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).
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
1

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.
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
2

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.
(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).
3

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.
(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.
(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.
4

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
5

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.
6

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.
(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 .
7

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.
(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.
(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
8

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.
(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
9

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.
(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
10

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
11

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.
(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
12

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.
13

(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
14

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
15

report – a lot of research, cutting, pasting, deleting and editing, the result of which is yet to be determined.
References:
 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.
16