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Animal cloning applications in agriculture

Article  in  IEEE Engineering in Medicine and Biology Magazine · March 2004

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 Animal Cloning

THE CLONING DEBATE

Applications in Agriculture

© COREL, 1991 AND 1999 PHOTODISC

The Potential for Making Improved
INC., 2001 IMAGE SOURCE LIMITED. SEE
PAGE 1 FOR DETAILS.
Agricultural Products to Benefit Consumers
BY RAYMOND L. PAGE AND
SAKTHIKUMAR AMBADY

he ability to replicate or duplicate the animal genome using a combination of chemicals that induce the biochemi-

T has had an enormous impact on the potential promise

of the agricultural animal biotechnology industry.
Prior to the publication of the techniques used to clone
the first animal, a sheep named Dolly, using cells derived
from mammary tissue of an adult sheep [1], it was thought
cal events that simulate the process of fertilization [15]. The
nuclear transfer derived embryo is then implanted into a sur-
rogate mother to produce the cloned animal. This process is
summarized in Figure 1.

that the developmental program acquired by cells as they

differentiate from early embryonic stages to form special-
ized tissues that make up the fetus was irreversible [2], [3].
In the following few years, researchers have rapidly shown Somatic Cell
that the same techniques, or with slight modifications, can Culture Mature
be used to clone other species including cattle [4], pigs [5], Oocyte
goats [6], mice [7], rabbits, [8], cats [9], endangered species
[10], equines [11], [12], and most recently rats [13]. The
rush to perfect these techniques in these other species is
reflective of the unique benefits of the basic technology and
Enucleated
its broad impact.
Somatic Cell Oocyte
The scope of this article will be to outline the potential
benefits of cloning technology to the animal agricultural
industry and highlight some of the possibilities for combin-
ing the tools offered by nuclear transfer, animal genomics,
Nuclear Transfer Unit
and genetic engineering to make improved animal agricul-
tural products.
Fusion/Activation
Cloning by Somatic Cell Nuclear Transfer
In normal fertilization, the sperm cell penetrates the egg cell Zygote Stage
and results in a zygote that contains a complete diploid
genome. During the process of fertilization, the sperm cell
also delivers a chemical signal to the egg, known as activa- In Vitro Culture
tion, which triggers the egg to resume the cell cycle and
begin development of the embryo. The zygote is referred to
as a totipotent cell, in that it is capable of forming a com- Two-Cell Embryo
plete organism. In nuclear transfer, the nuclear genome
(DNA) of one cell is replaced with that of another.
Typically, the removal of maternal DNA from the oocyte is
accomplished under a microscope equipped with microma-
nipulators and, subsequently, the donor DNA is introduced
Blastocyst Stage
by electrofusion [14]. For the nuclear transfer event to result
in a live animal, the donor cell DNA is transferred into a
mature oocyte, from which the DNA has been removed,
forming a nuclear transfer unit. Activation of the newly
formed nuclear transfer unit is accomplished artificially Fig. 1. Somatic cell nuclear transfer process.

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 Ultimately, economic factors will determine if
cloning technology will gain widespread use in
agricultural animal production.
Preservation and Dissemination of Genetic Value
The donor cells used in nuclear transfer to create a copy of
an animal can be propagated in vitro to produce millions of
cells that can be stored frozen indefinitely. Therefore, in
theory, cloning could be used to generate many copies of
valuable animals at any time in the future. The ability to
gain maximum benefit from this attribute relies on the fore-
sight of individuals that manage genetically valuable ani-
mals to cryopreserve cells. Furthermore, progress made in
animal genomics in the future may be capitalized on by the
availability of a frozen gene pool from which to screen for
valuable traits identified by genomic markers. For example,
if genetic markers are identified that correlate to traits such
as meat tenderness, parasite resistance, reproductive sound-
ness, or other desired attributes, the screening process could
be applied to a genetic base that far exceeds the number of
animals alive at the time. In fact, this can be viewed as a
potential method for broadening the gene pool to incorpo-
rate desired traits into breeding programs, as opposed to
narrowing it.
Traditionally, worldwide dissemination of genetic value
has been accomplished using artificial insemination and
embryo transfer. These techniques are limited by the amount
of material from the donor animals, thus the full distribution-
al potential of the animals of greatest value cannot be real-
ized when demand far exceeds supply. To solve this
problem, genetically important founder animals may be
cloned to increase the supply of semen or embryos in order
to achieve a broader distribution. In addition, a copy of the
founder animal may be delivered to the distribution site by
Fig. 2. Four somatic cell clones from the same cell line.
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shipping frozen cloned embryos, or by shipping cells to be
used for nuclear transfer at other market sites. Instead of pro-
ducing tens of embryos from a valuable donor cow, using
conventional embryo transfer, hundreds or thousands of
cloned embryos can be produced. For export markets, this
allows breeding programs to be established based on an
actual donor animal instead of offspring alone. This, in turn,
facilitates the expeditious evaluation of breeding interactions
between imported donor animals and animals indigenous to
the region.
Animal Genomics
Agricultural animals that exhibit desirable traits, such as
high milk yield, rapid weight gain, meat tenderness (natural
marbling), efficient nutrient utilization (feed efficiency),
product consistency, and disease resistance are of economic
importance to the food production industry. Traditionally,
gains toward increasing value by exploiting animals having
these traits has been accomplished by selective breeding
based on actual performance data. The problem with this
approach is that many breeding combinations must be
attempted and studied in order to establish which animals
pass the desirable traits most efficiently. In the dairy indus-
try, for example, this is called progeny testing, and it takes
up to five years to establish which bulls produce daughters
producing the most or best quality milk. Another drawback
is that the bull himself may not display the phenotype of an
undesirable recessive trait that could be passed along to his
progeny. Hence, there is a need for a better method of select-
ing the seed stock animals from which to generate the future
generations of production animals. Could there be informa-
tion in the genetic code (DNA) that can be used to make
more informed breeding decisions? The answer lies in the
promise of animal genomics and DNA mapping.
In the last decade, much attention has been given to the
subject of animal genomics [16]–[18]. For example,
researchers have begun to develop correlations between
specific gene loci (e.g., myostatin) and the characteristics
of meat such as marbling and tenderness [19]–[22].
Genomics, as it relates to animal breeding, can be broadly
defined as the use of genetic information at the DNA level
to develop correlations to performance criteria established
for particular traits. More importantly, genomic informa-
tion could be used to predict the ability of the desired traits
to be passed along to the progeny, thus to future genera-
tions. The power of this technology is immense. What if
information from tests conducted on the DNA from a blood
MARCH/APRIL 2004
 The ability to clone an animal also offers
the unique capability to alter the
gernome to greatly accelerate the rate
at which desirable traits are acquired.
sample taken from a potential breeding bull could predict
the milk yield of his daughters, whether he carries any
recessive genes that will impact his daughters health or
longevity, if he has genes that lead to a better defense
against diseases or parasites, etc? Now, what if this bull or
series of bulls like him were to be cloned? This would
enable a much more rapid and immediate transfer of those
valuable traits to the production herds and, ultimately, to
the consumer.
Benefits of Cloning and Animal Genomics
to the Consumer
In order to outline the benefits of cloning to the food con-
sumer, it is important to first have an understanding of the
reason a producer would choose to use cloning technology in
a breeding program. Ultimately, economic factors will deter-
mine if cloning technology will gain widespread use in agri-
cultural animal production. The economics are largely
dictated by the quantity and quality of the product, because
consumers desire the best product for the lowest price.
Quality food products are produced from animals that are
free from disease and have good nutrient utilization. These
factors translate into more product(s) from fewer animals,
which has an impact on the environment from a waste man-
agement perspective. Fewer animals mean lower production
cost and, ultimately, a lower food bill. On the beef side, if
more of the production animals are derived from seed stock
animals that yield a superior quality carcass (i.e., well mar-
bled, tender, less fat, etc.), the consumer will realize these
benefits at the meat counter. Animals with better disease and
parasite resistance traits translate into food derived from
healthier animals, as opposed to those slaughtered prema-
turely due to poor health or infections, etc. Furthermore,
heartier animals require less treatment with antibiotics and
other drugs.
Genetic Modifications
Thus far, this discussion has centered on using cloning and
genomics to capitalize on genetic traits that occur through
natural breeding or mutations induced by the environment.
The ability to clone an animal also offers the unique capabil-
ity to alter the genome to greatly accelerate the rate at which
desirable traits are acquired. This is accomplished using
transgenic technology.
The production of transgenic animals, by various meth-
ods, heralded an era of hopes and promises. However, many
of the promises remain unfulfilled due to technical and
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logistic problems inherent in the system, whereby
researchers were unable to control the site and number of
DNA insertions and the expression level of genes intro-
duced into a transgenic animal. This is because the methods
of gene delivery that yield very precise genetic modifica-
tions in cultured cells cannot be applied to embryos. Using
gene transfer techniques that function in embryos, it is only
possible to add genes, not to remove them. Therefore, the
utilization of techniques for targeted transgene integration
was restricted to mouse studies, where specialized cells
called embryonic stem cells (ES cells) were available, could
be genetically modified, and then used to generate an ani-
mal. The advent of cloning technologies using somatic cells
has now created the opportunity to introduce site-specific
genetic changes in the genome of livestock species. A dis-
cussion of the genetic engineering technologies that are
expected to have profound impact on the development of
improved agricultural animals follows.
There are numerous methods of delivering DNA into cells
in vitro. These include pronuclear injection in embryos [23],
[24], microinjection [25], electroporation [26], cationic lipids
[27], use of viral delivery systems such as retroviruses [28],
adenoviruses [29], adeno-associated viruses [30], lentiviruses
[31] and bacterial vectors [32], transposon-based systems
[33], sperm-mediated transgenesis [34], and receptor-
mediated endocytosis [35]. Among these methods, pronuclear
injection, viral delivery systems, bactofection, and sperm-
mediated DNA delivery are not useful for targeting the DNA
to precise chromosomal loci. Even if targeting vectors were
used, technical limitations inherent in pronuclear injection and
sperm-mediated gene transfer techniques prevent these meth-
ods from being employed to produce gene-targeted transgenic
animals. Viral and bacterial vectors are objected to as DNA
from the vectors themselves is also incorporated into the ani-
mal genome [36]. Electroporation, cationic lipids, microinjec-
tion, and receptor-mediated endocytosis are methods that can
be used to deliver DNA into cultured cells without adding
undesirable genetic components to the genome. The genetical-
ly engineered cells can then be used as donor cells to make
cloned transgenic animals using nuclear transfer technology.
Methods developed to clone livestock from adult somatic
cells have now paved the way to generate animals with
desired genetic modifications. The first transgenic cloned cat-
tle [37] and sheep [38], in which the DNA was inserted into
cells randomly, was rapidly followed by production of trans-
genic sheep [39]–[41] and pigs [42], [43] in which the cells
were precisely modified using homologous recombination.
MARCH/APRIL 2004 29
 Recently, bovine cells in which the function of the α-1,3-
galactosyltransferase gene has been removed through gene tar-
geting has been reported [44]. Cloned cattle engineered with
an artificial chromosome containing the genetic information to
make human antibodies has also been reported [45]. Most of
the transgenic livestock produced thus far are intended for bio-
pharmaceutical applications with the exception of the prion
protein [which is responsible for mad cow disease (Bovine
Spongiform Encephalopathy)] knockout sheep [40]. However,
recently cloning technology has been used to produce trans-
genic cows that produce higher concentrations of casein pro-
teins in their milk, which may yield superior cheese products
[46]. The same technologies could be used to produce agricul-
tural animals having improved meat or wool quality, carcass
uniformity, higher feed efficiency, and improved immunity.
These potential modifications will bring benefits to livestock
producers, consumers, and the environment [47].
The recent development of small interfering RNA
(SiRNA) technology, first discovered in plants and later in
mammals, offers additional possibilities due to their
unique ability to down-regulate gene expression at the
RNA level [48]. This technique might be used to attenuate
the expression level of specific proteins that have broad
implications for specifically modifying the content of ani-
mal products. Additionally, transgenic animals engineered
to carry SiRNA targeted against disease organisms are
envisioned. Foot and mouth disease and bovine viral diar-
rhea (BVD) are examples of pathologies for which this
strategy may be used. Other possibilities for transgenesis
include the introduction of point mutations in the host
genome for traits that are controlled by single copy genes,
for example, the bovine kappa casein gene [49]. This
could be accomplished by the use of RNA-DNA hybrid
oligonucleotides [50] and triple helix forming oligos
(TFOs) [51] to introduce point mutations at the target site,
taking advantage of the DNA repair mechanism of the
host cells.
Implications for Animal Agriculture
The potential benefits of transgenic technology in agri-
cultural animals has been reviewed extensively
[52]–[57]. Possibilities include: modification of milk
composition for improved cheese processing [58] and
infant formula [59], increasing desired proteins and
removing or lowering the concentration of undesirable
ones, removing potential allergy causing proteins from
milk [60], and improving the antibacterial properties of
milk [61], [62].
As genetic alteration technologies continue to gain
sophistication and precision, it is envisioned that either
whole chromosomes, or large fragments containing clusters
of genes, implicated in conferring specific traits may be
moved between breeds of animals. This approach will
enable the transfer of highly desirable traits, such as heat
tolerance, into high beef or milk production animals without
compromising the desired production traits, as would likely
result if conventional cross breeding were used.
Furthermore, using homologous recombination strategies in
somatic cells, specific genes can be removed from the
genome. Application of this approach could be used to
remove the prion gene in cattle generating animals that can-
not acquire mad cow disease. In theory, receptors on the
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surface of cells, which are present in certain animals, make
them susceptible to viral disease could be altered to elimi-
nate the possibility for infection.
Conclusion
Cloning technology offers the unique ability to replicate high-
quality production animals and disseminate their genetic value
more broadly than ever before possible. It also affords the
ability to preserve genetic seed stock indefinitely for potential
use in the future. These attributes have value in their own
right. However, cloning also represents a tool with which to
capitalize on the knowledge gleaned from animal genomics
research and the power of transgenic technology. Genetic
gains have been realized through conventional selective breed-
ing and culling of domestic animals for centuries. However,
those improvements are based strictly on trial and error due to
the nature of chromosome recombination events during meio-
sis, thus, offspring that have desirable traits are produced
alongside those that do not. Applying these new biotechnolo-
gies in the agricultural animal industry will permit deliberate
and precise modifications with highly predictable outcomes
avoiding the trial and error of selective breeding programs.
Raymond L. Page received the B.S.
degree in 1987 and the M.S. degree in
1989, both in chemical engineering from
West Virginia University, Morgantown,
and the Ph.D. degree in chemical engi-
neering from Virginia Tech University,
Blacksburg, in 1992 in the field of genetic
engineering and molecular embryology.
He joined PPL Therapeutics as a research scientist in 1992,
where he worked on production of human therapeutic pro-
teins in the milk of transgenic farm animals. He then joined
Advanced Cell Technology in 2001, where he studied cell
differentiation. In early 2002, he joined a spin-out agriculture
biotechnology company, Cyagra Inc., as its chief scientific
officer. Cyagra is focused on the use of cloning and genetic
engineering technology to create improved livestock prod-
ucts worldwide.
Sakthikumar Ambady obtained his
DVM degree in 1984 from Andhra
Pradesh Agricultural University, India,
and doctorate degree in 1996 from the
laboratory of F. Abel Ponce de Leon in
the Department of Veterinary & Animal
Science, University of Massachusetts at
Amherst, U.S.A. He was instrumental in
the development chromosome-specific libraries in a variety
of farm animals for use in the isolation and development of
microsatellite markers for generating genetic maps. He then
continued as a postdoctoral research associate in the same
laboratory working on gene mapping in chicken. In 1998,
he joined Advanced Cell Technology as a Research
Scientist to work on developing an in vitro culture system
for chicken primordial germ cells. In early 2002, he joined
an agricultural biotechnology company Cyagra, Inc. as a
research scientist where he is currently the principal scien-
tist. Cyagra, Inc. is focused on the use of cloning and
genetic engineering technology to create improved live-
stock products worldwide.
MARCH/APRIL 2004
 Address for Correspondence: Raymond L. Page, Cyagra
Research and Development, Building 21, 200 Westboro
Road, North Grafton, MA 01536 USA. E-mail:
rpage@cyagra.com.
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