):253-9
7) AgBiotechNet. 2001. Cloning hopes for extinct specie
8) Allmendinger, U. 2001. One small hop for Alba, one large hop for mankind. NY
Arts Mag 6 (May 3)

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