Download Cloning in Livestock Animal

CHAPTER 1
INTRODUCTION
Biotechnology (sometimes shortened to "biotech") is a field of applied biology that
involves the use of living organisms and bioprocesses in engineering, technology, medicine and
other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing
purpose. Modern use of similar terms includes genetic engineering as well as cell- and tissue
culture technologies. The concept encompasses a wide range of procedures (and history) for
modifying living organisms according to human purposes — going back to domestication of
animals, cultivation of plants, and "improvements" to these through breeding programs that
employ artificial selection and hybridization. By comparison to biotechnology, bioengineering is
generally thought of as a related field with its emphasis more on higher systems approaches (not
necessarily altering or using biological materials directly) for interfacing with and utilizing living
things.
The United Nations Convention on Biological Diversity defines biotechnology as: "Any
technological application that uses biological systems, living organisms, or derivatives thereof, to
make or modify products or processes for specific use." In other terms: "Application of scientific
and technical advances in life science to develop commercial products" is biotechnology.
Biotechnology draws on the pure biological sciences (genetics, microbiology, animal cell
culture, molecular biology, biochemistry, embryology, cell biology) and in many instances is
also dependent on knowledge and methods from outside the sphere of biology (chemical
engineering, bioprocess engineering, information technology, biorobotics). Conversely, modern
biological sciences (including even concepts such as molecular ecology) are intimately entwined
and dependent on the methods developed through biotechnology and what is commonly thought
of as the life sciences industry.
One example of biotechnology is cloning. We have been cloning plants for centuries. Each
time a leaf is excised from a violet plant and placed in soil to grow a new plant, cloning has
occurred. Today, we are not only doing the physical manipulation at the visual level but also on
the molecular level. In modern or molecular biotechnology, we physically select the desired
characteristic at the molecular level and add it to the organism's genetic makeup.
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What is cloning?
Cloning is a complex process that lets one exactly copy the genetic, or inherited, traits of
an animal (the donor). Animals can be cloned by embryo splitting or nuclear transfer. Embryo
splitting involves splitting the multicellular embryo at an early stage of development to generate
“twins”. This type of cloning occurs naturally and has also been performed in the laboratory with
a number of species. Cloning can also be achieved by nuclear transfer where the genetic material
in the nucleus from one cell is placed into a “recipient” unfertilized egg that has had its genetic
material (nucleus) removed by a process called enucleation. It is then necessary to activate the
egg to start dividing as if it had been fertilized. In mammals the egg must then be artificially
placed into the womb of a surrogate mother where it will grow until birth. The first mammals
cloned by this process were born during the mid 1980s, almost 30 years after the initial
successful experiments with frogs. Numerous mammalian species have been cloned via this
procedure, including mice, rats, rabbits, pigs, goats, sheep, cattle, horse, and rhesus monkeys.
There are no documented cases of human cloning.
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CHAPTER 2
BIOTECHNOLOGY IN ANIMALS
1. Biotechnology in animals
a. Artificial Insemination
Artificial insemination, or AI, is the process by which sperm is placed into the reproductive
tract of a female for the purpose of impregnating the female by using means other than sexual
intercourse or natural insemination. In humans, it is used as assisted reproductive technology,
using either sperm from the woman's male partner or sperm from a sperm donor (donor sperm)
in cases where the male partner produces no sperm or the woman has no male partner (i.e., single
women, lesbians). In cases where donor sperm is used the woman is the gestational and genetic
mother of the child produced, and the sperm donor is the genetic or biological father of the child.
Artificial insemination is widely used for livestock breeding, especially for dairy cattle and
pigs.
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b.
Embryo Transfer
Embryo transfer refers to a step in the process of assisted reproduction in which embryos
are placed into the uterus of a female with the intent to establish a pregnancy. This technique
(which is often used in connection with in vitro fertilization (IVF)), may be used in humans or in
animals, in which situations the goals may vary.
c.
Semen Presessing
(parting spermatozoa X and Y)
This method can make farmer controls the gender of their livestock animals. The search
begins with conditioning female livestock’s reproduction organ so that the environment will be
better for spermatozoa X than spermatozoa Y, or vice versa.
d.
In Vitro Fertilization
In Vitro Fertilization is commonly referred to as IVF. IVF is the process of fertilization by
manually combining an egg and sperm in a laboratory dish. When the IVF procedure is
successful, the process is combined with a procedure known as embryo transfer, which is used to
physically place the embryo in the uterus.
e.
Cloning
A clone is a genetic copy of another living organism. The genetic material of a cloned
offspring is drawn from a single source, rather than being a combination of sperm and egg genes.
In sexual reproduction, half of the genetic material of an individual comes from a female and
half from a male.
Two processes can be used to obtain clones. The first process called “embryo splitting” is
similar to what happens naturally when identical twins are produced. In the laboratory, an
embryo is obtained by joining a sperm cell from a male donor with an egg cell from a female
donor. When this embryo starts to divide into two cells, these are separated and implanted in
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different foster mothers. For cattle, the embryo can be at the 4- or 8-cell division stage when it is
separated into individual cells. This provides the potential to create 4 or 8 clones simultaneously.
This method can also be used for primates.
The second method is called nuclear transfer (NT) or cell nuclear replacement (CNR) and
it was used to produce Dolly the sheep, the first animal cloned from a cell taken from an adult
animal. In this technique, the donor nucleus containing the genetic material of a cell is
introduced into an unfertilized egg cell from which the nucleus has been removed. An electrical
pulse is used to fuse the donor nucleus and the egg cell together and to activate the development
of the “reconstructed embryo”. The embryo is cultured a few days in the laboratory and if it
develops normally, it is then implanted in a foster mother. The genetic make-up of this embryo is
identical to that of the donor of the nucleus. The donor nucleus can be obtained from embryonic
cells that are not fully differentiated into skin, heart, brain, etc. cells; this technique has been
used for the cloning of livestock animals for about 12 years. The donor nucleus can also
originate from the differentiated cells of an adult organism. Prior to the birth of Dolly in 1996,
scientists thought that differentiated adult cells could not revert back and be reprogrammed to
develop into a new embryo. Success rates for NT are low.
f. Transgenic Animal
There are various definitions for the term transgenic animal. The Federation of European
Laboratory Animal Associations defines the term as an animal in which there has been a
deliberate modification of its genome, the genetic makeup of an organism responsible for
inherited characteristics.
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.
The majority of transgenic animals produced so far are mice, the animal that pioneered the
technology. The first successful transgenic animal was a mouse. A few years later, it was
followed by rabbits, pigs, sheep, and cattle.
Why are these animals being produced?
The two most common reasons are:
1.
Some transgenic animals are produced for specific economic traits. For example,
transgenic cattle were created to produce milk containing particular human proteins, which may
help in the treatment of human emphysema.
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2.
Other transgenic animals are produced as disease models (animals genetically
manipulated to exhibit disease symptoms so that effective treatment can be studied). For
example, Harvard scientists made a major scientific breakthrough when they received a U.S.
patent (the company DuPont holds exclusive rights to its use) for a genetically engineered
mouse, called OncoMouse® or the Harvard mouse, carrying a gene that promotes the
development of various human cancers.
How are transgenic animals produced?
Since the discovery of the molecular structure of DNA by Watson and Crick in 1953,
molecular biology research has gained momentum. Molecular biology technology combines
techniques and expertise from biochemistry, genetics, cell biology, developmental biology, and
microbiology.
Scientists can now produce transgenic animals because, since Watson and Crick’s
discovery, there have been breakthroughs in:
1.
2.
3.
4.
Recombinant DNA (artificially-produced DNA)
Genetic cloning
Analysis of gene expression (the process by which a gene gives rise to a protein)
Genomic mapping
The underlying principle in the production of transgenic animals is the introduction of a
foreign gene or genes into an animal (the inserted genes are called transgenes). The foreign genes
“must be transmitted through the germ line, so that every cell, including germ cells, of the animal
contain the same modified genetic material.” (Germ cells are cells whose function is to transmit
genes to an organism’s offspring.)
To date, there are three basic methods of producing transgenic animals:
1.
2.
3.
DNA microinjection
Retrovirus-mediated gene transfer
Embryonic stem cell-mediated gene transfer
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.
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1.
DNA Microinjection
The mouse was the first animal to undergo successful gene transfer using DNA
microinjection. This method involves:
a.
transfer of a desired gene construct (of a single gene or a combination of genes that
are recombined and then cloned) from another member of the same species or from
a different species into the pronucleus of a reproductive cell
b.
the manipulated cell, which first must be cultured in vitro (in a lab, not in a live
animal) to develop to a specific embryonic phase, is then transferred to the recipient
female
2.
Retrovirus-Mediated Gene Transfer
A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA.
This method involves:
a.
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
b.
chimeras are inbred for as many as 20 generations until homozygous (carrying the
desired transgene in every cell) transgenic offspring are born
The method was successfully used in 1974 when a simian virus was inserted into mice
embryos, resulting in mice carrying this DNA.
3.
Embryonic Stem Cell-Mediated Gene Transfer
This method involves:
a.
isolation of totipotent stem cells (stem cells that can develop into any type of
specialized cell) from embryos
b.
the desired gene is inserted into these cells
c.
cells containing the desired DNA are incorporated into the host’s embryo,
resulting in a chimeric animal
Unlike the other two 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.
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How do transgenic animals contribute to human welfare?
The benefits of these animals to human welfare can be grouped into areas:
1.
2.
3.
Agriculture
Medicine
Industry
The examples below are not intended to be complete but only to provide a sampling of the
benefits.
1.
Agricultural Applications
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.
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 influenzaresistant 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)
Xenotransplantation
Patients die every year for lack of a replacement heart, liver, or kidney. For example,
about 5,000 organs are needed each year in the United Kingdom alone. 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
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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
nutritional or digestive needs. Rosie’s milk contains the human gene alphalactalbumin.
c)
3.
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.
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.
What are the ethical concerns surrounding transgenesis?
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.
g.
Gamete and Embryo Cryopreservation
Cryopreservation—the ability to freeze and thaw with retention of viability—provides
flexibility in human infertility therapy when gametes or embryos are handled in vitro because
frozen tissue can be stored indefinitely in liquid nitrogen at -196°C. The freezing of human
sperm is an established procedure that has resulted in the birth of thousands of progeny, as many
as 30,000 per year. Not only can partner or donor sperm be frozen for therapeutic insemination
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(TI) at a subsequent date, but sperm banking provides the assisted reproductive technologies
(ART) couple with a backup option if a sample cannot be collected on demand or if sample
quality is poor on the day eggs are available. In the case of donor insemination, samples are
quarantined for 6 months before use, thereby minimizing the risk of infectious disease
transmission. In other words, the donor is screened for sexually transmitted diseases at the time
of collection, and the frozen sample is released for use 6 months later only after the donor passes
rescreening. Pregnancies have been established with sperm stored at low temperatures for more
than 10 years; however, it is generally recognized that fecundity using cryopreserved sperm is
lower than that obtained with nonfrozen sperm with the possible exception of cryopreserved
sperm delivered by intracytoplasmic sperm injection (ICSI).
The cryopreservation of human ova would be advantageous to the patient with ovarian
disease or any other condition that limits egg production. Moreover, freezing oocytes
circumvents the ethical issues associated with embryo freezing. Unfortunately, the technology
has not yet been perfected for egg freezing on a routine basis, despite the reports of successful
pregnancies. In contrast, embryo cryopreservation is an established procedure that has been
employed successfully for several years. Embryo banking was originally designed to provide
alternatives for the ART couple with an embarrassment of riches. Because the risk of multiple
pregnancy restricts the number of embryos that can be transferred during the treatment cycle,
cryopreservation was begun to store surplus embryos for transfer at a later date to either the egg
donor or to other women in the context of an embryo donor program. Additionally, embryo
banking may be appropriate when embryos cannot be transferred during the egg pickup cycle.
Another advantage to embryo freezing is that embryo thaw and transfer can be conducted in a
natural or regulated cycle when the patient has not been subjected to controlled ovarian
stimulation with exogenous hormones.
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CHAPTER 3
CLONING
1. History in Cloning
The first theories and experiments with cloning began in the late 1880s as scientists sought
to prove their theories about how the genetic material inside cells worked. The earliest
experiments involved splitting the embryos of frogs and salamanders to see how the resulting
animals would develop. As chromosomes became better understood, more experiments were
done with cloning, resulting in the first cloned animals in 1952.
a. History
Discoveries about the nature of DNA in the 1940s made it possible for cloning
experiments to progress. In 1944 it was discovered that genetic information for each
cell was kept in the cell's DNA. When Oswald Avery found this genetic information, it
gave scientists new ways to try to clone animals by using that genetic blueprint.
b. Types
The first cloned animals were northern leopard frogs that were cloned in 1952. Thomas
J. King and Robert Briggs cloned 35 frog embryos and saw 27 tadpoles hatch. This first
successful cloning taught scientists more about what cells needed to be used in the
cloning process. King and Briggs believed, based on their clones, that young cells were
more viable for the cloning process. Cells that were taken from adults resulted in
abnormally developed tadpoles.
c. Significance
The next successful cloning experiments also resulted in cloned frogs. John Gurdon
cloned South African frogs in 1962. His use of adult cells disproved the prior theory
that only young cells could be used in the cloning process with success. From 1962 to
1965, Thomas King, Robert McKinnell and Marie Di Berardino created more frog
clones from adult frog cells.
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d. Considerations
While animal cloning had been the focus of cloning experiments, the 1960s also saw
other types of cloning. In 1964 F.C. Steward took an adult root cell from a carrot plant
and successfully cloned the plant. Throughout the rest of the 1960s, scientists
continued to clone frogs and to discover more about DNA. The first gene was
discovered in 1969.
e. Potential
In 1977, the first cloned mice were created. Mouse cloning research continued, and
new cloned mice were created in 1979. The first mammal was cloned in 1984. The
cloned sheep was quickly followed in 1985 with cloned cattle embryos. A cow clone
was created in 1986 and several calves in 1993. That same year, human embryos were
cloned for the first time. In 1995 and 1996, sheep were cloned, including the famous
Dolly. Since Dolly's creation, the cloning of mice and other small animals has
continued, but human cloning research has been banned in many countries.
f. Cloning Timeline
1885
August Weismann, professor of zoology and comparative anatomy at the
University of Freiberg, theorized that the genetic information of a cell would
diminish as the cell went through differentiation.
1888
Wilhelm Roux tested the germ plasm theory for the first time. One cell of a 2-cell
frog embryo was destroyed with a hot needle; the result was a half-embryo,
supporting Weismann's theory.
1894
Hans Dreisch isolated blastomeres from 2- and 4-cell sea urchin embryos and
observed their development into small larvae. These experiments were regarded as
refutations of the Weismann-Roux theory.
1901
Hans Spemann split a 2-cell newt embryo into two parts, resulting in the
development of two complete larvae.
1902
Walter Sutton published "On the Morphology of the Chromosome Group in
Brachyotola magna", hypothesizing that chromosomes carry the inheritance and
that they occur in distinct pairs within a cell's nucleus. Sutton also argued that how
chromosomes act when sex cells divide was the basis for the Mendelian Law of
Heredity.
1902
German embryologist Hans Spemann split a 2-celled salamander embryo and
each cell grew to adulthood, providing proof that early embryo cells carry
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necessary genetic information. This finally disproved Weismann's 1885 theory
that the amount of genetic information in cells decreases with each division.
1914
Hans Spermann conducted and early nuclear transfer experiment.
1928
Hans Spemann performed further, successful nuclear transfer experiments.
1938
Hans Spemann published the results of his 1928 primitive nuclear transfer
experiments involving salamander embryos in the book "Embryonic Development
and Induction." Spemann argued the next step for research should be the cloning
organisms by extracting the nucleus of a differentiated cell and putting it into an
enucleated egg.
1944
Oswald Avery found that a cell's genetic information was carried in DNA.
1950
First successful freezing of bull semen at -79°C for later insemination of cows
was accomplished.
1952
First animal cloning: Robert Briggs and Thomas J. King cloned northern leopard
frogs.
1953
Francis Crick and James Watson ,working at Cambridge's Cavendish Laboratory,
discovered the structure of DNA.
1962
Biologist John Gurdon announced that he had cloned South African frogs using
the nucleus of fully differentiated adult intestinal cells. This demonstrated that
cells' genetic potential do not diminish as the cell became specialized.
1962 - 65
Robert G. McKinnell, Thomas J. King, and Marie A. Di Berardino
produced swimming larvae from enucleated oocytes that had been injected with
adult frog kidney carcinoma cell nuclei.
1963
Biologist J.B.S. Haldane coined the term "clone" in a speech entitled "Biological
Possibilities for the Human Species of the Next Ten-Thousand Years."
1964
F.C. Steward grew a complete carrot plant from a fully differentiated carrot root
cell.
1966
Marshall Niremberg, Heinrich Mathaei, and Severo Ochoa broke the genetic
code, discovering what codon sequences specified each of the twenty amino acids.
1966
John B. Gurdon and V. Uehlinger grew adult frogs after injecting tadpole
intestinal cell nuclei into enucleated oocytes.
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1967
DNA ligase, the enzyme responsible for binding together strands of DNA, was
isolated.
1969
James Shapiero and Johnathan Beckwith announced that they had isolated the
first gene.
1970
Howard Temin and David Baltimore each independently isolated the first
restriction enzyme.
1972
Paul Berg combined the DNA of two different organisms, thus creating the first
recombinant DNA molecules.
1973
Stanley Cohen and Herbert Boyer created the first recombinant DNA organism
using recombinant DNA techniques pioneered by Paul Berg. Also known as gene
splicing, this technique that allows scientists to manipulate the DNA of an
organism - the basis of genetic engineering.
1977
Karl Illmensee and Peter Hoppe created mice with only a single parent.
1978
David Rorvik published the novel In His Image: The Cloning of a Man.
1978
Baby Louise, the first child conceived through in vitro fertilization, was born.
1979
Karl Illmensee claimed to have cloned three mice.
1980
In the case Diamond v. Chakrabarty, the United States Supreme Court ruled that
a "live, human made microorganism is patentable material."
1983
Kary B. Mullis developed the polymerase chain reaction (PCR) in 1983. This
process allows for the rapid synthesis of designated fragments of DNA.
1983
Davor Solter and David McGrath tried to clone mice using their own version of
the nuclear transfer method.
1983
The first human mother-to-mother embryo transfer was completed.
1983 - 86
Marie A. Di Berardino, Nancy H. Orr, and Robert McKinnell transplanted
nuclei of adult frog erythrocytes, thus obtained pre-feeding and feeding tadpoles.
1984
Steen Willadsen cloned a sheep from embryo cells, the first verified example of
mammal cloning using the process of nuclear transfer.
1985
Steen Willadsen used his cloning technique to duplicate prize cattle embryos.
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1985
Ralph Brinster created the first transgenic livestock: pigs that produced human
growth hormone.
1986
Using differentiated, one week old embryo cells, Steen Willadsen cloned a cow.
1986
Artificially inseminated surrogate mother Mary Beth Whitehead gave birth to
Baby M. She tried and failed to retain custody.
1986
Neal First, Randal Prather, and Willard Eyestone used early embryo cells to clone
a cow.
October 1990 The National Institutes of Health officially launched the Human Genome
Project to locate the 50,000 to 100,000 genes and sequence the estimated 3 billion
nucleotides of the human genome.
1993
M. Sims and N.L. First reported the creation of calves by transfer of nuclei from
cultured embryonic cells.
1993
Human embryos were first cloned.
July 1995
Ian Wilmut and Keith Campbell used differentiated embryo cells to clone
two sheep, named Megan and Morag.
July 5, 1996
Dolly, the first organism ever to be cloned from adult cells, was born.
February 23, 1997
Scientists at the Roslin Institute in Scotland officially announced
the birth of "Dolly"
March 4, 1997 President Clinton proposed a five year moratorium on federal and
privately funded human cloning research.
July 1997
Ian Wilmut and Keith Campbell, the scientists who created Dolly, also
created Polly, a Poll Dorset lamb cloned from skin cells grown in a lab and
genetically altered to contain a human gene.
August 1997
President Clinton proposed legislation to ban the cloning of humans for at
least 5 years.
September 1997
Thousands of biologists and physicians signed a voluntary fiveyear moratorium on human cloning in the United States.
December 5, 1997
Richard Seed announced that he intended to clone a human before
federal laws could effectively prohibit the process.
early January 1998
Nineteen European nations signed a ban on human cloning.
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January 20, 1998
The Food and Drug Administration announced that it had
authority over human cloning.
July 1998
Ryuzo Yanagimachi, Toni Perry, and Teruhiko Wakayama announced
that they had cloned 50 mice from adult cells since October, 1997.
January 1998 Botechnology firm Perkin-Elmer Corporation announced that it would
work with gene sequencing expert J. Craig Venture to privately map the human
genome.
2. Types of Cloning
a. Molecular Cloning
Molecular cloning refers to the process of making multiple molecules. Cloning is
commonly used to amplify DNA fragments containing whole genes, but it can also
be used to amplify any DNA sequence such as promoters, non-coding sequences
and randomly fragmented DNA. It is used in a wide array of biological experiments
and practical applications ranging from genetic fingerprinting to large scale protein
production. Occasionally, the term cloning is misleadingly used to refer to the
identification of the chromosomal location of a gene associated with a particular
phenotype of interest, such as in positional cloning. In practice, localization of the
gene to a chromosome or genomic region does not necessarily enable one to isolate
or amplify the relevant genomic sequence. To amplify any DNA sequence in a
living organism, that sequence must be linked to an origin of replication, which is a
sequence of DNA capable of directing the propagation of itself and any linked
sequence. However, a number of other features are needed and a variety of
specialised cloning vectors (small piece of DNA into which a foreign DNA
fragment can be inserted) exist that allow protein expression, tagging, single
stranded RNA and DNA production and a host of other manipulations
b. Cellular Cloning
1. Unicellular Organisms
Cloning a cell means to derive a population of cells from a single cell. In the
case of unicellular organisms such as bacteria and yeast, this process is
remarkably simple and essentially only requires the inoculation of the
appropriate medium. However, in the case of cell cultures from multi-cellular
organisms, cell cloning is an arduous task as these cells will not readily grow in
standard media.
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A useful tissue culture technique used to clone distinct lineages of cell lines
involves the use of cloning rings (cylinders). According to this technique, a
single-cell suspension of cells that have been exposed to a mutagenic agent or
drug used to drive selection is plated at high dilution to create isolated colonies;
each arising from a single and potentially clonal distinct cell. At an early
growth stage when colonies consist of only a few of cells, sterile polystyrene
rings (cloning rings), which have been dipped in grease are placed over an
individual colony and a small amount of trypsin is added. Cloned cells are
collected from inside the ring and transferred to a new vessel for further growth.
2. Cloning in Stem Cell Research
Somatic cell nuclear transfer, known as SCNT, can also be used to create
embryos for research or therapeutic purposes. The most likely purpose for this
is to produce embryos for use in stem cell research. This process is also called
"research cloning" or "therapeutic cloning." The goal is not to create cloned
human beings (called "reproductive cloning"), but rather to harvest stem cells
that can be used to study human development and to potentially treat disease.
While a clonal human blastocyst has been created, stem cell lines are yet to be
isolated from a clonal source.
Therapeutic cloning is achieved by creating embryonic stem cells in the hopes
of treating diseases such as diabetes and Alzheimer’s. The process begins by
taking out the nucleus that contains the DNA from an egg and putting it in a
nucleus from an adult. In the case of someone with Alzheimer’s disease, the
nucleus from a skin cell of that patient is placed into an empty egg. The
reprogrammed cell begins to develop into an embryo because the egg reacts
with the transferred nucleus. The embryo will become genetically identical to
the patient. The embryo will then form a blastocyst which has the potential to
form/become any cell in the body.
The reason why SCNT is used for cloning is because somatic cells can be easily
acquired and cultured in the lab. This process can either add or delete specific
genomes of farm animals. A key point to remember is that cloning is achieved
when the oocyte maintains its normal functions and instead of using sperm and
egg genomes to replicate, the oocyte is inserted into the donor’s somatic cell
nucleus. The oocyte will react on the somatic cell nucleus, the same way it
would on sperm cells.
In SCNT, not all of the donor cell's genetic information is transferred, as the
donor cell's mitochondria that contain their own mitochondrial DNA are left
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behind. The resulting hybrid cells retain those mitochondrial structures which
originally belonged to the egg. As a consequence, clones such as Dolly that are
born from SCNT are not perfect copies of the donor of the nucleus.
3. Techniques of Cloning
a. Somatic Cell Nuclear Transfer
Somatic cell nuclear transfer, (SCNT) uses a different approach than artificial
embryo twinning, but it produces the same result: an exact clone, or genetic copy,
of an individual. This was the method used to create Dolly the Sheep.
What does SCNT mean?
Somatic cell: A somatic cell is any cell in the body other than the two types of
reproductive cells, sperm and egg. Sperm and egg are also called germ cells. In
mammals, every somatic cell has two complete sets of chromosomes, whereas the
germ cells only have one complete set.
Nuclear: The nucleus is like the cell's brain. It's an enclosed compartment that
contains all the information that cells need to form an organism. This information
comes in the form of DNA. It's the differences in our DNA that make each of us
unique.
Transfer: Moving an object from one place to another.
To make Dolly, researchers isolated a somatic cell from an adult female sheep.
Next, they transferred the nucleus from that cell to an egg cell from which the
nucleus had been removed. After a couple of chemical tweaks, the egg cell, with its
new nucleus, was behaving just like a freshly fertilized zygote. It developed into an
embryo, which was implanted into a surrogate mother and carried to term.
The lamb, Dolly, was an exact genetic replica of the adult female sheep that
donated the somatic cell nucleus to the egg. She was the first-ever mammal to be
cloned from an adult somatic cell.
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How does SCNT differ from the natural way of making an embryo?
The fertilization of an egg by a sperm and the SCNT cloning method both result in
the same thing: a dividing ball of cells, called an embryo. So what exactly is the
difference between these methods?
An embryo is composed of cells that contain two complete sets of chromosomes.
The difference between fertilization and SCNT lies in where those two sets
originated.
In fertilization, the sperm and egg both contain one set of chromosomes. When the
sperm and egg join, the resulting zygote ends up with two sets - one from the father
(sperm) and one from the mother (egg).
In SCNT, the egg cell's single set of chromosomes is removed. It is replaced by the
nucleus from a somatic cell, which already contains two complete sets of
chromosomes. Therefore, in the resulting embryo, both sets of chromosomes come
from the somatic cell.
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b. Honolulu technique
In July of 1998, a team of scientists at the University of Hawaii announced that
they had produced three generations of genetically identical cloned mice.
The technique is accredited to Teruhiko Wakayama and Ryuzo Yanagimachi of the
University of Hawaii. Mice had long been held to be one of the most difficult
mammals to clone due to the fact that almost immediately after a mouse egg is
fertilized, it begins dividing. Sheep were used in the Roslin technique because their
eggs wait several hours before dividing, possibly giving the egg time to reprogram
its new nucleus. Even without this luxury, Wakayama and Yanagimachi were able
to clone with a much higher success rate (three clones out of every one-hundred
attempts) than Ian Wilmut (one in 277).
Wakayama approached the problem of synchronizing cell cycles differently than
Wilmut. Wilmut used udder cells, which had to be forced into the G0 stage.
Wakayama initially used three types of cells, Sertoli cells, brain cells, and cumulus
cells. Sertoli and brain cells both remain in the G0 state naturally and cumulus cells
are almost always in either the G0 or G1 state.
Unfertilized mouse egg cells were used as the recipients of the donor nuclei. After
being enucleated, the egg cells had donor nuclei inserted into them. The donor
nuclei were taken from cells within minutes of the cell’s extraction from a mouse.
Unlike the process used to create Dolly, no in vitro, or outside of an animal,
culturing was done on the cells. After one hour, the cells had accepted the new
nucleus. After an additional five hours, the egg cell was then placed in a chemical
culture to jumpstart the cell’s growth, just as fertilization does in nature.
In the culture was a substance (cytochalasin B) which stopped the formation of a
polar body, a second cell which normally forms before fertilization. The polar body
would take half of the genes of the cell, preparing the other cell to receive genes
from sperm.
After being jumpstarted, the cells develop into embryos. These embryos can then
be transplanted into surrogate mothers and carried to term. The most successful of
the cells for the process were cumulus cells, so research was concentrated on cells
of that type.
After proving that the technique was viable, Wakayama also made clones of clones
and allowed the original clones to give birth normally to prove that they had full
reproductive functions. At the time he released his results, Wakayama had created
fifty clones.
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This new technique allows for further research into exactly how an egg reprograms
a nucleus, since the cell functions and genomes of mice are some of the best
understood. Mice also reproduce within months, much more rapidly than sheep.
This aids in researching long term results.
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c. Roslin technique
The cloning of Dolly has been the most important event in cloning history. Not
only did it spark public interest in the subject, but it also proved that the cloning of
adult animals could be accomplished. Previously, it was not known if an adult
nucleus was still able to produce a completely new animal. Genetic damage and the
simple deactivation of genes in cells were both considered possibly irreversible.
The realization that this was not the case came after the discovery by Ian Wilmut
and Keith Cambell of a method with which to synchronize the cell cycles of the
donor cell and the egg cell. Without synchronized cell cycles, the nucleus would
not be in the correct state for the embryo to accept it. Somehow the donor cell had
to be forced into the Gap Zero, or G0 cell stage, or the dormant cell stage.
First, a cell (the donor cell) was selected from the udder cells of a Finn Dorset
sheep to provide the genetic information for the clone. For this experiment, the
researchers allowed the cell to divide and form a culture in vitro, or outside of an
animal. This produced multiple copies of the same nucleus. This step only becomes
useful when the DNA is altered, such as in the case of Polly, because then the
changes can be studied to make sure that they have taken effect.
A donor cell was taken from the culture and then starved in a mixture which had
only enough nutrients to keep the cell alive. This caused the cell to begin shutting
down all active genes and enter the G0 stage. The egg cell of a Blackface ewe was
then enucleated and placed next to the donor cell. One to eight hours after the
removal of the egg cell, an electric pulse was used to fuse the two cells together
and, at the same time, activate the development of an embryo. This technique for
mimicking the activation provided by sperm is not completely correct, since only a
few electrically activated cells survive long enough to produce an embryo.
If the embryo survives, it is allowed to grow for about six days, incubating in a
sheep's oviduct. It has been found that cells placed in oviducts early in their
development are much more likely to survive than those incubated in the lab.
Finally, the embryo is placed into the uterus of a surrogate mother ewe. That ewe
then carries the clone until it is ready to give birth. Assuming nothing goes wrong,
an exact copy of the donor animal is born.
This newborn sheep has all of the same characteristics of a normal newborn sheep.
It has yet to be seen if any adverse effects, such as a higher risk of cancer or other
genetic diseases that occur with the gradual damage to DNA over time, are present
in Dolly or other animals cloned with this method.
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4. The Benefits of Cloning
a.
Cloning for medical purposes
Of all the reasons, cloning for medical purposes has the most potential to benefit large
numbers of people.
1. Cloning animal models of disease
Much of what researchers learn about human disease comes from studying animal models
such as mice. Often, animal models are genetically engineered to carry disease-causing
mutations in their genes. Creating these transgenic animals is a time-intensive process that
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requires trial-and-error and several generations of breeding. Cloning technologies might reduce
the time needed to make a transgenic animal model, and the result would be a population of
genetically identical animals for study.
2. Cloning stem cells for research
Stem cells are the body's building blocks, responsible for developing, maintaining and
repairing the body throughout life. As a result, they might be used to repair damaged or diseased
organs and tissues. Researchers are currently looking toward cloning as a way to create
genetically defined human stem cells for research and medical purposes.
3. "Pharming" for drug production
Farm animals such as cows, sheep and goats are currently being genetically engineered to
produce drugs or proteins that are useful in medicine. Just like creating animal models of disease,
cloning might be a faster way to produce large herds of genetically engineered animals.
b. Cloning for agriculture purposes
Another benefit from modern cloning is in agriculture. Farmers and ranchers can now have
their strongest crops and animals twinned so that they are less likely to contract diseases.
c.
Reviving Endangered or Extinct Species
Just like what had been pictured in ‘Jurassic Park’ movie. In this feature film, scientists use
DNA preserved for tens of millions of years to clone dinosaurs. They find trouble, however,
when they realize that the cloned creatures are smarter and fiercer than expected.
Could we really clone dinosaurs?
In theory? Yes.
What would you need to do this?
A well-preserved source of DNA from the extinct dinosaur, and
A closely related species, currently living, that could serve as a surrogate mother
In reality? Probably not.
It's not likely that dinosaur DNA could survive undamaged for such a long time. However,
scientists have tried to clone species that became extinct more recently, using DNA from wellpreserved tissue samples.
d. Reproducing a Deceased Pet
If we really wanted to, and if there is enough money, cloning our beloved family cat is not
a dream. At least one biotechnology company in the United States offers cat cloning services for
the privileged and bereaved, and they are now working to clone dogs.
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e.
Cloning Humans?
To clone or not to clone: that is the question. The prospect of cloning humans is highly
controversial and raises a number of ethical, legal and social challenges that need to be
considered.
Why would anyone want to clone humans? Some reasons include:
- To help infertile couples have children
- To replace a deceased child
5. Pros and cons in cloning
When Dolly, the first cloned sheep came in the news, cloning interested the masses. Not
only researchers but even common people became interested in knowing about how cloning is
done and what pros and cons it has. Everyone became more curious about how cloning could
benefit the common man. Most of us want to know the pros and cons of cloning, its advantages
and its potential risks to mankind. Let us understand them.
a. Pros of Cloning
Cloning finds applications in genetic fingerprinting, amplification of DNA and alteration
of the genetic makeup of organisms. It can be used to bring about desired changes in the genetic
makeup of individuals thereby introducing psitive traits in them, as also for elimination of
negative traits. Cloning can also be applied to plants to remove or alter defective genes, thereby
making them resistant to diseases. Cloning may find applications in development of human
organs, thus making human life safer. Here we look at some of the potential advantages of
cloning.
1. Organ Replacement: If the vital organs of the human body can be cloned, they can
serve as backup systems for human beings. Cloning body parts can serve as a lifesaver.
When a body organ such as a kidney or heart fails to function, it may be possible to
replace it with the cloned body organ.
2. Substitute for Natural Reproduction: Cloning in human beings can prove to be a
solution to infertility. Cloning can serve as an option for producing children. With
cloning, it would be possible to produce certain desired traits in human beings. We
might be able to produce children with certain qualities. Wouldn't that be close to
creating a man-made being?!
3. Help in Genetic Research: Cloning technologies can prove helpful to researchers in
genetics. They might be able to understand the composition of genes and the effects of
genetic constituents on human traits, in a better manner. They will be able to alter
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genetic constituents in cloned human beings, thus simplifying their analysis of genes.
Cloning may also help us combat a wide range of genetic diseases.
4. Obtain Specific Traits in Organisms: Cloning can make it possible for us to obtain
customized organisms and harness them for the benefit of society. Cloning can serve as
the best means to replicate animals that can be used for research purposes. Cloning can
enable the genetic alteration of plants and animals. If positive changes can be brought
about in living beings with the help of cloning, it will indeed be a boon to mankind.
b. Cons of Cloning
Like every coin has two sides, cloning has its flip side too. Though cloning may work
wonders in genetics, it has potential disadvantages. Cloning, as you know, is copying or
replicating biological traits in organisms. Thus it might reduce the diversity in nature. Imagine
multiple living entities like one another! Another con of cloning is that it is not clear whether we
will be able to bring all the potential uses of cloning into reality. Plus, there's a big question of
whether the common man will afford harnessing cloning technologies to his benefit. Here we
look at the potential disadvantages of cloning.
1. Detrimental to Genetic Diversity: Cloning creates identical genes. It is a process of
replicating a genetic constitution, thus hampering the diversity in genes. While
lessening the diversity in genes, we weaken our ability of adaptation. Cloning is also
detrimental to the beauty that lies in diversity.
2. Inivitation to Malpractices: While cloning allows man to tamper with genetics in
human beings, it also makes deliberate reproduction of undesirable traits, a probability.
Cloning of body organs might invite malpractices in society.
3. Will this Technology Reach the Common Man?: In cloning human organs and using
them for transplant, or in cloning human beings themselves, technical and economic
barriers will have to be considered. Will cloned organs be cost-effective? Will cloning
techniques really reach the common man?
4. Man, a Man-made Being?: Moreover, cloning will put human and animal rights at
stake. Will cloning fit into our ethical and moral principles? Cloning will make man
just another man-made being. Won't it devalue mankind? Won't it demean the value of
human life?
6. Cloning in Livestock Production
Why use cloning in livestock production?
Researchers around the world are investigating the potential for using cloned animals in
livestock production. Cloning allows breeders to take animals with desirable traits and
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successfully have these new traits reproduced in the offspring. Selective breeding using
traditional practices does not always result in offspring with the desired traits.
Cloning could be used for a dairy cow that produces milk with an unusually high milk
protein content (important in cheese manufacture) or an unusually low saturated fat content
(potential human health benefits), for example. Cloning could also be used for a sheep identified
as superior for a particular type of wool. Researchers have also suggested that cloning could be
used to preserve a species nearing extinction or to enhance livestock resistance to illnesses such
as foot-and-mouth disease. The ethical and moral implications of using cloning for livestock
production are also being considered. Concerns have been raised about the low success rate of
the NT cloning technique and that cloning could reduce genetic diversity.
Why clone?
The main use of clones is to produce breeding stock, not food. Clones allow farmers to
upgrade the overall quality of their herds by providing more copies of the best animals in the
herd. These animals are then used for conventional breeding, and the sexually reproduced
offspring become the food-producing animals. Just as farmers wouldn’t use their best
conventionally bred breeding animals as sources of food, they are equally unlikely to do so for
clones.
Some examples of desirable characteristics in livestock that breeders might want in their
herds include the following:
a. Disease resistance: Sick animals are expensive for farmers. Veterinary bills add up,
and unhealthy animals don’t produce as much meat or milk. A
herd that is resistant to disease is extremely valuable because it
doesn’t lose any production time to illness, and doesn’t cost the
farmer extra money for veterinary treatment.
b. Suitability to climate: Different types of livestock grow well in different climates.
Some of this is natural and some results from selective breeding.
For instance, Brahma cattle can cope with the heat and humidity
of weather in the southwestern United States, but they often do
not produce very high grades of meat. Cloning could allow
breeders to select those cattle that can produce high quality meat
or milk and thrive in extreme climates and use them to breed
more cattle to be used for food production. Similarly, pork
production has traditionally been centered in the eastern United
States, but is moving to different regions of the United States
(e.g., Utah). Cloning could allow breeders to select those pigs that
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naturally do well in the new climate, and use them to breed more
pigs to be used for food production.
c. Quality body type: Farmers naturally want an animal whose body is well suited to its
production function. For example, a dairy cow should have a
large, well-attached udder so that she can produce lots of milk.
She should also be able to carry and deliver calves easily. For
animals that produce meat, farmers breed for strong, heavymuscled, quick-maturing animals that will yield high quality meat
in the shortest time possible. The most desirable bulls produce
offspring that are relatively small at birth (so that they are easier
for the female to carry and deliver), but that grow rapidly and are
healthy after birth.
d. Fertility: Quality dairy cows should be very fertile, because a cow that doesn’t get
pregnant and bear calves won’t produce milk. Male fertility is just
important as that of the female. The more sperm he can produce,
the more females a bull can inseminate, and the more animals will
be born. Beef cattle or other meat-producing animals such as pigs
need to have high fertility rates in order to replace animals that
are sent to slaughter. Cloning allows farmers and breeders to
clone those animals with high fertility rates so that they could
bear offspring that would also tend to be very fertile.
e. Market preference: Farmers or ranchers may also want to breed livestock to meet the
changing tastes of consumers. The traits the producers are looking
for include leanness, tenderness, color, and size of various cuts.
Preferences also vary by culture, and cloning may help tailor
products to the preferences of various international markets and
ethnic groups.
How does cloning help get these characteristics into the herd more quickly?
As we’ve previously said, cloning allows the breeder to increase the number of breeding
animals available to make the actual food production animals. So, if a producer wanted to
introduce disease resistance into a herd rapidly, cloning could be used to produce a number of
breeding animals that carry the gene for disease resistance, rather than just one. Likewise, if a
breeder wants to pass on the genes of a female animal, cloning could result in multiples of that
female to breed, rather than just one.
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Is it safe to eat food from clones?
It’s important to remember that the purpose of clones is for breeding, not eating. Dairy,
beef, or pork clones will make up a tiny fraction of the total number of food producing animals
in the United States. Instead, their offspring will be the animals actually producing meat or milk
for the food supply.
Dairy clones will produce milk after they give birth, and the dairy farmers will want to be
able to drink that milk or put it in the food supply. Once clones used for breeding meatproducing animals can no longer reproduce, their breeders will also want to be able to put them
into the food supply.
In order to determine whether there would be any risk involved in eating meat or milk
from clones or their offspring, in 1999 FDA asked the National Academy of Sciences (NAS) to
identify science-based concerns associated with animal biotechnology, including cloning. The
NAS gathered an independent group of top, peer-selected scientists from across the country to
conduct this study. The scientists delivered their report in the fall of 2002. That report stated that
theoretically there were no concerns for the safety of meat or milk from clones. On the other
hand, the report expressed a low level of concern due to a lack of information on the clones at
that time, and not for any specific scientific reasons. The report also stated that the meat and milk
from the offspring of clones posed no unique food safety concerns.
Meanwhile, FDA itself began the most comprehensive examination of the health of
livestock clones that has been conducted. The evaluation has taken more than four years. This
examination formed the basis of a Draft Risk Assessment to determine whether cloning posed a
risk to animal health or to humans eating food from clones or their offspring. FDA conducted a
thorough search of the scientific literature on clones, and identified hundreds of peer-reviewed
scientific journal articles, which it then reviewed. They were also able to obtain health records
and blood samples from almost all of the cattle clones that have been produced in the United
States and data from clones produced in other countries. FDA compared these health records,
and the independently analyzed blood results with similar samples from conventional animals of
the same age and breed that were raised on the same farms.
After reviewing all this information, FDA found that it could not tell a healthy clone from
a healthy conventionally bred animal. All of the blood values, overall health records, and
behaviors were in the same range for clones and conventional animals of the same breed raised
on the same farms. FDA also saw that milk from dairy clones does not differ significantly in
composition from milk from conventionally bred animals.
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In the Draft Risk Assessment, FDA concluded that meat and milk from cattle, swine, and
goat clones would be as safe as food we eat from those species now. It did not have enough
information to make a decision on the safety of food from sheep clones.
For another study similar to the one conducted on cow clones, the Agency also evaluated
the health of offspring sexually derived from swine clones, as well as the composition of their
meat. After reviewing this very large data set, the Agency concluded that all of the blood values,
overall health records, and meat composition profiles of the progeny of clones were in the same
range as for very closely genetically related conventionally bred swine. Based on these results,
other studies from scientific journals, and our understanding of the biological processes involved
in cloning, the Agency agreed with NAS that food from the sexually reproduced offspring of
clones is as safe as food that we eat every day. These offspring animals will produce almost all
of the food from the overall cloning/breeding process.
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CHAPTER 4
CONCLUSION AND ADVICE
Internet Resources
http://animalscience.ucdavis.edu/animalbiotech/Outreach/Livestock_cloning.pdf
http://www.fda.gov/AnimalVeterinary/NewsEvents/FDAVeterinarianNewsletter/ucm1081
31.htm
http://learn.genetics.utah.edu/content/tech/cloning/whyclone/
http://en.wikipedia.org/wiki/
http://chestofbooks.com/animals/horses/Health-Disease-Treatment-4/ArtificialInsemination.html
http://www.buzzle.com/articles/pros-and-cons-of-cloning.html
http://www.actionbioscience.org/biotech/margawati.html
http://www.glowm.com/index.html?p=glowm.cml/section_view&articleid=365
http://robby.nstemp.com/shopping_page.html
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