IQP-43-DSA-1172 IQP-43-DSA-2345

TRANSGENIC ANIMALS

An Interactive Qualifying Project Report

Submitted to the Faculty of

W ORCESTER POLYTECHNIC INSTITUTE

In partial fulfillment of the requirements for the

Degree of Bachelor of Science

By:

______Robert Brooks Nathan Levesque

October 22, 2007

APPROVED:

______Prof. David S. Adams, Ph.D. W PI Project Advisor

1 ABSTRACT

Transgenic animals are genetically altered to express traits or behaviors not normally present in that species by inserting a foreign gene into their genome. The purpose of this IQP was to study the potential of this technology and its effects on society. The scope of our research incorporates a description of transgenic animal creation, a distribution of animal types, and a discussion of current ethical and legal debates. The conclusion formed from this research indicates that regulation and cautious deliberation of animal treatment can offer minimal animal suffering, while providing maximum benefit for society and humanity.

2 TABLE OF CONTENTS

Signature Page … … … … … … … … … … … … … … … … … … … … … … … … … … … ...1

Abstract … … … … … … … … … … … … … … … … … … … … … … … … … … … … … … ..2

Table of Contents … … … … … … … … … … … … … … … … … … … … … … … … … … ...3

Project Objective … … … … … … … … … … … … … … ...… … … … … … … … … … … … .4

Chapter-1: Description and Construction of Transgenic Animals… … … … … … … … 5

Chapter-2: Transgenic Applications ..… … … … … … … … … … … … … … … … … … . 13

Chapter-3: Transgenic Ethics … … … … … … … … … … … … … … ..… … … … … … … 26

Chapter-4: Transgenic Legalities … … … … … … … … … … … … … … … … … … … … . 34

Conclusions … … … … … … … … … … … … … … … … … … … … … … … … … … … … ... 37

Bibliography … … … … … … … … … … … … … … … … … … .… … … … … … … … … … . 39

3 PROJECT OBJECTIVES

The objective of this IQP was to examine the complexities of controversial new transgenic animal technologies, the intricacies involved with the ethics, and document the benefits or effects on society. The objective was accomplished by defining the methods for creating transgenic animals, categorizing and describing the different classes of applied technologies, and investigating the benefits to society. An analysis of the ethical and legal debates surrounding transgenic animals provided the backing for our conclusions about this technology and its role in society.

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Chapter 1: Description and Construction Of Transgenic Animals

A transgenic animal is an animal whose genome has been modified deliberately to contain a foreign gene from a different species. The genome is the genetic makeup of the animal and is responsible for its inherited characteristics. In every living organism the genome consists of DNA which is divided into genes (that code for proteins and regulatory RNAs), and some junk DNA (that seems to encode nothing useful). Genes contain information that regulates how the organism functions. Genes can be altered artificially so that some of the characteristics of an animal are changed. The genome is altered by having a gene from another source introduced into it, usually for the purpose of giving the animal a new feature, such as the ability to produce a life saving drug, or the ability to produce organs histocompatible with humans for organ transplants. Animals that have their DNA manipulated in this way are considered transgenic animals, and the purpose of this chapter is to describe how the DNA is manipulated, and how such animals are created.

Creating the Transgene

The first step in creating a transgenic animal is to create the DNA to be inserted.

The foreign gene or genes which are inserted into an animal are called transgenes. These transgenes must be initially cloned (to make copies) then inserted into the nucleus of the egg to be transmitted through the germ line so that every cell of the animal contains the same modified genetic material. Transgenes are linear pieces of DNA made up of three

5 separate parts. The first part of the transgene is the promoter sequence. The promoter is responsible for the function of the transgene by determining the tissue in which the transgene will be expressed. The best promoter for a given transgene will depend on the exact aims of the research. For example, when creating an animal that produces a life saving drug in the milk would use the casein promoter to ensure the drug is produced only in the milk. Tissue-specific promoters can be used to limit the spatial expression pattern, while inducible promoters are used to control the timing of the expression (UCI-

Transgenic Mouse Facility, 2003). The second important part of the transgene is the structural gene of interest. This part contains DNA sequences encoding the foreign protein to be expressed, such as human growth hormone or insulin. The third part of the transgene is the polyadenylation signal, also known as the termination sequence, because it dictates the end of the RNA encoding the protein.

Microinjection into the Male Pronucleus

Once the transgene has been cloned, it must be inserted into the genome of an animal. There are a few different ways to do this. The most popular method is to microinject the DNA into the male pronucleus. This method involves using a very fine needle (usually a thin glass hollow tube) to inject the cloned transgene into the pronucleus of a reproductive cell. The first step is to harvest freshly fertilized eggs by super-ovulating female animals by injecting them with specific hormones. These eggs are then fertilized in vitro, meaning the sperm and egg are mixed in a test tube not in the live animal. The sperm head containing the male pronucleus penetrates the egg. Before the male pronucleus fuses with the female pronucleus of the egg, the DNA is injected into

6 the male pronucleus (it is larger and easier to inject into). The injection process is performed by holding the egg with a microtube suction device and injecting a solution containing the transgene into the male pronucleus using a micropipette (Figure-1).

Figure 1: DNA Microinjection into the Male Pronucleus. On the left, the microtube suction device holds the egg and the micropipette (shown on the right) inserts the transgene into the egg. (http://www.research.uci.edu/tmf/dnaMicro.htm)

The fertilized and injected egg is then cultured. W hen the two pronuclei have fused to form the diploid zygote nucleus, the zygote will divide by mitosis to form a two- cell embryo, etc. The embryo is usually cultured to the blastocyst stage, about 5 days, where the embryo is a hollow ball of cells about the size of the period at the end of this sentence. The blastocyst is then implanted into the uterus of a pseudopregnant mother

(prepared by mating a female mouse with a vasectomized male). W ith microinjection, the insertion of DNA is random, so no control exists as to where it inserts. If it inserts within a required gene, the process can be fatal to the animal. Following the birth of the potentially transgenic pups, they are screened to determine whether they are transgenic

(discussed below). If they are transgenic, positives are often mated with other positives to increase expression of the transgene. However, the overall success rate of producing a

7 transgenic animal is very low, when using mice no more than 10-20% will have the gene, and when using farm animals no more than 0.1 œ 1.0% will be positive (Transgenic

Animals, 2003).

Embryonic Stem Cell Gene Transfer

Another frequently used method for creating transgenic animals involves embryonic stem cell gene transfer. Embryonic stem (ES) cell lines are established from the inner cell mass of blastocysts. These ES cells can be cultured and manipulated in vitro, and will resume normal development when implanted into a recipient blastocyst.

Introducing the foreign gene into these cells can be done by microinjection (as discussed above), by a virus, or using chemicals. Thus the use of these cells allows the use of a greater variety of techniques for inserting the transgene than for pronuclear micro- injection. Delivering the transgene virally is based on inserting the transgene into a virus then using the virus to infect the ES cells. Usually the virus used is mutated so it does not cause disease or multiply in infected cells. The most widely used type of virus is called a lentivirus. Lentiviruses are known as slow viruses because symptoms do no appear until long after the initial infection. These viruses (including HIV) have the ability to naturally infect both dividing and non-dividing cells, which makes them efficient for delivering genes into embryonic stem cells, and others. In developing lentiviral vectors, the DNA encoding some or all of the viral genes is removed and replaced with the foreign gene.

Thus, the viral vector is designed to be able to enter the cell, deliver the gene, but does not have the ability to replicate or cause disease once inside.

8 Chemical methods have also been developed for non-viral delivery of the transgene. These methods are based on coating the DNA with lipids, polymers, or proteins. For example, micelles are formed when DNA is coated with phospholipids.

The micelles can efficiently fuse with the ES cells to deliver the DNA. The lipids are useful because they are positively charged and help when the negatively charged plasmid

DNA makes contact with the negatively charged cell surface.

Once the specific transgene has been inserted, the ES cells containing the transgene can be selected for, which is a second strong reason for using ES cells for creating transgenic animals (Figure-2). Selection of positives can be accomplished by inserting an antibiotic resistance gene into the transgene, then exposing the treated ES cells to the antibiotic. Only those containing the transgene will survive. This selection step greatly increases the efficiency of the procedure. From here on, the procedure is similar to microinjection into the male pronucleus with respect to embryo implantation, and screening of pups.

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Figure 2: Comparison of DNA Microinjection into the Pronucleus versus Transformation of ES Cells. Method 1 shows DNA being transformed into ES cells (upper left) then injection of the ES cells into a blastocyst (upper right). Method 2 shows the DNA (red) being injected into the male pronucleus (blue). (Transgenic Animals, 2003)

Homologous Recombination with ES Cells

Another method to deliver DNA into ES cells uses the natural biological process of homologous recombination. Homologous recombination is used when the objective is to replace a specific host gene with the engineered transgene, so the transgene does not insert randomly, and the procedure does not affect any other part of the genome. To perform this method one must know the DNA sequence of the host gene that is to be replaced (Figure-3). After the transgene has been constructed, it is inserted in the middle of a host gene (if you want to perform a host knockout) (middle panel in the figure), or inserted adjacent to the host gene (if you do not wish to knock out the host gene function). The DNA is added to host ES cells which contain the targeted gene of interest.

The transgene‘s surrounding host DNA (yellow in the diagram) finds the targeted gene, and recombination takes place between the homologous sequences. The recombination

10 may take place anywhere within the host flanking DNA sequences, and the exact location is determined by the position of the targeted host gene, not the transgene. Once the cells have performed their part of the procedure, the end result is a new piece of DNA inserted into the chromosome (lower portion of diagram). The rest of the genome is unaltered, but the single targeted locus has been replaced with the engineered construct and some of its flanking DNA.

Figure 3: Inserting a Transgene by Homologous Recombination. The first step is to locate the gene of interest in the genome. Next, insert the transgene into the targeted area. This results in the genome containing the engineered transgene. (Genoway… .2003)

Screening for Transgenic Animals

Once the process for creating transgenic animals is complete, the animals are screened to find those that have incorporated the transgene. Most pups are evaluated for the presence of the required transgenic DNA at about three weeks of age. The most common site for tissue collection is the end of the tail. Ear tissue and white blood cells are also sometimes used to obtain genomic DNA for analysis.

11 The most common screening methods are polymerase chain reaction (PCR) and

Southern blot analysis. These tests help find information such as presence or absence of the transgene, transgene rearrangement or deletion, and correct transgene expression.

Failure to obtain positive transgenic pups is usually caused by poorly prepared DNA or improper embryo transfers.

12 Chapter-2: An Overview of Transgenic Applications

The purpose of this chapter is to document and categorize the types of transgenic animals that have been created to date, focusing on their benefits to society.

Documentation of these benefits will serve as a prelude to the ethical discussions of chapter 3.

Disease Models

Traditionally, researchers have had to use naturally occurring models of genetic disease. The observation of large numbers of animals was necessary to try to isolate specific examples which could serve as a model of a particular human genetic disease.

For some diseases, no naturally occurring mutants can be found. But with the development of transgenic animals, it is now possible to produce animal models of a particular human genetic disease. Recent research involving genetically manipulated animals has produced a greater understanding of specific human diseases, and brought us closer to finding a cure. Some notable disease models include Alzheimer‘s mouse,

Oncomouse, and the Parkinson‘s fly (discussed below).

Alzheimer‘s Mouse

Alzheimer‘s disease (AD) is a neurodegenerative disease that results in memory loss and disorientation, and is eventually fatal. Currently there is no cure. In AD, highly toxic G-amyloid (AG) protein accumulates in specific areas of the brain, and this protein eventually aggregates to form senile plaques. AG is cleaved from amyloid precursor

13 protein (APP). In some cases of early onset AD, there is a genetic mutation in APP that results in an accelerated production of AG.

Scientists have now successfully created a transgenic mouse carrying a gene for early onset human Alzheimer‘s disease, which will provide researchers with an important new tool in the search for effective AD treatments. Dr. David Adams (W PI) and his colleagues at the former Transgenic Sciences Inc (W orcester) were the first to insert the human gene for APP in mice to demonstrate substantial plaque accumulation and true neurodegenration (Games et al, 1995). Amyloid plaques in Alzheimer‘s mouse were shown to form extracellularly in the brain and develop neurofibrillary tangles within neurons (www.alz.org, 1994). These plaques that develop have been shown to impede brain function. These mice were shown to display behavioral traits of Alzheimer‘s patients, including memory loss in a maze test circuit.

Since the creation of the first true Alzheimer‘s mouse, several other models have been created by other researchers. A mouse showing —a five-fold increase in AG (1-40) and a 14-fold increase in AG (1-42/43)“ was created (Hsiao, et al 1996). The younger mice in the experiment showed typical learning patterns up to 3 months, however after 9 months memory loss was apparent.

True benefits to society have already been achieved with AD mouse models. In

1999, researchers at Elan Pharmaecuticals used the W orcester mouse model discussed above (Games et al., 1995), now termed the PDAPP mouse, to try to develop a vaccine for this disease. They discovered that immunization of the PDAPP mice with human AG produced antibodies against AG that cleared the plaques from the brain (Schenk et al,

1999), and also restored mental functioning on a maze test. Human clinical trials are

14 currently underway to see whether decreasing levels of amyloid plaques in the human brain allows restoration of brain function (Jones, 2000).

Oncomouse

Finding cures for cancer has been one of the major crusades for researchers and treatment specialists for several decades now. An extremely controversial transgenic animal, Oncomouse (a.k.a. the Harvard Mouse) was developed specifically for cancer research. Oncomouse contains several human oncogenes known to cause cancer (Leder,

1984). Oncomouse has already provided society with an important model for the

development of cancer treatment and

medicines. This mouse model is being

used world wide for cancer studies and

has lead to prevention of a certain type

of blood cancer in Africa. Oncomouse will be discussed in more detail in Chapter-4 in discussions of animal patenting.

Parkinson‘s Fly

A strain of transgenic fruit flies was developed by Dr. Mel Feany and Dr.

W elcome Bender at Harvard Medical School. These flies

display signs of Parkinson‘s disease, and are hoped to expedite

research for a cure for the disabling disease. The Parkinson‘s

strain of flies produce the human protein alpha-synuclien.

Synuclien has been shown to cause nerve damage in association

15 with Parkinson‘s disease. Lewy bodies, or dense fibrous plaques of protein, form in the brains of the flies when synuclien is expressed (Feany and Bender, 2000). The

Parkinson‘s flies display symptoms that are strikingly similar to the disease in humans.

Flies in the experiment demonstrated progressive loss of dopamine neurons and loss of motor control. These symptoms are caused by the death of dopamine nerve cells in the region of the brain called the substantia nigra. The role of Lewy bodies in the brain and the affect of synuclien on Lewy bodies is hoped to be revealed by this new fly model.

The fly model provides an inexpensive and convenient model to work with versus a mammal model. Thousands of flies can be bred within a short period of time, and there is less resistance to testing on these non-mammals. New pharmaceutical compounds can be tested on the large sample base. The hope is to find a compound that can suppress or enhance the toxicity of Lewy bodies (Feany and Bender, 2000). This is a good target for the development of new therapies for the prevention of Parkinson‘s disease if indeed,

Lewy bodies are the root cause.

Xenotransplanters

Xenotransplanters are transgenic animals developed to produce organs for transplant into humans. A significant amount of research within this field has focused on transgenic pigs that are capable of growing hearts suitable for human transplant. Organ rejection is the major hurdle in xenotransplantation. For example, pigs normally produce specific sugars on the surface of their hearts that are viewed as foreign to our immune systems, which causes organ rejection (Butler, 2002). Xenotransplanters have been engineered to lack the genes necessary to manufacture these sugars. PPL Therapeutics

16 Inc. first developed five female knockout pigs, missing the genes to produce alpha-1,3- galactosyl. This gene encodes the enzyme responsible for the presence of alpha-1,3- galactosyl on the surface of pig cells. These animals are not yet ready for transplantation experiments because the presence of sugar in the subject pig cells still occurs; there are two copies of each gene in all animals, and only one copy of alpha-1,3-galactosyl was knocked out in the transgenic subjects. The hope is to breed the female pigs with similarly deactivated alpha-1,3-galactosyl genes and achieve offspring that have both copies of the gene deactivated thus making a major step toward viable xenotransplanters

(Lai et al, 2002).

A system is now being developed to temporarily disable the patient‘s immune system, then slowly allow recovery so the patient‘s immune system will recognize the pig organ cells as —self“ (Butler 2002). This will be essential, since T-cell-mediated chronic rejection is a major concern in transplants (Butler, 2002). During xenotransplantations, great care must be made to screen the donated organs for animal viruses. Pigs frequently carry infectious diseases that could harm the recipient. In addition, there is fear of creating man-made pandemics using the practice.

Food Sources

Transgenic food sources are animals that have been modified to produce more product (food) for the investment (food), in other words, animals that require less feed or time to grow to a given size.

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Superfish

Future demand for seafood could possibly double by 2040. Many of the fish we already eat have already been selectively bred by classic breeding experiments to over produce growth hormone. To create Superfish, researchers microinjected a salmon gene construct overexpressing growth hormone into eggs of rainbow trout of a slow-growing wild strain (Devlin et al, 2001). These fish grew faster (but not larger) than their wild

type counterparts. Devlin et al. also

expressed concern that all the transgenic

fish with the inserted growth hormone

died before sexual maturation due to

cranial abnormalities. The authors

demonstrated that the transgenics achieved no larger a size than traditionally bred strains, but since they matured faster this method may still provide some advantage in aquaculture. This will allow shorter batch times at salmon farms due to the increased maturation rates thus producing more food in a shorter period of time.

Super Tilapia

Greater success than the trout was achieved with a different fish species. In

Havana, Dr. Mario Garcia altered an African freshwater fish, Tilapia to produce a key promoter of growth hormone. This modified Tilapia has been observed to grow twice as

18 fast as unmodified specimens (Stokstad, 2002). British researchers found that —on average the transgenic tilapia were three times heavier than nontransgenics at harvest“

(Stokstad, 2002). Tilapia commercialization is very near.

Superpig

W ith the goal of producing larger pigs to provide more pork, Rurel et al (1989) created a transgenic pig that expressed the human hrowth hormone (hGH). The pig grew

to an abnormally large size, much like

with the human disease gigantism. The

animals increased feed efficiency by 15%,

increased weight by 15%, and reduced fat

by 18% (Rollin, 1995). This achieved the

initial goal of the researchers which was to

produce a more efficient food source.

Unfortunately, as with the human affliction, the pig developed major medical problems, which included heart disease and major joint problems. This caused the animals to be euthanized. Eventually, a self imposed moratorium was placed on hGH mammalian research within the scientific community (Rollin, 1995), leaving us only with talapia as a viable transgenic food model for now.

Transpharmers

Another class of transgenic animals is called transpharmers. Initially, research in this field involved production of human therapeutic proteins in the animal‘s blood;

19 however these proteins sometimes affected the animal‘s physiology, so the limitations in this process were overcome by switching production to the animals‘ milk. Most transpharmer animals now produce societal benefits in their milk. Transpharming results in easy product purification, low production costs, and the elimination of drug refrigeration. Researchers creating a transpharmer insert a human gene encoding a useful drug into the animal, then the expressed product is produced within the breast epithelium and secreted into the animal‘s milk. Little if any is found in the bloodstream of the animal, allowing a greater variety of drugs to be transpharmed since their physiological effect on the animal is not a variable. Using this method, large production scales can be maintained, and the drug can sometimes be delivered to a patient directly via milk consumption.

Transpharmer Sheep

PPL Therapeutics created a transgenic sheep expressing alpha-1-antitrypsin

(AAT) (Ezzel, 1991). AAT deficiencies in

humans have been shown to affect the lungs

and respiratory functions. The average age

of survival of this deficiency is about forty-

four (W hite, 1999). AAT is normally produced in the liver and it protects the lungs by stimulating infection fighting bacteria which also gets rid of dead lung tissue. People lacking this protein are prone to lung damage and emphysema.

20 Transgenic sheep expressing this protein in their milk have already provided an affordable alternative to the only alternative treatment via plasma donations since the transpharmed drug is now FDA approved for U.S. use (W hite 1999). Plasma treatments for this disease cost as much as $80,000 (W hite, 1999). AAT is the only FDA approved transpharmer drug to date.

Another notable transgenic sheep produces human clotting factor IX (FIX) in its mammary glands. FIX is crucial to blood coagulation, and its deficiency can cause hemophilia B (Schnieke et al. 1997). The only current alternative treatment for this disease is also plasma donations which are inherently expensive. Although problems with final FIX delivery remain to be solved, the project was one of the first successful of its type.

Transpharmer Cows

Another important transphamer is Herman the Bull. Herman was engineered to carry a lactoferrin gene. Lactoferrin is an important iron transporting, antimicrobial agent

found in human milk. The substance is essential

for child development but is not normally found

in bovine milk that children consume. Herman,

being a male can not produce milk but he can

produce female offspring. Herman fathered

eight claves in 1994 (Biotech Notes 1994), and the females from his offspring expressed lactoferrin in their milk at levels high enough to meet the demands of human children. These animals are being bred in Leiden,

21 Netherlands for distribution to developing nations to supplement or provide an alternative to mother‘s milk (Biotech Notes, 1994.)

Transpharmer Goats

Genetically altered goats have been able to produce human antithrombin III

(hAT), which is a serum glycoprotein (Baguisi et

al, 1999). hAT-III controls blood clots, and

inactivates thrombin, tryspin, and chrymotrysin

(Phake et al, 1998). hAT has applications mostly

in the medical field for patients undergoing

surgery or for heart disease treatment. —Pre- clinical trials proved successful and were approved by the FDA and clinical trials are now underway. The market for hAT-III is $200 million annually, and this amount of the transgenic protein can be produced by a herd of less than 100 goats, each initially costing a half to $1 million per doe“ (Gaffney 2003). These transpharmers will prove both financially and medically beneficial.

Another financially beneficial transpharmer goat model has been produced by

Nexia Biotechnologies. This transpharmer produces spider silk proteins in the goats‘ milk. Applications for industrial silk are numerous; silk is three times stronger than

Kevlar, and can withstand up to 600,000 pounds per square inch of pressure (Gaffeny

2003). The major advantages of using goats rather than spiders to produce silk is that the goats are easier to domesticate as wells as produce larger volumes.

22 Transgenic Biological Models

Biological or scientific models are transgenic animals developed to help elucidate biological processes or mechanisms of development. These animals are created to help demonstrate the cause and effect of specific genes. The negation of, increase, or decrease of a specific gene may be used to create scientific models. These modifications also help scientists understand how genes are regulated and how they affect development.

Supermouse

Supermouse was created to help understand a number of issues that also relate to disease models. Rat growth hormone genes were cloned and microinjected into fertilized mouse eggs, and a large portion of the litter grew to a large size. The model provided insight into the effects of increased growth hormone, a disease model for gigantism, and possible methods for farming valuable gene products (Palimiter et al, 1982). Supermouse was the first transgenic animal and paved the way for the development of bioengineering.

ANDi

Researchers created ANDi (Inserted DNA backwards) was the first successful transgenic primate. —The major obstacle in producing transgenic nonhuman primates has been the low efficiency of conventional gene transfer protocols. By adapting a pseudotyped vector system, efficient at up to 100% in cattle, we circumvented problems in traditional gene transfer methodology to produce transgenic primates“ (Chan et al,

2001). The transgenic gene expressed in ANDi is a fluorescent gene marker from a

23 jellyfish, green fluorescent protein. ANDi actually appears green under fluorescent light.

The point of the research was not to create a color changing primate but to prove that transgenic monkeys could be created that will eventually provide insight into more complex diseases that are difficult to study in rodents. —For questions that are difficult to study in rodents, such as those related to aging, neurodegenerative diseases, immunology, and behavior, transgenic primates could prove a plus“ (Vogel, 2001).

Smart Mouse

Smart Mouse a.k.a. Doogie, was created in 1999 as a transgenic mouse with genes to improve learning ability and memory. —Neurobiologist Joe Tsien, with collaborators at

MIT and W ashington University, found that adding a single gene to mice significantly boosted the animals‘ ability to solve maze tasks, learn from objects and sounds in their environment, and to retain that knowledge. This strain of mice, named Doogie, also retained into adulthood certain brain features of juvenile mice, which, like young humans, are widely believed to be better than adults at grasping large amounts of new information“ (Harmon 1999). Through this study, some of the biological processes that are involved in creating associations in the brain were revealed which may lead to medical and therapeutic advancements in the future. The study may also eventually lead to genetic modifications in patients with learning disabilities.

Youth Mouse

Youth Mouse might aid in answering many questions about longevity, lifespan, and life-long health in humans. This transgenic model was created to overproduce

24 urokinase-type plasminogen activator (uPA) in many brain sites. —In alpha MUPA mice, overproduction of uPA in brain sites controlling feeding leads to reduced food consumption that, in turn, results in retardation of growth and body weight, and also in increased longevity“ (Miskina et al, 1997). Youth mouse has provided researchers with another tool for development of anti-aging and possible longevity treatments.

Chapter Conclusions

There are many opportunities to learn from various categories of transgenic animals. These opportunities would not be available without the creation of these models.

Transgenic animals have the potential to help cure some major and persistent diseases that humans have struggled with throughout history. Some transgenic animals have already provided a strong medical benefit to society, for example Alzheimer‘s mouse with its facilitation of a human Alz vaccine in phase II clicincal trials, and FDA approved transpharmed AAT for treating emphysema. Although not all transgenic animals are successful, most are worthy of attention and offer a great deal toward improving both society and health care. Nevertheless, there are limits and boundaries that must be considered prior to the creation and use of these transgenic animals regardless of the benefit to humans, which are the subjects of the next two chapters.

25 Chapter-3: Transgenic Ethics

Humans have been manipulating genetics in animals since the first domestication of wild dogs. Desired traits have been bred into animals for an extremely broad spectrum of applications. But it is only recently that a direct modification of an animals‘ genetics with transgenic animals has been possible. This new development has raised the question: should we create such animals?

Ethical issues involved with transgenic animals are very complex, however the different factions can be roughly divided into those who believe that engineered animals are cruel, unnatural and/or go against —God‘s will“ versus those who counter that there are immense medical and societal benefits from the production of these animals. W e believe that both debate sides must be considered on a case by case basis for each type of transgenic animal. The question of the manipulation of life must be addressed, and this manipulation must be weighed against the benefit to society and the quality of life of the animal. To better understand the complexities of this situation, it helps to discuss transgenic animals by type rather than using a broad all encompassing judgment.

Religion and Transgenic Ethics

Due to many traditional religious teachings, there is a large faction of people that feel that humans are obligated not to inflict suffering or harm to animals. Some religions even idolize specific animals such as Bovine worship among Hindus. This platform is morally based, and is a strong naysayer of transgenesis for cows. Hindus and Buddhists are known to practice external simplicity which advocates avoidance of undesirable

26 activities, costs, or side effects. This advocates the attitude that animals should not be modified, especially for the purpose of consumption because alternate protein sources are

simpler and cheaper (Sager, 2003). But what if a transgenic

experiment does not alter the behavior of the animal, as with

transpharmers, yet can save lives. Even in this situation, likely

Hindus would argue against making transgenic cattle, but the

authors of this IQP are not Hindu, and believe such experiments

should be allowed.

Animal Rights Ethics

A governing group on transgenic research is animal rights activists. This faction believes they are to defend rights that animals are unable to defend for themselves.

Disease models are particularly atrocious to this group because the sole creation of the animal is to live a diseased life. The divisions among animal rights groups remain large and varied, but most want to see a strong justification for the treatment of the animals and a minimization of animal suffering. In fact, the authors of this report believe that regulation of transgenic animals is strongly desirable. As long as there is a careful consideration of animal suffering versus the medical benefit to society, the authors of this

IQP regard the animal rights opinions as relevant to transgenic animal questions.

Disease Model Ethics

Disease models are designed to mimic human diseases (or certain aspects of the disease) for study. Generally, these animals make drug and vaccine testing possible, or

27 allow help prove that a specific gene can contribute towards a disease. Some of these animals have already provided great medical benefits with little or no animal suffering, while others offer medical benefits accompanied by much pain.

The creation of Alzheimer‘s mouse was a milestone accomplishment created by

Dr. Adams and his colleagues at W PI. The mouse model provided researchers with an animal model of a disease that was not naturally occurring in mice. Further experiments with the model allowed the development of an Alzheimer‘s vaccine currently being tested in human patients. This treatment clears plaques in older animals and also prevents plaques from forming in younger animals (Schenk et al, 1999). The vaccine represented the first effective treatment of Alzheimer‘s disease in a transgenic animal (Check, 2002).

This vaccine would not have been possible without Alzheimer‘s mouse. The ethical hang-ups with this mouse models are few because there is no apparent suffering in the animal; the mice learn slower in a maze test and are euthanized prior to advanced progression of the disease. In this case, the authors of this IQP believe the benefit to society far outweighs the harm to the animal.

However, other transgenic models endure a large amount of pain and suffering.

An example of such a model is Oncomouse. Oncomouse was created to help determine the specific genes that cause cancer. Ethically, Oncomouse falls into the —gray area“ due to the fact that full term tumors are sometimes allowed to develop in the animals, and the models can suffer if no pain medication is used. However, the medical benefits are strong due to the potential to identify specific human cancer genes without having to sacrifice a human (Society, Religion and Technology Project, 2001). Oncomouse has strong ethical considerations and it considered an unacceptable model for research by some factions,

28 however, our opinion is that the medical benefits outweigh the animal‘s suffering, especially if all attempts are made to mimimize the suffering using pain killers and by sacrificing prior to advanced tumor formation. The authors of this paper support the use of Oncomouse for cancer research for the preceding reasons.

Xenotransplanter Ethics

The demand for human organs increases annually, and efforts to satisfy the demand has been futile. Only a fraction of patients in need of transplants receive donor organs. Brian Carnell wrote an article to address many of the activist‘s concerns. He first stressed the incredible gains xenotransplantation has to offer, stating —13 people in the

United States die every day waiting for an organ transplant, and any advance that utilizes animal tissues or organs would save many lives“ (Carnell, 2000). Xenotransplanters would provide a solution to the organ deficit and would allow —customization“ of the animal to the patient. Because the animals would be specifically created and selected for organ donation, the care that they receive during their existence would be excellent.

There are some major risks associated with the practice of xenotransplantation including the potential transfer or transformation of viruses between humans and animals.

Influenza often infects humans via transfer from pigs and birds. In addition, research has demonstrated that a retrovirus carried by pigs can infect human cell lines (Carnell, 2000).

Most scientists admit that these are legitimate concerns, however, —the risk is not great enough to forego the advantages of this technology“ (Carnell, 2000), especially if rigorous testing is performed on the organs prior to transplant. No major medical procedure is risk free, but the minimization of risks is a valid action to counter skeptical

29 factions. Xenotransplanters could be kept in disease free (or as close to possible) environments to minimize infections. —Animals intended for xenotransplantation use will be special breeding populations that are kept under special clean laboratory conditions“

(Carnell, 2000) and frequently assayed for viruses.

Although many people agree that Xenotransplanters are a valid method of organ donation, research for alternatives should not stop. For example, advancement in artificial hearts may make xenotransplantation of hearts unnecessary (Society, Religion, and

Technology Project, 2001). This technology would avoid the debate over animal sacrifice altogether.

The potential benefit to society of xenotransplantation is immense. This classification of animal can be compared to food sources due to the fact that the animal is being created for beneficial sacrifice for humans. W hile alternatives may exist, there is anormous gap that Xenotransplanter could fill in the wait for other organ technologies to catch up. It is the view of the authors of this paper that Xenotransplanters are acceptable models, provided that other technologies are not an option and the care and sterility of the animals are high.

Food Source Ethics

Genetically modified foods have been on the market for a number of years now.

W ith the development of transgenic animal food sources, the issues that surround these foods are magnified. A bad experiment of this type is Superpig. The growth hormone increases in the animal caused major joint and organ failures. The pain and suffering of the animal far outweighed any potential benefits to society of a larger pig for

30 consumption. The pig had to be sacrificed before the experiment was over. The ethical point is well confirmed by the statement, —If you want more meat, just breed more pigs“.

An example of a —good“ transgenic animal food source is exemplified by

Superfish, especially the talipia. There is no evidence that Superfish suffered at all, and the approach of the researchers was influenced by the failure of Superpig, therefore, in essence, Superpig was not a complete failure. Superfish have some potentially negative points, but they are controllable. If poorly maintained breeding pens are kept, there is a concern that these Superfish could escape and destroy (out breed) natural populations of native fish. As previously stated, this is a controllable risk, and regulation of the transgenic fish could minimize or eliminate this risk altogether.

Hunger is a constant problem in developing nations and these —super“ animals could provide the answers. Because these animals reach the slaughter size with less food and less time, they could prove to be a major success for the fight against hunger. The world is only growing, and the hunger problem is directly proportional to population.

Transgenic fish could potentially offer solutions to these problems, providing large amounts of food relatively cheaply (Stokstad, 2002).

Transgenic animal food sources may provide society with a range of benefits. It is the author‘s opinion that responsible management and regulation of these animals could help provide a solution to a major hunger problem. There are however —good“ food sources such as Superfish and —bad“ sources exemplified by Superpig. It is our view that animal suffering should not occur during the life of these food sources.

31 Transpharming Ethics

Transpharmers are genetically altered to secrete human drugs (products) in their milk. This category of animal is relatively low on the transgenic controversy scale because no direct harm or pain is encountered by the animals since the product is not produced in the blood, and there is a strong societal and ethical benefit created by these animals (Society, Religion and Technology Project, 2001). The benefits of these animals are immense: easy purification of product, high product production, low operating cost, and no animal suffering. Goats have already been made to produce silk fibers for industrial application, and one transpharmed drug has already been approved by the FDA.

Sheep have been made to produce clotting factors. These animals show great prospective advantages to society.

Cows, however have been a target for criticism by some religious groups. As transpharmers, cows represent the best means of product production, they produce large amounts of milk and have long life-spans. But Hindus believe that cows are sacred.

Although Hindus hold cows with great reverence, not all of us are Hindus. Domesticated cows in the United States are already given hormones to produce more milk, tinkering around with cows is not news in some countries. Animals have been used by humans for food since the dawn of man. Since our existence, man has manipulated breeding and lactation. These are already established ethical practices in our society, so it is the author‘s opinion that responsible, regulated transpharming is ethical and falls in to the

—good“ transgenic animal category.

32 Scientific Model Ethics

Biological transgenic animal models are created to test the over expression, under expression or knockout of specific genes. These animals are closely monitored for specific behaviors and quickly sacrificed if suffering occurs. In cases such as

Smartmouse, no harm was caused to the animal, they simply learn maze tests faster, and there is no suffering by any measurable criterion. Yet the experiment allowed the identification of a potential —smart gene“. The benefit could be immense and exciting for the application of this research in humans.

ANDi, the transgenic monkey also encountered no suffering since he was only engineered to express green flourescent protein as a test of primate transgenesis. The jellyfish marker gene that he possessed was simply a test to see if primates could be potential models for transgenesis. This test opened doors to future research on primates for the development of real benefits, afterall primates are better models for human diseases than rodents. ANDi demonstrates the essence of scientific models in that he breaks open the path for new research and creates conclusions that can only be achieved by the creation of scientific models. Because of these facts, the authors of this paper consider purposeful, planned scientific transgenic models to be worth continuing due to their unique potential for research.

33 Chapter 4: Transgenic Legalities

The production of transgenic animals not only raises ethical issues, but legal concerns as well. Questions regarding animal patentability are the main concern, and there is always the possibility of governments outlawing any technology considered unacceptable at any stage of research and development. W ith respect to the legal system, many controversial new technologies only surface publicly when they reach the patent office. This is likely the case for oncomouse. As discussed in Chapters 2 and 3,

Oncomouse was the first transgenic animal patented and it eventually became well known to the public due to the controversy of patenting it. This chapter will focus on the different views of patenting transgenic animals and the resolution of the Oncomouse court case.

Harvard‘s Oncomouse

In the early 1980‘s, Harvard Medical School produced a genetically modified mouse that was highly susceptible to cancer. This was accomplished by introducing an oncogene that could trigger the growth of tumors. The oncomouse became a valuable means of furthering cancer research, serving as a test system for therapies. Harvard sought patent protection in the United States and several other countries.

The Oncomouse Case

A patent is a legal right given to reward inventors for their invention. For

Harvard to get its patent, it had to prove that Oncomouse, according to the Patent Act,

34 could be considered to be —any new and useful art, process, machine, manufacture or composition of matter, or any new and useful improvement in any art, process, machine manufacture or composition of matter“ (Cruel Science- Oncomouse, 2003). The case raised a key issue regarding the patent system of whether patents should be granted at all for animals, particularly for higher-order animals such as mammals.

The Oncomouse Case was resolved differently by the patent authorities of different countries. In 1988, the United States granted a patent to Harvard College claiming —a transgenic non-human mammal whose germ cells and somatic cells contain a recombinant activated oncogene sequence introduced into said mammal“ (Today in

Science- United States Patent, 1988). The European Patent Office also granted the patent to Harvard after a lengthy case. The case was only resolved just recently in 2004.

Canada, however, initially rejected claims to transgenic animals on the basis that they were not included in the definition of an invention, but allowed claims on the process for obtaining Oncomouse. The country‘s commissioner for patents decided that the mouse did not meet the Canadian patent law‘s requirements of —manufacture“ and —composition of matter.“ In 1997, a federal court agreed with this decision.

However, a Canadian appeals court ruled in 2000 that transgenic animals, such as

Oncomouse, are considered a composition of matter and therefore can be patentable under certain conditions. On December 5th, 2002, the Supreme Court of Canada rejected the patent application. In a 5-4 decision, the court ruled that transgenic animals do not meet the Canadian Patent Act‘s definition of —composition of matter“ and therefore cannot be considered as an invention. According to the court‘s interpretation, the definition was not intended to cover higher life forms. The court made it clear that its

35 decision was based on technical and not moral considerations. The decision means that the animal itself cannot be patented. However, a patent had already been granted for the cancer-prone gene used in Oncomouse and the process by which the mouse is developed.

Chapter-4 Conclusions

The difficulty of patenting animals is great even when not factoring in moral considerations. The definition of a patent is clear, but when it involves a mammal it becomes unclear. The success of the Oncomouse case in the United States was based on earlier successes patenting lower life forms in the now famous Diamond versus

Chakrabarty (1972) case in which a microbe capable of digesting oil was patented for use in cleaning up oil slicks. Since the Oncomouse U.S. case, hundreds of patents have been awarded to transgenic animals.

The Canadian court eventually ruled against the patent because of what the

Canadian Parliament defined as a patent in the 1800‘s. W hat makes the Oncomouse Case so intriguing is the fact that Parliament could not have known the types of advances made in the scientific world by this transgenic model, but the Canadian Supreme Court did not take this into consideration when making their final decision.

36 CONCLUSIONS

The conclusions formed from this IQP research indicates that regulation and cautious deliberation of animal treatment can provide minimal animal suffering, while allowing maximum medical benefit for society. Certain transgenic animal types have been deemed acceptable by the authors of this IQP (see below), while others are not.

However, strict regulation and moral consideration should be applied when considering any new transgenic model. W e believe the development of methods for creating transgenic animals have grown much more efficient, and have provided a great advancement to the scientific world.

W e consider transpharmers to be one of the best types of transgenic animals because the animals do not suffer at all, and the medical benefits of their products are immense. W e also consider xenotransplanters to be a good type of transgenic model because there is no animal suffering during the animal‘s life, but alternatives that could replace the model should always be researched in parallel. W e also condone the creation of specific disease models, provided animal suffering is minimized (using pain killers or early euthanasia if needed) and the model brings strong medical benefits to outweigh any suffering that may occur. Food sources are also an area of research and development that we regard as valuable, as long as HGH research is limited to non-mammalian species.

Finally, we believe that scientific models offer benefits and insight into areas of research that would not otherwise be able to be studied, but these models should be allowed to be created only as long as pain is controlled.

37 The Oncomouse case shows just how controversial the legal issues surrounding patenting new transgenic animals are in society. It is difficult to receive patents for new scientific achievements, such as transgenic animals, and until certain patent laws are changed, it will remain that way.

38 BIBLIOGRAPHY

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40

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