Report on Human Manipulations of Genetic
Transfer and its Biological Implications
Introduction
Dairy farming is very important to New Zealand as evidenced by the hit our
economy is currently taking due in part to the unanticipated slump in global
dairy prices. Many diary farmers are suffering (and even the absurb “marketbased” $4.18 million annual salary paid Fonterra’s CEO could be in jeopardy).
Dairying has seen better days, but in the longer term our use of biotechnology
may provide us with positive opportunities for this innovative industry.
For example, Kiwi farmers have been docking cows’ and lambs’ tails for
centuries, yet these appendages, unwanted by our farmers, still arrive intact with
each generation. Maybe a more permanent solution to this tail issue lies with
genetic engineering (GE). Interestingly, New Zealand has already genetically
engineered a cow that had no tail, albeit an unexpected outcome of a ground
breaking cloning experiment. With the 2012 announcement of “Daisy” the calf,
bred in captivity by AgResearch from a cloned embryo, New Zealand scored an
impressive first in this rapidly developing science.
Driven by curiosity and concern, given the early death of Dolly the cloned sheep,
last week I contacted AgResearch scientist Kamla Chandar and contrary to
pessimistic predictions, Daisy the cow remains alive and well, securely contained
and most unlikely to enter our food chain.
If Kiwi anxiety caused over “Corngate” (a New Zealand political scandal in 2002
about the suspected local release of genetically modified corn) is any indication,
Kiwi resistance to GE is strongly entrenched. We are thus unlikely to find milk
from cloned cows at Pak ‘n Save, Countdown or New World supermarkets any
time soon, albeit these enterprises seem to be “milking” us. Yet surprisingly
perhaps, our government may soon approve the development and propagation
of genetically modified forestry products in the Hawkes Bay region (The
Dominion Post, 10 August 2015).
Purpose and Structure
The purpose of this report is to discuss the processes and implications of human
manipulation of genetic transfer when modifying the expression of existing
genes and when cloning mammals, with particular reference to Daisy the cow.
After a brief description of mammal cell structure, this report is presented in the
following two parts:
Part One: Ribonucleic Acid Interference (RNAi) or gene knockdown as
used for the creation of Daisy the cow. Since its discovery in 1998 this
technique, which was hailed by Science magazine as the break-through of
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the year in 2003, has largely been used to “knock out” or “knock down”
genes in order to better understand their function.
Part Two: A cow can be cloned by embryo splitting or by nuclear
transfer. This report addresses nuclear transfer or somatic cell nuclear
transfer (SCNT) as used for cloning Dolly the sheep and could be used for
cloning cows or any other mammal. Transgenic embryoes can be made
with this technique.
Cell Biology
To help understand the two GE processes described in this report, readers are
reminded that within the cell nucleus reside microscopic chromosomes that are
composed of genes made of deoxyribonucleic acid (DNA). See Figure 2. DNA is
the molecule that contains the genetic code for life forms and is comprised of two
long, twisted chains made up of nucleotides, each of which contains one base,
one phosphate molecule and the sugar molecule deoxyribose.
Figure 2: The cell nucleus contains most of the cell's
genetic material in the form of DNA molecules arranged
as chromosomes.
All biological behaviour, whether plant or animal, originates in the genes via the
proteins they express. For a cow, DNA provides the set of instructions (genes)
that are needed in order to grow, produce milk, breed, etc. The genome for a
cow contains 60 chromosomes and some 22,000 genes of which about
80 percent are the same as human genes. Genes are inherited – they are passed
from parents to their offspring. Mammals each inherit two copies of each gene,
called alleles, one from each parent. These inherited alleles determine the
particular characteristic or traits that mammals have, although genes can change
somewhat over time, due to natural selection and ecological pressures, as they
pass from one generation to the next.
Genes serve as instructions for making functional molecules such as ribonucleic
acid (RNA) and proteins. Proteins are the machines within cells that make all
living things function. While genes seem to get a lot of attention, it is
proteins that perform most life functions. Each gene in DNA encodes
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information about how to make a particular protein. While proteins provide a
body's main building materials, they cannot make copies of themselves. When a
cell needs more proteins, it uses the instructions coded in DNA whereby
“daughter” cells inherit the genes of “mother” cells.
Genetic engineering (GE) or genetic modification (GM), is the introduction of
genes that provide the code for a particular trait inserted into DNA. The process
involves the artificial addition, deletion or re-arrangement of DNA to achieve the
desired characteristic. DNA is used as a template to make a messenger RNA
(mRNA). This message molecule is then used to build protein molecules.
Techniques for modifying the DNA of living organisms such as cows include gene
knockdown and nuclear transfer, which are described and examined in this
report. First – RNA interference or gene knockdown as used by AgResearch to
create Daisy the cow.
PART ONE: RNA INTERFERENCE (GENE KNOCKDOWN)
A description of the RNA interference (Requirement 1 in Assignment
Instruction). RNAi is a natural process that scientists are now beginning to better
understand, but as yet cannot convincingly control. RNAi, gene knockdown or
gene silencing is about selectively turning off genes or reducing their
expression. Using a boxing metaphor, “knockdown” means the gene is
significantly depleted (70% to 90%), which is not to be confused with
“knockout” when the target gene is completely erased from the cow’s genome. In
research, gene knockdown is possibly more useful than gene knockout, since
knockdown allows scientists to see what least expression is needed for the result
they desire.
Figure 3: A photo of “Daisy”, that AgResearch
created by gene knockdown that is also called
Ribonucleic Acid Interference (RNAi).
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It was this process of gene knockdown that New Zealand AgResearch scientists
used to bred Daisy (Figure 3) the genetically engineered female calf designed to
produce milk that contained less beta-lactoglobulin (BLG) protein, which is a
milk whey protein known to be allergenic for some 2 or 3 percent of human
infants. BLG is not naturally present in human breast milk. The RNAi process is
explained later in this report.
A description of two biological implications of RNA interference
(Requirement 3 in the Assignment Instruction). Two biological implications of
RNAi or gene knockdown concern genetic biodiversity and ecosystem survival,
each of which is briefly described here:
1. Threat and promise for biodiversity. Biodiversity of the bovine
species helps ensure the species’ survival given a changing environment.
An implication of RNAi is that it has the potential to either decrease or
increase the species’ biodiversity. Some traits are not necessarily
“intelligently” designed and natural selection can backfire.
2. Ecosystem survival imperilled or revived. Cows should not be seen
as isolated members of an ecosystem, but rather as genetically connected
members of a very complex interacting community. Thus, another
implication is that genetically altered cows, as a consequence of RNAi,
could upset or help restore an ecosystem’s delicate balance.
These two biological implications of RNAi are explained in the following section
of this report.
An explanation of two biological implications for RNA
interference (Requirement 7 in Assignment Instruction). Two biological
implications of gene knockdown concern biodiversity and a species’ survival, and
ecosystem survival, each separately explained here:
1. Biodiversity and Survival
Dairy farmers need a broad gene pool to draw upon if they are to improve the
characteristics of their animals under changing conditions. RNAi may help or
hinder such biodiversity. If RNAi is used to develop all cows with the same traits
we may put the survival of this species at risk in a changing environment. Also,
natural selection can backfire and result in evolutionary convergence rather than
divergence.
The Global Databank for Farm Animal Genetic Resources compiled by the UN
Food and Agriculture Organisation estimates that over 99% of all species (ie,
over five billion species) that have ever lived on this planet are now extinct.
Also, in the past 15 years, 190 breeds of farm animals (including cow breeds)
have become extinct and some 1,500 are at risk of becoming extinct (1). The
World Life Fund reported that in 2014 Earth had lost 52% of its biodiversity.
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Footnote (1). Kunin, W.E.; Gaston, Kevin, eds. (31 December 1996). The Biology of Rarity: Causes and
consequences of rare—common differences. ISBN 978-0412633805. Retrieved 26 May 2015
Genetic diversity is important to the continuing development of bovine varieties
that are resistant to new pests, diseases, and changing climatic and
environmental conditions. Ecologists argue that diversity means ecological
resilience – an ability to adapt to future unknowns.
With the unpredictable effects of global warming for example, diversity within
the genetic structure of the bovine species seems essential. Artificial gene
knockdown may help arrest this problem as we create organisms with traits
designed to survive our changing habitat. Ironically, some would argue that
cows are the cause of global warming.
When diversity is very low, all the individuals are nearly identical. If a new
environmental pressure, such as a disease, comes along, all of the individuals
within the population may get the disease and die. But in a population with
greater genetic diversity, assisted through selective gene knockdown, chances
are better that some individuals will have a genetic makeup that allows them to
survive. These individuals will reproduce and the population will survive.
and also to create variety within a species that helps with a species’ survival.
Species need to evolve as their habitat changes. Carefully applied, gene
knockdown, coupled with selective breeding, could create genetic biodiversity of
the cow gene pool to help ensure a variety of traits among the individuals of that
population. This variety of traits then provides a greater chance of the bovine
species’ survival in our changing environment.
Thus, genetic biodiversity (or biological diversity) is an important factor in the
survival and adaptability of the bovine species. We also need to recognise that
many species depend on each other. Habitat destruction might be caused by
nature or by man. With little or no genetic diversity our dairy industry is not
only more vulnerable to environmental changes, but is also more vulnerable to
diseases and problems arising from inbreeding. Interestingly, barriers such as
rivers, seas, mountains and deserts also enable diversity through separate
evolution either side of the barrier.
However, gene knockdown may also have the reverse effect. While US cow
cloning companies may promise the production of identical, high-quality
animals, scientists warn that this could be a recipe for disaster. Concern has
been raised that bovine genetic diversity will decrease because breeding
programmes will concentrate on a smaller number of high value cow types that
have had currently undesireable traits removed through RNAi manipulation.
2. Ecosystem’s Survival
Cows in which genes have been knocked down may help or hinder an
ecosystem’s survival. A species should not be seen as isolated members of an
ecosystem, but rather as genetically connected members of a very complex
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interacting community. An ecosystem is a group of living organisms and nonliving elements (weather, earth, sun, soil, climate, atmosphere) living in a
particular environment of indefinite size and interacting as a system where
changes in genetic traits to one species can influence ecological interactions and
vice versa.
Ecosystems can be of any size but are usually limited to a specific space. Some
would argue that our entire planet is an ecosystem. Ecosystems are dynamic and
the introduction of variety in species through gene knockdown, artificial or
natural, can have both positive and negative implications for the ecosystem’s
operation and evolution, the study of which is called “ecological genomics” a field
very early in its development that recognises genetic variations influence
ecological variables. Ecosystems can have very different characteristics because
of the different species they accommodate and the introduction of new or
genetically modified species can cause changes in ecosystem function. Used
responsibly, gene knockdown could considerably benefit cows, humans and the
environment (2). If an external factor such as rise in temperature or genetically
altered cow is introduced into an ecosystem, this could dramatically affect that
ecosystem.
Arguably, an introduced genetically altered cow as a consequence of gene
knockdown could distort the natural balance of an ecosystem and possibly harm
or even destroy the ecosystem. One biotic factor, such as a genetically altered
cow through gene knockdown could affect all parties in the ecosystem even in
ways we do not yet know about. As the naturalist, writer, and environmental
activist John Muir stated, "When we try to pick out anything by itself we find that it
is bound fast by a thousand invisible cords that cannot be broken, to everything in
the universe.”
Yet, one small change to the bovine species through gene knockdown could
perhaps save and restore a natural ecosystem and provides the genetic variation
needed to increase food production, enhance its quality and adapt it to our everchanging environmental and socio-economic conditions. Also, gene knockdown
might benefit biodiversity by reducing or even eradicating environmentally
damaging invasive species such as rabbits that compete with cows for grass.
Perhaps the introduction of genetically engineered cows into our local
ecosystems would be a dangerous experiment with nature and evolution. On
balance these genetically manipulated cows could reproduce and interbreed
with natural cows, thereby spreading to new environments and future
generations in unpredictable and uncontrollable ways. If a transgenic cow is
mated with a non-transgenic bull, her offspring will have only a 50% chance of
being transgenic, but if we were to mate two transgenic cows, there is a 75%
chance of transgenic offspring.
Footnote (2) Chapin, F. Stuart; Pamela A. Matson; Harold A. Mooney (2002). Principles of Terrestrial
Ecosystem Ecology. New York: Springer.
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separate from regular herds. The reason transgenic or genetically
modified organisms in New Zealand need to be contained is because they are
classed as a new organism, so they are foreign to New Zealand.
An explanation of how RNA interference effects target gene
expression (Requirement 5 in Assignment Instruction)
“Gene expression” is the process whereby genes are first converted to
messenger RNA and then into proteins. Genes are expressed by being
transcribed into RNA, which may then be translated into protein. Over the past
decade RNA interference (RNAi) has emerged as the preferred biological process
for knocking down the expression of targeted genes. RNAi inhibits gene
expression by causing the destruction of specific messenger RNA (mRNA)
molecules. Advantages of RNAi over more classical methods include the higher
efficiency of gene knockdown and the ability to more easily target the gene of
interest. And RNAi provides the scientist with the ability to knockdown virtually
any gene. The process reduces the activity of targeted genes without eliminating
it completely.
Central to the RNAi interference process are two small ribonucleic acid (RNA)
molecules – microRNA (miRNA) and small interfering RNA (siRNA). Small
interfering RNA (siRNA) is sometimes also known as short interfering RNA or
silencing RNA. RNA molecules bind to other specific messenger RNA (mRNA)
molecules that convey genetic information from DNA to the ribosome (consisting
of RNA and associated proteins found in the cytoplasm of cells), where they
specify the amino acid sequence of the protein products of gene expression and
either increase or decrease their activity, thus preventing an mRNA from
producing a protein. Each siRNA is unwound into two single-stranded RNAs
(ssRNAs), the “passenger” strand and the “guide” strand. The passenger strand is
degraded and the guide strand is incorporated into the RNA-induced silencing
complex (RISC). See Figure 4.
Figure 4: Gene knockdown or RNA interference (RNAi)
is a process in which RNA molecules inhibit gene
expression.
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Figure 4 depicts double-stranded RNA digested into siRNA’s by an enzyme called
Dicer. Next, each siRNA is split into single strands that are incorporated into a
protein complex. This complex binds to the viral target gene, silencing the gene
or limiting its expression.
An evaluation of the effectiveness of RNA interference to deliver
its intended outcomes. (Requirement 8 of Assignment Instruction)
RNAi has most successfully been applied in plants, but some practical obstacles
need to be overcome before it becomes entirely effective for the genetic
modification of cows. One significant concern is “off-target effects.”
Complications can arise with gene knockdown when a gene closely related to the
target gene is destroyed. RNAi has potential for this cross-interference where
RNAi of one gene inhibits the intended target gene as well as a closely related
gene. So far, there is no hard and fast rule for predicting cross interference side
effects. Reduction of gene expression as a result of these off-target effects could
result in outcomes that appear to be related to changes in the antiviral pathways.
Off-target effects are of concern because of the potentially catastrophic results
such as the accidental targeting of a gene necessary for survival. Such off-target
effects represent a significant roadblock to RNAi technologies in research,
therapeutic, and diagnostic situations. Daisy’s missing tail may have been an offtarget effect.
The second part of this report follows. It concerns the process of cloning a cow
using the SCNT technique, which technique could be combined with RNAi to
produce ongoing generations with identical genetic modifications? The
production of cloned animals following nuclear transfer, represents a
remarkable feat of developmental biology.
PART TWO: THE SCNT CLONING TECHNIQUE
Description of cloning a cow by the SCNT technique (Requirement 2
of Assignment Instruction). There is a multitude of techniques to make
transgenic animals and cloning is just one of them. Employing cloning to produce
transgenic animals involves the use of genetically modified cells as DNA donors
instead of unmodified cells. SCNT produces an exact genetic copy, or clone. In
natural fertilisation, the sperm and egg have one set of chromosomes each and
when the sperm and egg join, they grow into an embryo with two sets of
chromosomes—one from the father's sperm and one from the mother's egg.
However, in SCNT, the egg cell's single set of chromosomes is removed and
replaced by the nucleus from a somatic cell. This cell already contains two
complete sets of chromosomes, such that in the resulting embryo, both sets of
chromosomes come from the somatic cell. The first successful nuclear transfer
was done with a frog in the 1970s. In 1996 “Dolly” the sheep was the first
mammal to be cloned from an adult somatic cell using SCNT and then cattle were
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the second mammal species to be cloned using this technique. Cattle are
currently the most widely used species for SCNT experiments and cloning a cow
by this technique is illustrated at Figure 5. The SCNT technique is explained later
in this report.
Figure 5: Basic SCNT cloning process to produce an
identical animal to the genetic donar cow.
A description of two biological implications of SCNT cloning
(Requirement 4 in Assignment Instruction). Two biological implications of SCNT
cloning are the uncertainty of the process and the outcome, and the promise of
beneficial outcomes for mankind, each of which implication is described here:
1. Implication of SCNT uncertainty. Biologically SCNT cloning is high
risk. The process has a very big chance of failure with potentially serious
consequences, since even in the very few successful clonings, problems
can arise later during the cow's life. Cloning through somatic cell nuclear
transfer is also enormously inefficient. The success rate ranges from 0.1
percent to 3 percent only. Also, DNA is pre-programmed so that selected
genes are "turned on," or expressed, at certain times while other genes
are "turned off." However, cloned cells may lack the programming
necessary to message genes when to turn on and off, which may cause
disorganised cell growth or improper cell functioning. Such
consequences could cause a cow’s death. Another biological implication
of SCNT cloning is that as cells divide, the DNA sequences that cap the end
of a chromosome, called telomeres, reduce in length every time the DNA
is copied causing early aging. Also, cloned animals that do survive are
often much bigger at birth than their natural counterparts, which can
result in breathing and blood flow problems.
2. SCNT benefits for mankind. As a customer we humans favour, or
should favour, nutritious and wholesome produce provided in a
repeatable, humane, ethical and reliable manner. SCNT provides us with
the ability to propagate the best cows. The process would enable us to
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determine the characteristics of diary cows, rather than taking the chance
with the normal breeding process that is slow and too hit and miss.
Cloning could also improve the welfare of cows by eliminating pain and
suffering from disease. We could expand the cow genome through
cloning and breed disease resistance into the population. Also, cloning
could reduce the number of unwanted cows, such as veal calves. Cloning
could ensure more female offspring for dairy production. Also, cloning
has the potential to eradicate mastitis (a painful disease of the cow’s
udder).
These two biological implications of SCNT are explained in the following section
of this report.
An explanation of two biological implications for SCNT cloning
(Requirement 7 in Assignment Instruction). Two biological implications of SCNT
cloning involve the uncertainty of the process and its potential benefit to
mankind as explained here:
1. Implication of uncertainty.
When we hear of SCNT cloning successes such as Dolly the sheep, we usually
hear about only the few attempts that worked. We do not hear about the large
number of cloning attempts that fail. Thus, biologically the process is high risk –
very big chance of failure with potentially considerable consequences, since even
in the very few successful clones, problems often arise later during the animal's
life.
Making changes to a cow’s genetic code is called epigenetic reprogramming and
this accounts for most cloning failures. The process is yet to be mastered. The
process allows the cell to develop into a new organism instead of continuing to
do its old specified cellular functions, which for example accounts for the
different fingerprint patterns of identical human twins.
SCNT cloning through somatic cell nuclear transfer is enormously inefficient. The
success rate ranges from 0.1 percent to 3 percent, which means that for every
1000 tries, only one to 30 clones are made. Or we can see it as 970 to 999
failures in 1000 tries. The reasons for such a failure rate include an
incompatibility between the enucleated egg and the transferred nucleus, an egg
with a newly transferred nucleus may not begin to divide or develop properly,
implantation of the cow embryo into the surrogate mother cow might fail, and
the pregnancy itself might fail.
Also, DNA is pre-programmed so that selected genes are "turned on," or
expressed, at certain times while other genes are "turned off." However, cloned
cells may lack the programming necessary to message genes when to turn on
and off, which may cause disorganised cell growth or improper cell functioning.
Both these consequences could cause a cow’s death.
Another biological implication of SCNT cloning is that as cells divide, their
chromosomes appear to get shorter. It is thought that this occurs because the
DNA sequences that cap the end of a chromosome, called telomeres, reduce in
length every time the DNA is copied. Telomeres have been compared with the
plastic tips on shoelaces, because they keep chromosome ends from fraying and
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sticking to each other, which would destroy or scramble an cow's genetic
information.
Thus the older the animal is, the shorter its telomeres will be, because the cells
have divided many times as a natural part of aging. Interestingly, Dolly lived for
only six years – much shorter life span than for naturally created sheep. Dolly
the sheep's chromosomes had shorter telomere lengths than normal. Her cells
appeared to aged faster than the cells from a normal sheep. Her cells could not
divide properly and soon not at all causing lung tumours and eventual death.
Also, cloned animals that do survive are often much bigger at birth than their
natural counterparts. This "Large Offspring Syndrome” (LOS) can lead to
breathing and blood flow problems, although some clones without LOS have
developed kidney, brain and immune system problems.
2. Benefits to mankind.
Cloning cows could be good or bad. Some would argue that commercial cloning
of cows would further erode this species diversity, because the protections
provided by genetic diversity would be lost in genetically identical herds. While
cloning might reduce biodiversity, cloning could also have potential benefits for
farmers, consumers, cows themselves and for our environment. As a customer
we favour, or should favour, nutritious milk provided in a repeatable, humane,
ethical and reliable manner. A diary farmer’s livelihood depends on providing
and selling consistently high-quality milk and related dairy products, for which
cloning offers a huge advantage. Cloning provides us with the ability to breed the
best cows that are fast-growing, have highly nutritious milk, and are diseaseresistant.
Also, cloning would enable us to better predict the characteristics of each cow,
rather than taking the chance with the normal selective breeding process that is
too uncertain. Selective breeding requires that two cows are chosen for their
dominant and favoured traits to produce offspring that show dominance
(phenotypically) in these favoured traits.
Through SCNT cloning even diseased cows could be cloned if a tissue sample is
preserved during their life or even within a short time after their death. Cloning
could also improve the welfare of cows by removing pain and suffering from
disease. We could expand the genome through cloning and breed disease
resistance into an overall cow population.
Cloning can also reduce the number of unwanted animals, such veal calves that
are surplus male offspring from dairy cows. Since the males do not produce
milk, they are turned into veal calves. Cloning would ensure more female
offspring for dairy production. Incidentally, US cows feed from genetically
enhanced grain are seemingly better tasting and more tender. And according
today’s Dominion (The Dominion Post, 11 August 2015) New Zealand’s top
quality grass-feed beef cattle and dairy cull cows are now being exported to the
US for blending with these US animals to make for bigger and better burgers.
Of course, cloning may increase the hold that Fonterra already has on milk
supply at the expense of small family farmers, who are less likely to afford the
costly technology. And with no genetic variability, a disease could affect all cows
simultaneously and potentially wipe out entire herds. A herd with genetic
differences will typically include some animals that possess natural resistance to
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certain diseases, but with genetically identical clones the protections that
diversity provides are lost. The commercialisation of cow cloning would make it
difficult if not impossible to reverse weaknesses or adverse effects bred into a
cow species. As a consequence of cloning, over 90 percent of US dairy cows are
now Holsteins.
Thus, in summary, while selective breeding has been a very successful in shaping
New Zealand’s cow livestock, it is a very slow and random process. It not only
produces desired phenotypes or traits, but often undesirable traits as well. But
cloning is a much more directed process, because we can immediately target a
specific trait – and only that trait – by introducing a new gene that holds
information for this trait, and we can, just in one generation, produce a huge leap
in a difference. An example might be the kappa casein in our casein cows, where
we have within one generation increased the production of kappa casein
threefold, which would be not possible within a selective breeding approach.
An explanation of how the process of SCNT cloning is achieved
(Requirement 6 in Assignment Instruction).
Cloned cows are genetically identical, younger twins of the cow whose cells were
used to replace the maternal genetic material from an unfertilised egg. Thus,
SCNT produces an exact genetic copy, or clone. In natural cow fertilisation, the
sperm and egg have one set of chromosomes each and when the sperm and egg
join, they grow into an embryo with two sets of chromosomes—one from the
bull’s sperm and one from the cow's egg. However, with SCNT, the egg cell's
single set of chromosomes is removed and replaced by the nucleus from a
somatic cell. This cell already contains two complete sets of chromosomes, such
that in the resulting cow embryo, both sets of chromosomes come from the
somatic cell. The first successful nuclear transfer was done with a frog in the
1970s and in 1996 “Dolly” the sheep was the first mammal to be cloned from an
adult somatic cell using SCNT and cows were the second mammal species to be
cloned using this technique. Cattle are currently the most widely used species
for SCNT experiments and cloning a cow by this technique is described in the
following paragraphs.
Somatic cell nuclear transfer (SCNT) or simply nuclear transfer, which involves
transferring genes from one cow to empty egg cell of another cow, then
implanting the embryo in a host female cow or surrogate mother. “Nuclear”
refers to the nucleus that contains the cell DNA and “transfer” refers to moving
the nucleus from the somatic cell to the egg cell. Figure 6 illustrates this process
diagrammatically.
The process is undertaken in dairy to increase the number of high quality
breeding animals, to preserve useful traits of a single animal after its death, and
to improve genetics of the dairy herd. The success rate is still low and the
process is costly. The process requires two types of cell as mentioned above:
A somatic cell, which is collected from the cow that is to be cloned,
called the genetic donor. This cell is any cell other than a sperm cell or
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egg cell. It contains the complete DNA of the cow it came from. In
mammals, somatic cells have two sets of chromosomes. For cloning
purposes, somatic cells are usually obtained by a skin biopsy performed
by a veterinarian.
An egg cell is also needed. This cell is collected from a female cow known
as the egg donor. A scientist removes the nucleus of the egg cell, inserts
the somatic cell from the genetic donor cow into the egg and fuses the two
together. Fusing can be done either chemically or by electric pulse. The
fused egg contains the genetic donor cow’s DNA.
The scientist then activates the egg, causing it to divide as an egg does if it had
been fertilised by a sperm cell in the conventional cow reproduction
process. The activated egg is placed in a culture medium where cell division
continues for several days and a blastocyst (early cow embryo) forms. After a
week or so, the blastocyst is transferred to the recipient female cow’s uterus
where development continues as a normal embryo. After full-term pregnancy,
the cow gives birth to a calf that is identical to the genetic donor.
Figure 6: The diagram shows the removal of the donor nucleus
during the SCNT process. Scientists may let the clone live—
reproductive cloning; or kill the clone for her stem cells—therapeutic
cloning.
Thus, in summary, SCNT is a GE technique used to make a transgenic embryo.
An oocyte with its chromosomes removed is fused with a transgenic bovine cell.
After artificially activating this single cell embryo so that it divides like a normal
embryo, it is grown for seven days till it reaches the blastocyst stage. Suitable
embryos are then transplanted into recipient cows.
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An evaluation/analysis of two biological implications of SCNT
cloning (Requirement 9 in Assignment Instruction). The two biological
implications of SCNT selected for evaluation/analysis are the high failure rate
associated with the process and the health of the cloned animal (cow), each
assessed here:
1. High failure rate. SCNT cloning has a high rate of failure. Success is only 0.1
percent to 3 percent. For example, Dolly the sheep was the only embryo of 277
to survive to adulthood and there is no reason to believe that other cloned cows
would live longer. One reason for this failure appears to be the incomplete
resetting of the somatic cell's DNA. During egg and sperm formation, DNA is
reset to a baseline or embryonic state. As the embryo develops, cells begin to
develop into different types of cells - muscle, liver, nerve, etc. Part of the
differentiation process involves adding and removing chemical tags on the DNA,
which keep genes turned on that are necessary for the function of that cell type
and keep others turned off. This high failure rate is attributed to causes such as
the enucleated egg and the transferred nucleus not being compatible, the egg
with the new nucleus not developing properly, the implanted embryo fails, or a
miscarriage might occur.
2. Cloned cow’s health. SCNT cloning may compromise a clone’s survival. For
example, Dolly only lived for some six years, whereas normally a Finn Dorset
sheep such as Dolly lives for 11 or 12 years. It was decided to euthanize Dolly
when she developed lung disease. However, it has been suggested that Dolly was
born with a genetic age of six years, the same age as the sheep from which she
was cloned. A post-mortem revealed that Dolly's telomeres were indeed shorter
than anticipated, suggesting that her cells had aged faster than the cells of a
normal sheep. As yet scientists are not sure why cloned animals show
differences in telomere length. An important issue is what happens to the clone
if the transferred nucleus is old? Will telomere condition determine a cloned
cow’s lifespan? Cloned embryos can be born with a variety of birth defects. For
example, at birth cloned cows may have much bigger organs than their natural
counterparts. This "Large Offspring Syndrome" (LOS) is common in clones of
cows, even among cows whose birth weights are within the normal range. A
cloned cow’s birth problems may be respiratory, cardiac, hepatic, renal,
umbilical, and immunologic. Other problems include organ dysfunction,
pulmonary and cardiovascular abnormalities, delayed time to suckle, delayed
time to stand, forelimb tendon contracture, hypoglycemia, enlarged umbilicus,
and patent urachus (inability to excrete urine). Studies show incidence of LOS as
high as 50% in cloned cows. Fortunately, food products derived from cloned
cows and their offspring seem to be as safe to eat as food from their non-clone
counterparts, unless the milk has been treated with the genetically engineered
growth hormone rBGH. This hormone can cause health problems in cows, and is
linked to cancer in humans
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Conclusions
GE is concerned with altering cows (and other animals and plants) by changing
the information encoded in their DNA, but has not yet done so in a meaningfully
commercial way. And at present there seems to be more speculation than fact
about the science and some material I perused seemed to be written from a
biased perspective. But consequences of GE probably will not be clear without
multigenerational studies. Meanwhile, many people believe that GE is
unethical. Some argue that scientists are wrong to “play God”.
While we have been altered the genomes of cows for hundreds of years
by selective breeding, achieving the ideal cow through GE would be faster and a
more assured way of getting this desired result. Two such techniques to achieve
an improved cow that are considered in this report are RNA interference (gene
knockdown) and the SCNT cloning process. RNA interference is a gene
knockdown technique that created Daisy the famous Kiwi cow. RNAi is often
preferred over the SCNT process insomuch as it is faster, less expensive, avoids
the likelihood of the cow’s embryonic death, and has reduced risk of
compensatory gene regulation.
Since the cloning of Dolly the sheep, GE has been used to clone cows, mice, goats,
pigs, rabbits, and cats. Cloning may reduce the number of cows needed. But it
could also have adverse effects on cow health. Calves and lambs produced
through cloning usually have higher birth weights and longer gestation periods.
This may lead to difficult births. Repeated exposure of cows to invasive
procedures in order to harvest oocytes for SCNT may cause them pain and
distress, but perhaps GE could also solve that issue. In addition, the survival rate
of cloned cow foetuses is low, and some survivors have on-going health
problems. Even the creator of Dolly the sheep, Ian Wilmut, now prefers another
methodology.
Interestingly, an article in the Dominion Post (18 August) reads “Crop spays that
mimic the effects of GE without changing the DNA promise a farming revolution.
Scientists predict that this will give many of the benefits of GE without the safety
concerns that manipulated genes would be passed on to future generations.” Could
this advancement also have relevance for the New Zealand dairy farming?
Those favouring GE argue that such technologies will improve animal welfare,
reduce disease (both animal and human), increase production, and create
enriched animal products that could help with our nutrition and feed our rapidly
growing population. While we have cloned farm animals such as Daisy the cow
without too much emotional turbulence, cloning humans would of course be
very controversial.
There would doubtlessly be massive clinical, religious, ethical, legal and social
issues involved in human GE, although it may have been helpful to have my clone
tackle this biology assignment. In fact, genetic GE seems a bit like writing this
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report – a lot of research, cutting, pasting, deleting and editing, the result of
which is yet to be determined.
References:
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Jaslow, R. (2012). “scientists produce a cow that produces hypoallergenic milk”. Available at:
http://www.cbsnews.com/news/scientists-create-cow-that-produces-hypoallergenic-milk/ (date acc:
10/08/15).
Center for food safety. (date unknown). “Not ready for prime time”. Available at:
http://www.centerforfoodsafety.org/files/notreadyforprimetime_execsummary.pdf (date acc: 11/08/15).
University of Utah. (2015). “What is a trait?” Available at:
http://learn.genetics.utah.edu/content/inheritance/traits/ (date acc: 11/08/15).
http://www.bewellbuzz.com/news/milk-anyone-genetically-engineered-cloned-cow/
University of Utah. (2015). “What are the risks of cloning?”. Available at:
http://learn.genetics.utah.edu/content/cloning/cloningrisks/ (date acc: 11/08/15).
Adams, B. (2015). “People aren’t ready for the imminent rise of genetic engineering”. Available at:
http://www.businessinsider.com/genetic-engineering-will-change-the-world-so-much-its-scary-but-hereswhy-the-future-is-bright-2015-4#ixzz3iD7b8VMV (date acc: 12/08/15).
http://www.agresearch.co.nz/news/a-scientific-world-first-at-agresearch/
Bren, L. (2003). “Cloning: Revolution or Evolution in Animal Production?” Available at:
http://www.fda.gov/AdvisoryCommittees/CommitteesMeetingMaterials/VeterinaryMedicineAdvisoryCommit
tee/ucm127249.htm (date acc: 12/08/15).
http://animalscience.ucdavis.edu/animalbiotech/Outreach/Livestock_cloning.pdf
http://animalscience.ucdavis.edu/animalbiotech/My_Laboratory/Presentations/2007/CCA2007.pdf
http://animalscience.ucdavis.edu/animalbiotech/My_Laboratory/Presentations/2007/CCA2007.pdf
Study.com. (date unknown). “PCR”. Available at: http://study.com/academy/lesson/pcr-synthesizing-dnausing-polymerase-chain-reaction.html#lesson date acc: 12/08/15).
BeWellBuzz (date unknown). “Milk anyone, from a genetically cloned cow?” Available at:
http://www.bewellbuzz.com/news/milk-anyone-genetically-engineered-cloned-cow/ (date acc: 13/08/15).
Wikipedia. (last updated 2013). “Gene knockdown”. Available at:
https://en.wikipedia.org/wiki/Gene_knockdown (date acc: 14/08/15).
https://www.genome.gov/27544125
Scitable. (date unknown). “Gene expression”. Available at:
http://www.nature.com/scitable/nated/topicpage/gene-expression-14121669 (date acc: 14/08/15).
http://www.nytimes.com/video/us/100000002496111/dolly-the-sheep.html
Esvelt, K. (2014). “Gene drives and CRISPR could revolutionize ecosystem management”. Available at:
http://blogs.scientificamerican.com/guest-blog/gene-drives-and-crispr-could-revolutionize-ecosystemmanagement/ (date acc: 14/08/15).
http://publications.nigms.nih.gov/thenewgenetics/chapter1.html
Variety of material (attached) obtained from AgResearch
Laible, G. (2010). “Nuclear transfer”. Available at:
http://biotechlearn.org.nz/focus_stories/transgenic_cows/video_clips/nuclear_transfer (date acc: 14/08/15).
Stokes, T. (2012). “Holy Cow! Daisy makes hypoallergenic milk”. Available at:
http://www.livescience.com/23615-transgenic-cow-hypoallergenic-milk-whey.html (date acc: 15/08/15).
http://www.sciencemag.org/search?site_area=sciencejournals&y=0&fulltext=genetic%20engineering&x=0&jo
urnalcode=sci&journalcode=sigtrans&journalcode=scitransmed&submit=yes
Akst, J. (2014). “Designer Livestock.” Available at: http://www.thescientist.com/?articles.view/articleNo/40081/title/Designer-Livestock/ (date acc: 18/08/15).
Telephone conversations and email exchanges over the period 10 – 19 August 2015 with Kamia Chandar,
AgScience, regarding Daisy and genetic transfer.
Articles in The Dominion Post dated 11 and 12 August 2015.
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Report on Human Manipulations of Genetic Transfer