Therapeutically Important Proteins - Transgenic Animals

Chapter 22
Transgenic Animals and their Applications

Shet Masih, Pooja Jain, Rasha El Baz and Zafar K. Khan
Department of Microbiology and Immunology, Drexel Institute for Biotechnology and Virology Research, Drexel University College of Medicine,
Doylestown, Pennsylvania, USA
Chapter Outline
Summary407 Transgenic Animals as Xenotransplanters 415
What You Can Expect to Know 407 Transgenic Animals as Food Sources 415
Introduction408 Superpig415
Creating Transgenic Animals 409 Superfish416
Construction of a Transgene 409 Transgenic Animals for Drug and Industrial Production 416
Microinjection409 Transgenic Animals’ Impact on the Environment 417
Embryonic Stem Cell Transfer 409 Patenting Transgenic Animals 418
Retrovirus-Mediated Gene Transfer 411 Ethical Issues 419
Gene Knockdown and RNA Interference 412 FDA Guidelines on Genetically Engineered Animals 420
Screening for Transgenic Positives 412 Translational Significance 420
Transgenesis Versus Cloning 412 World Wide Web Resources 420
Transgenic Animals as Disease Models 413 References421
OncoMouse413 Further Reading 422
AIDS Mouse 413 Glossary422
Alzheimer’s Mouse 413 Abbreviations423
Parkinson’s Fly 414 Long Answer Questions 423
Transgenic Animals as Biological Models 414 Short Answer Questions 423
ANDi (Monkey) 414 Answers to Short Answer Questions 423
Doogie (Smart Mouse) 414
Supermouse414
Youth Mouse 414
Influenza-Resistant Mouse 415
SUMMARY transplant. These proteins have significant therapeutic

potential for the treatment of cystic fibrosis, hemophilia,
Transgenic animals are the result of manipulation of spe- osteoporosis, arthritis, and parasitic and infectious diseases
cific genes of interest by creating animal models and biore- such as malaria and HIV. Transgenic animals can also pro-
actors to (for example) produce vaccines and antibodies that duce monoclonal antibodies (specifically targeted towards
can be used to help save millions of lives. These animals disease proteins) that are used in vaccine development to
with altered genomes must therefore be used under strict meet global demands.
ethical control. This chapter has been framed in a manner to educate
the reader about transgenic animals, methods of creat-
ing them, their effects on the environment, and associated
WHAT YOU CAN EXPECT TO KNOW socio-ethical concerns. Readers will also get a glimpse of
Transgenic animals are animals that have been genetically the history of transgenic animal production and the implica-
transformed by splicing and inserting foreign (animal or tions for pharmaceutical research and industry. The over-
human) genes into their chromosomes. When inserted suc- all purpose of selection and production of animal models
cessfully, these genes enable an animal to produce (for required for human welfare has been described. In medical
example) pharmaceutical proteins in its milk, urine, blood, research, animal models are required to understand various
sperm, or eggs, or to grow rejection-resistant organs for cellular and molecular pathways; these are explained here
Animal Biotechnology. http://dx.doi.org/10.1016/B978-0-12-416002-6.00022-5

Copyright © 2014 Elsevier Inc. All rights reserved. 407
408 SECTION | III Animal Biotechnology: Applications and Concerns
with examples. Some of the disease models that are used Generally, three parts are required to construct a trans-
vigorously in the scientific community to study alterations gene: (1) a promoter sequence that determines which tissue
in normal physiology of the host that lead to a diseases or should express the recombinant protein; (2) the sequence of
disorders are also discussed. This chapter also includes a the gene coding for amino acids of the desired recombinant
section on the engineering of animals as food sources or protein; and (3) the sequence responsible for the termina-
to produce human tissues to be used in histocompatible tion of the expressed protein. The most common method
transplantations. Finally, ethical issues related to genetic of producing transgenic animals is microinjection, in which
engineering and the U.S. Food and Drug Administration’s the constructed transgene is inserted into the male pro-
(FDA) ethical guidelines regarding the creation of trans- nucleus of a freshly fertilized egg. Generally, several cop-
genic animals are covered. ies of the transgene are inserted into the male pronucleus,
which is larger than the female pronucleus. The other com-
mon method for producing transgenic animals is embryonic
INTRODUCTION
stem (ES) cell manipulation. In this method a transgene is
The practice of carefully selecting and reproducing animals inserted into the stem cells of the blastocyst with the help of
with new combinations of genes is not new. In nature, how- microinjection, chemicals, or viral transduction. The ease
ever, new gene combinations are found only in the same or of screening of embryonic stem cells carrying transgenes
similar species. Transgenic or genetically engineered ani- is the main advantage leading to the high efficiency of the
mals are the result of novel gene combinations thoughtfully technique. Polymerase chain reaction (PCR) and Southern
manipulated and implemented by scientists. Because DNA blot hybridization are two main techniques for screening the
contains the universal genetic code for all living organisms, animals carrying the desired transgene.
it can be easily altered and transferred between two com- Different categories of transgenic animals include those
pletely unrelated organisms to produce a combination of produced as disease models, xenotransplanters, trans-
characteristics that would not otherwise be possible. pharmers, food sources, and scientific research models.
The term “transgenic” was coined in 1981 to describe Through genetic engineering various disease models have
an animal in which an exogenous gene had been introduced been developed to mimic the human symptoms of disease.
into the genome (Gordon and Ruddle, 1981). A geneti- Examples of such models include the OncoMouse (mouse
cally engineered or “transgenic” animal carries a known model for cancer study), the AIDS mouse, the Alzheimer’s
sequence of recombinant DNA in its cells and passes the mouse, the HLA-A2.1/Tg mouse (to study the presentation
recombinant DNA on to its offspring. In the late 1980s, the of antigens that are normally not presented by the surface
term “transgenic” was extended to gene-targeting experi- of mouse antigen-presenting cells), and Parkinson’s fly.
mentation and the production of chimeric or “knockout” Animals that are engineered to express a desired protein
mice in which a gene has been selectively removed from in their milk are known as transpharmers; for example,
the host genome (Beardmore, 1997). Recombinant DNA mice, sheep, goats, and cows have been engineered by this
refers to fragments of DNA sequences that have been joined method. Also, there are animals that have been engineered
together in a molecular biology laboratory. The DNA to to produce histocompatible organs that can be implanted in
express a desired protein is constructed in such a manner humans without fear of rejection by the human body. This
that it can express the functional protein when inserted in technique has been used for producing pigs as xenotrans-
the nucleus of a transgenic animal. The gene encoded in planters, but the use of those organs has not been approved
the resultant DNA construct is capable of producing the yet. Similarly, to meet the daily increasing demand of food,
same kind of protein no matter which animal or microbe animals have been engineered that grow larger than their
(or even plant) is producing it. This extra desired protein wild-type counterparts without requiring extra food. Two
synthesis is the only difference between transgenic and examples of transgenic food sources are Superfish and
non-transgenic animals; otherwise the two are identical in Superpig. Superfish is a promising food source, but Super-
behavior and appearance. Some of the proteins, particularly pig proved futile because the animals developed multiple
for therapeutic purposes, have been produced in transgenic health issues.
animals (Echelard et al., 2006; Lillico et al., 2007). Some Transgenic animal models are generally produced for
of these engineered proteins provide the animals with better scientific research by inserting a transgene in their DNA to
disease resistance than their wild-type counterparts. Other study the effect of overexpression of that particular gene on
examples are proteins that provide healthier milk, meat, or the animal’s physiological processes. Sometimes the gene
eggs (Lai et al., 2006). under investigation is knocked out to determine its effect
Transgenic animals can be produced through various on normal body metabolism. Well-known examples of sci-
methods, but in all procedures the first requirement is to entific research models are ANDi, the transgenic monkey;
generate a transgene (i.e. the DNA sequence that encodes for smart mouse; youth mouse; Supermouse; and influenza-
a particular protein along with other necessary sequences). resistant mouse.
Chapter | 22 Transgenic Animals and their Applications 409
Many ethical issues are associated with the production pronuclear-microinjection technique primarily involves five
of transgenic animals. First, is it ethical to generate trans- major steps that are given in Flow Chart 22.1. Another com-
genic animals? It seems clear that altering the genome of an mon technique is embryonic stem cell transfer. Other meth-
animal to create artistic effects (e.g. the green fluorescent ods include transfer of transgenes into embryonic stem cells
rabbits) is unnecessary and cannot be recommended. If the with the help of a chemical or virus, homologous recombi-
creation of an animal would increase scientific knowledge nation, or gene knockdown.
and possibly help understand a disease condition, then alter-
ation of the genome can be accepted by most. Although, in
most transgenic experiments some animals die, the number Construction of a Transgene
of human lives saved as a result may be much greater than Although the genetic code is almost the same for all organ-
the animals’ suffering. Further, it is recommended that the isms, the fine details of gene control are different. For
suffering of the transgenic animals should be reduced as example, a gene from a bacterium will often not work cor-
much as possible. The production of all transgenic animals rectly if it is introduced unmodified into an animal cell.
cannot be justified, but each experimental instance should Therefore, genetic engineers first construct a transgene
be judged on case-to-case basis. Alzheimer’s mouse, for containing the gene of interest plus extra DNA sequences
example, feels no pain due to its transgene, and its creation that correctly control the function of the gene in the new
is justified because studying this mouse model might lead animal (Figure 22.1). When constructing a transgene, sci-
to new Alzheimer’s treatments. Conversely, the Beltsville entists generally substitute the donor’s promoter sequence
pig or so-called Superpig experienced much suffering for a with one that is specially designed to ensure that the gene
remote possibility of helping meet world food demand, so will function in the correct tissues of the recipient animal;
this experiment was rightly terminated. Another important for example, if the gene needs to be expressed in the milk
example is OncoMouse, a model that straddles this ethical of a mammal, the promoter specific for mammary tissues is
edge because the mouse does suffer tremendously and even- used in the transgene (Murphy et al., 1993).
tually dies of cancer, but the knowledge gained from this
transgenic model is highly valuable in the fight against can-
cer in human beings. Therefore, the creation of OncoMouse
Microinjection
is justified, but measures must be applied to reduce the ani- Microinjection is the most popular technique for creating
mals’ pain; or they can be sacrificed before advanced tumor transgenic animals (Cho et al., 2009). In this technique
development causes them unbearable suffering. Although recombinant DNA (transgene) is inserted into the male
animal physiology is not identical to human physiology, the pronucleus. First, eggs are collected from super-ovulating
knowledge obtained from living disease models is impor- female animals and then fertilized in vitro. The freshly fer-
tant and useful, thus justifying the creation of transgenic tilized eggs are held stable by a microtube suction device
animals as disease models. (Figure 22.2), and a solution containing 200 to 300 copies
Another concern is that transgenic animals will escape of the recombinant DNA is injected using a micropipette.
and outbreed with their wild-type natural cousins. Despite The recombinant DNA is injected into the male pronucleus
this concern, transgenic animals can be reared and used because it is larger than the female pronucleus (Wong et al.,
with restrictive security measures in place. Established 2000). Ethical concerns arise because a very small percent-
regulatory authorities such as universities’ Institutional Ani- age of animals are born with the desired transgene while a
mal Care and Use Committees (IACUC) monitor scientific huge number of eggs are wasted in the experiment.
research and require research scientists to justify animal use
for each experiment. The creation of transgenic animals per
se is opposed by many religious groups and by some volun-
Embryonic Stem Cell Transfer
tary and nongovernmental organizations that feel the prac- Another commonly used technique of creating transgenic
tice interferes with nature. Most, however, have no problem animals is by transferring the transgene to embryonic stem
if the suffering of the animals is minimized and the experi- (ES) cells. This technique is generally used when a particu-
ment is important scientifically. lar gene must be altered in a particular tissue. A transgene
is constructed to target a specific site in the genome of the
animal to be created. For embryonic stem cells, a blastocyst
CREATING TRANSGENIC ANIMALS
is harvested from a female animal or by in vitro fertiliza-
Transgenic animals can be created using various techniques tion, and the inner cell mass is collected (Jacenko, 1997).
(Cho et al., 2009). One of the most popular techniques is Sometimes chemicals or steroids can be given to an animal
microinjection of a transgene into the male pronucleus of to prevent implantation of the embryo. The transgene can
a freshly fertilized egg in vitro. A brief protocol/method be introduced into the embryonic stem cells with the help
for generation of transgenic mice has been described. The of a chemical, by a virus. Embryonic cells are used because
FLOW CHART 22.1

1. Purification of transgenic DNA constructs. Purification of the construct is a critical step for a healthy embryo. A sucrose gradient or a gel-purification
method can be adopted to obtain purified and clean DNA that is dissolved in a microinjection buffer.
2. Harvesting zygotes/fertilized eggs from donor female mice. First, egg donor mice (the widely used strain FVB/N that has a large pronucleus in
embryos) are superovulated by injecting (ip) pregnant mare’s serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) to obtain a
maximum number of zygotes for microinjection before mating. Then, isolate the ovary and oviduct through surgical procedures and release the eggs
into M2 medium dishes. Finally, separate and wash the zygotes in medium and transfer the dishes at 37°C in a 5% CO2 incubator.
3. Microinjection of construct. Purified DNA (conc. 2 ng μl−1) is gently microinjected into the zygote pronucleus with a constant flow rate of 50 hPa
(hectopascal) using an automated microinjector in order to initiate integration of the transgene and cell division. Transfer the zygotes in an M16 cul-
ture medium dish and maintain at 37°C in a 5% CO2 incubator until implantation into the recipient mice.
4. Implantation of microinjected zygotes into recipient mice. Implantation of microinjected zygotes into pseudo-pregnant recipient mice is done
through surgical steps. First, locate the infundibulum, then open an oviduct into which the microinjected zygotes can be carefully transferred, tuck the
oviduct and ovary into the body cavity of the mice, stitch up the skin, and, finally, transfer the animals into separate cages.
5. Screening of pups for transgenic positives. The recipient mice are normally pregnant and deliver pups 20 days post-transfer of the embryos. The pups
are genotyped 10 days after birth for positives (from tail clippings of ∼0.5 cm) for transgenic DNA by standard procedure.
FIGURE 22.1 Transgene. The promoter (indigo) dictates in which tissue the transgene will be expressed. The transgene (orange) contains cDNA
sequences encoding the protein to be expressed (e.g. a human therapeutic protein to be produced in the animal’s milk). There is evidence that intron splic-
ing plays a role in gene expression, so an intron (purple) is used (not necessarily from the gene of interest) between the cDNA sequences. The untranslated
region (UTR) termination sequence (green) dictates the termination of transcription. The construct is linearized before injection (Murphy et al., 1993).
FIGURE 22.2 Microinjection. After fertilization, the egg is held stable by a microtube suction device, while a solution containing many copies of the trans-
gene (green) is injected into the male pronucleus (blue) using a micropipette (because it is larger than the female pronucleus) (indigo) (Wong et al., 2000).
FIGURE 22.3 Embryonic stem cell transfer. Once the transgene is incorporated into the embryonic stem cells, those cells can be left to divide in vitro,
or they can be injected into a blastocyst and implanted into a host’s uterus to grow normally (Jacenko, 1997).
they are capable of differentiating into any of the three such as calcium phosphate or rubidium chloride, or even
germ layers. Therefore, if the embryonic stem cells carry- with microinjection, as all of these techniques insert the
ing the recombinant gene are injected into an embryo, that transgene randomly into the genome.
embryo, when implanted into the uterus of a host animal,
will develop into an animal carrying that particular trans-
Retrovirus-Mediated Gene Transfer
gene (Figure 22.3). Once the transgene is inside the embry-
onic stem cells, with the division of the cells the transgene Retroviral vectors have also been used to transfer genes of
incorporates itself by homologous recombination. This interest into animal genomes (van der Putten et al., 1985).
natural technique involves crossing over between paired Although, embryos can be used up to mid-gestation, 4- to
“sister” chromatids, which can lead to a new recombinant 16-cell stage embryos are generally used for infection with
sister chromatid. These homologous sequences are consid- one or more retroviruses carrying recombinant DNA (effec-
ered when the transgene is designed in order to determine tively transducing only mitotically active cells). Immedi-
where to integrate the desired gene. The flanking sequences ately following infection, the retrovirus produces a DNA
of that particular area are chosen and added to both flanking copy of its RNA genome using the viral enzyme reverse
sides of the transgene. Thus, via this technique a transgene transcriptase (Figure 22.4). Usually without any deletions or
can be integrated at a particular position; this is not possible rearrangements, the DNA copy of the viral genome, or pro-
using general techniques of viral transduction, chemicals virus, integrates randomly into the host cell genome. Very
the result of complementary sequence homology (Fire

et al., 1998). From an evolutionary point of view, RNAi
appears to protect the cell against foreign (e.g. viral) RNA
invasion. The mechanism of RNAi is thought to involve a
double-stranded (sometimes hairpin) RNA molecule that is
cleaved into small fragments of ∼22 nucleotides in length
and assembled into a ribonucleoprotein complex referred
to as the RNA-inducing silencing complex (RISC) (Reid
et al., 2001). The RISC then binds to homologous mRNA
and performs its endonucleolytic cleavage (Dykxhoorn
et al., 2003). Recently, it has been shown that altera-
tions in the complementary oligonucleotide (i.e. length
and nucleotide composition) can have a significant effect
on both the degree and the duration of “gene silencing”
(Lamberton and Christian, 2003). These oligonucleotides
that silence gene expression (i.e. protein synthesis) are
referred to as siRNAs. Small interfering RNA has been
shown to be a potent inhibitor of gene function in vivo
FIGURE 22.4 Retrovirus-mediated gene transfer via transduction. After (Fire et al., 1998). Mouse and rat models harboring small
viral transduction of the host cell, the viral RNA genome is reverse tran- hairpin RNA (shRNA) transgenes, following shRNA tran-
scribed into DNA by reverse transcriptase (delivered into the cell along
with viral RNA), and this cDNA is then integrated into the host cell
scription, produced lower levels of the homologous protein
genome (Han et al., 1999). when compared with controls (Hasuwa et al., 2002). Gene
silencing and knockdown technology has potential medi-
cal and agricultural applications, including the inhibition
high rates of gene transfer, approaching 100% efficiency, of viral gene transcription and of endogenous genes cod-
have been achieved with the use of retroviruses (Han et al., ing for deleterious gene products (Novina et al., 2002). In
1999). However, as with gene transfer by microinjection, small animals such as the mouse, RNAi has several advan-
integration events are random. For safety reasons, retrovi- tages over homologous recombination and embryonic
ruses are frequently modified by removing structural genes stem cell-mediated gene knockout methods. RNAi can be
such as gag, pol, and env (which support viral particle for- chemically synthesized directly, thus avoiding laborious
mation). However, most retroviral lines used in transgenic cloning steps. Therefore, this methodology is the most sig-
animal experiments are ecotropic, meaning that they infect nificant advance since nuclear transfer in effecting efficient
only the model systems (e.g. mice or rats); hence, rodent loss-of-function modeling in mammalian species (particu-
cell lines, rather than humans, could be at risk of contami- larly for non-murine species in which embryonic stem cell
nation if correct precautions are not met. Disadvantages of transgenesis has not been successful).
retrovirus-mediated gene transfer include low copy number
integration; the additional steps required to produce retrovi-
ruses in comparison with microinjection or embryonic stem
Screening for Transgenic Positives
cell-based techniques; a general limitation on the length of After the transgenic animals are born, they are screened
the foreign DNA insert (usually < 15 kb); a potential for either by polymerase chain reaction or by Southern blot
undesired genetic recombination that might alter the repli- analysis. The expression of transgene depends on the site
cative characteristics of the retrovirus; a high frequency of of integration of the transgene; some transgenes may not be
mosaicism; and finally, possible interference of retroviral expressed if they are integrated into a site that is transcrip-
sequences on recombinant gene expression. tionally inactive. A common practice therefore is to breed
the animals further to obtain the optimal expression of the
Gene Knockdown and RNA Interference desired protein.
Efforts at gain-of-function and loss-of-function modeling

Transgenesis Versus Cloning
have usually concentrated on introducing specific muta-
tions into the nuclear genome. RNA interference (RNAi) Transgenesis should not be confused with cloning. Clon-
technology has broadened the possibilities for creating loss- ing refers to the reproduction of an exact replica of a liv-
of-function animal models. Short interfering RNA (siRNA) ing organism using the DNA (without manipulation) of that
exists in a double-stranded state and inhibits endogenous organism, whereas transgenesis (genetic engineering) refers
genes (and/or exogenous sequences, as in viral genes) as to the human manipulation of genetic material in a manner
that does not occur in nature. In 1997, Dolly the sheep was AIDS Mouse
born as a result of cloning experiments at Roslin Institute
AIDS mouse is another good example of a disease model.
(Midlothian, Scotland, UK) using a somatic cell from an
AIDS mouse was created in 2001 at the University of Mary-
adult sheep.
land by microinjecting the genome of HIV-1 into fertilized
mouse eggs. The recombinant DNA of HIV-1 (the transgenic
TRANSGENIC ANIMALS AS DISEASE genome) was altered by deletion of the two genes that cause
MODELS the virus to become infectious (Reid et al., 2001). Thus,
HIV-1 mouse cannot transmit the virus to humans, making
Many animal models have been created for studying the
handling of the animal relatively safe while still allowing
mechanisms of disease or changes in physiology that char-
study of the HIV biology. This model allows researchers to
acterize some disorders. A few important examples are dis-
study early-stage symptoms to better diagnose the disease
cussed here. Many other animal disease models have been
in humans. It also allows researchers to track chronic con-
created or are on researchers’ lists to be generated. Small
ditions associated with AIDS and to test numerous treat-
animal models such as mouse models are favored because
ments in the search for a cure for HIV disease (Kohn, 2001).
of the animals’ relatively short gestation time and easy
Previous reports showed that chimpanzees were capable of
manipulation and housing. However, many researchers like
supporting HIV replication, but no small animal developed
pigs as disease models for their anatomical and physiologi-
the virus, and use of chimpanzees as a disease model is very
cal similarities to humans in studying inflammation, heart
expensive. An original female mouse that possessed the
disease, Lou Gehrig’s disease, sickle cell disease, and many
modified viral gene was bred with a healthy male mouse
other diseases.
to produce HIV gene-bearing pups. Skin lesions were also
seen in the AIDS mouse, similar to those seen in Kaposi’s
OncoMouse sarcoma, which often occurs in AIDS patients. These obser-
vations indicated that HIV may indeed be a cause of can-
An important disease model is OncoMouse, a mouse model
cer (Vogel et al., 1988). Thus, AIDS mouse was a big step
created to study cancer. Cancers develop in different ways
towards finding treatments to prevent or cure the disease.
from different causes; therefore, there are many ways to
make a model for studying cancer. One way is to create
mice that do not possess the p53 allele, the tumor suppres-
Alzheimer’s Mouse
sor gene that is involved in most cancers and that is crucial
in checking the uncontrolled growth characteristic of can- Alzheimer’s mouse is another important disease model.
cer. Removal of this allele leaves mice susceptible to many Alzheimer’s disease has been linked to the formation of
types of cancer, most frequently lymphoma (Harvey et al., beta-amyloid plaques in the brain, places where fibers have
1993). OncoMouse was created in 1984 by replacement of developed tangles that can block and degrade neurons dur-
the normal mouse myc gene, which is believed to regulate ing the progression of the disease. Similar to humans with
expression of 15% of all genes by acting as a transcription the disease, Alzheimer’s mouse overproduces a protein that
factor, with a virus tumor promoter/myc fusion recombinant forms these amyloid plaques, and it displays both the symp-
gene. The mice and their offspring developed carcinomas. toms and diagnostic characteristics of Alzheimer’s disease
This first mouse was created at Harvard Medical School in (Duff et al., 1996). Alzheimer’s mouse was generated in
Boston for DuPont (Stewart et al., 1984). The researchers 1995 by a collaborative experiment between Worcester
applied for, and in 1988 received, a patent on the process of Polytechnic Institute and Transgenic Sciences, Inc. (which
creating the animal (Leder and Stewart, 1984), and on the became Athena, then Exemplar Corp., then Elan Pharma-
mouse itself, making OncoMouse the world’s first patented ceuticals). This mouse line was generated by overexpress-
animal. This caused a considerable stir in the scientific ing a mutation that causes an aggressive early-onset form of
community (as will be discussed further in the “Ethics” sec- Alzheimer’s disease (Games et al., 1995). Scientists studying
tion), because to study this cancer model, or to create a new Alzheimer’s disease wanted an animal model for some time
model using Harvard’s techniques of producing the Onco- before this breakthrough occurred. The first Alzheimer’s
Mouse, obtaining a license was required. DuPont argued vaccine, which almost entirely prevented the creation
that the patent covered any transgenic animal predisposed of amyloid plaques in young mice and even reduced the
to cancer (Marshall, 2002). Since then, the company has damage of the plaques already allowed to develop in older
allowed researchers working with the U.S. National Insti- mice, was developed from research using Alzheimer’s
tutes of Health to work on the mouse for nonprofit research mouse (Schenk et al., 1999). This vaccine moved to human
(Smaglik, 2000). Various experiments continue on Onco- clinical trials in 2000, but was cancelled in 2001 because
Mouse that may lead to preventing and curing multiple a minority of patients developed brain inflammation; a
forms of cancer. second-generation vaccine by the same company is now
in Phase II human clinical trials and no deleterious side Therefore, ANDi proved that transgenic primates could be
effects have been observed. Thus far, no vaccine has been created and could express a foreign gene if delivered into
approved by the FDA for the treatment of Alzheimer’s dis- their genome. ANDi opened the door for the creation of
ease in humans, but on the basis of the scientific credibility other primate biological models for the study of primates’
established using the mouse model, researchers may be on natural physiology and behavior.
track to preventing and curing Alzheimer’s disease entirely.
Doogie (Smart Mouse)
Parkinson’s Fly In 1999 another important biological model was created at
Studying neurological diseases without a model is very Princeton University. The transgenic mouse “Doogie” (the
difficult, but animal models produced through transgenic smart mouse) was engineered to overexpress NR2B recep-
approaches have yielded significant progress. A Drosoph- tors in synaptic pathways. Overexpression of these receptors
ila fly was created at Harvard Medical School in 2000 as a makes the mice learn faster, like juveniles, throughout their
model for Parkinson’s disease. This fly has a mutation of the lives. When tested for learning and memory the “Doogie”
α-synuclein gene linked to inheritable Parkinson’s disease. mice performed better than their wild-type counterparts
Parkinson’s fly shows disease-specific characteristics that (Tang et al., 1999). To test the memory of a mouse, two
are seen in humans during the progression of the disease, objects are presented to the mouse in a cage for exploration.
such as loss of motion control and loss of dopamine. This Then one object is replaced with another and the mouse is
fly carries a much simpler genome and serves as an excel- again allowed to explore the objects. If the mouse spends
lent model for studying Parkinson’s disease at the genetic more time paying attention to the new object, that is a good
level (Feany and Bender, 2000). Many previously unobserv- sign that it remembers the old one. If the mouse explores
able characteristics in the progression of the disease have each object equally, the mouse has probably forgotten the old
been studied with the help of this fly. The symptoms of Par- object that it explored previously. Doogie mice perform con-
kinson’s generally do not become visible in humans until an sistently better on these tests as they grow older. In the near
estimated 60% to 80% of dopamine nerve cells have already future, this research may lead to improvements in learning
died (Vatalaro, 2000). Parkinson’s fly allows scientists to and memory in humans and other animals. The fact that over-
understand early-onset symptoms, which could lead to ear- expression of this gene improves memory confirms an old
lier diagnosis and ultimately to a treatment or cure of the theory about how mammals think and learn (Harman, 1999).
disease in humans. Further research on the Doogie mice can provide valuable
information on human development, learning, and memory.
TRANSGENIC ANIMALS AS BIOLOGICAL
MODELS Supermouse
Transgenic animals are also created as biological models Supermouse is a transgenic biological model developed in
with the aim of increasing knowledge about genetics and 1982 in which the gene of a rat growth hormone is microin-
expression of genes during certain natural and physiologi- jected into fertilized eggs. These mice grew noticeably larger
cal conditions. Some of the important biological models than their wild-type littermates and became the world’s
that have been produced through transgenesis are discussed first expressing transgenic animals and the first ones with a
here. noticeable phenotypic response to the transgene. Research-
ers hoped to use these mice to study the effects of growth
hormone, accelerated animal growth, gigantism, and as a
ANDi (Monkey) means of correcting genetic defects related to the growth
The first transgenic monkey, ANDi (“inserted DNA” pattern of animals and humans (Palmiter et al., 1982). This
spelled backwards), was born in 2000. ANDi is one of most mouse model was also used in research aiming to create
important biological models (Chan et al., 2001). A harm- food-producing animals with accelerated growth. Correction
less gene for green fluorescence protein (GFP) was inserted of dwarfism is the most obvious application of these animals.
into ANDi’s rhesus genome using an engineered virus. The
eyes and fingernails of two other monkeys in the project,
Youth Mouse
stillborn twins, glowed under ultraviolet light, although
ANDi himself did not. The GFP gene was chosen for two In 1997 the Weizmann Institute of Science in Rehovot,
reasons: first, it would have very little effect on the monkey, Israel, created a mouse model called “youth mouse.” These
and detecting whether the transgene had been transmitted mice characteristically overexpress urokinase-type plas-
properly would be easy. ANDi is the only monkey of 40 fer- minogen activator, primarily thought to be helpful in dis-
tilized eggs to be born alive and to express the gene for GFP. solving blood clots. These mice are smaller, eat less, and
live about 20% longer than normal wild-type mice (Miskin system attacks the transplanted organ, causing a cascade of
and Masos, 1997). Overexpression of the clot dissolver problems (most notably blood clotting) that are dangerous
extends life by preventing atherosclerosis, a process in to the already weakened patient.
which plaques develop in the arteries of an animal as it ages Xenotransplanters are animals engineered not to express
and which can lead to clots, hemorrhages, and heart attacks. those antigens that are recognized by the host immune sys-
Of the four lines of transgenic mice attempted, only one tem. The only animal currently chosen for xenotransplant
kind autonomously ate less and lived longer, but it also dis- research is the pig because its physiology closely matches
played infrequent muscle tremors. This transgenic mouse that of humans, and pigs are much less expensive to study
line, dubbed Alpha MUPA, shows the same characteristics than monkeys or other primates. In the pig, a sugar called
as normal wild-type counterparts on restricted diets (Miskin alpha-1,3-galactosyltransferase present on the surface of the
et al., 1999). Therefore, “youth mouse” promises to be a cells needed to be knocked out. In 2002 at the University of
useful biological model for studying development processes Missouri, four pigs were produced whose transferase genes
and aging, especially in relation to diet. had been knocked out (Lai et al., 2002). A null gene was
introduced into the pig embryos using the nuclear transfer
technique. A second copy of the blank gene was introduced
Influenza-Resistant Mouse
by nuclear transfer into the embryos from these adult pigs,
Influenza-resistant mice were created to study the use of which resulted in piglets with both copies of the gene that
genetic alterations for effecting disease resistance against encode the antigen knocked out. Previously, the organs
(for example) influenza. These mice overproduce Mx pro- from transgenic pigs designed in this way were transplanted
tein, which is known to act as an antiviral agent. These into baboons and no rejection was seen (Logan and Sharma,
mice are significantly more resistant to influenza and other 1999). Human trials have not yet been approved for organs
orthomyxoviruses compared with their normal wild-type from transgenic xenotransplanters from fear that the pig
littermates (Staeheli et al., 1986). If these findings were organs will allow the crossover of viruses from animals
applied to farm animals such as pigs and ducks, the ani- to humans (zoonotic infection), especially to humans with
mals’ chances of infection with avian strains of influenza weakened immune systems like patients waiting for a trans-
and other viruses, and also their chances of passing these plant. The most clear-cut example of zoonotic infection is
infections to humans, might be lowered. The rate of evo- influenza, which is often transmitted from pigs to humans
lution of these viruses in the animal hosts might also be even without organ transplantation. Human trials may begin
lowered, thus helping humans retain an immunoprotective soon, but debate is ongoing about the issue of transmission
response against future viral outbreaks. of infections.
TRANSGENIC ANIMALS AS TRANSGENIC ANIMALS AS FOOD SOURCES

XENOTRANSPLANTERS
Another purpose of producing transgenic animals is to cre-
Organ transplantation is necessary in those cases when the ate food sources to meet increasing global food require-
whole-self organ fails to function (e.g. liver or kidney). ments. Following are examples of the transgenic animals
However, organ transplantation can be performed only if a that have been created to be used as food sources.
donor’s organ is determined to be histocompatible with the
patient to take over the function of the diseased or failed
Superpig
organ. Generally a small percentage of donated organs are
found to be histocompatible with any given patient, and Growing animals more efficiently and with less food would
such matched organs are in extremely short supply. Thus, be very beneficial to society. “Superpig,” one of the food
immunosuppressive drugs are frequently given to lower the strains created, was a pig engineered to grow bigger and
patient’s immune response against the transplanted organ in faster, thus producing a more efficient food source. Many
order to avoid graft rejection. However, this forced decrease of the transgenic superpigs were created by microinjection
response of the immune system leaves the patient vulnera- of the transgene for a growth hormone, whether porcine,
ble to opportunistic infections. Therefore, to solve the organ ovine, bovine, or even rat (Pursel et al., 1997). The popular
shortage and histocompatibility problem, xenotransplanters Beltsville pig was created in Beltsville, Maryland, under the
are being engineered to provide animal organs that are his- supervision of the U.S. Department of Agriculture. Human
tocompatible with humans. Generally, the recipient rejects or bovine growth hormones were expressed in these pigs
organs that come from other tissue types, even from the (Miller et al., 1989). Unfortunately, the Beltsville pigs har-
same species. The well-known cause of this rejection is the bored many health problems, most commonly arthritis. Ani-
repertoire of antigens on the organ’s surface that inform the mal rights groups even claimed that the pig was impotent
host that the organ is non-self. Thus, the host’s own immune and had ulcers along with heart problems, lameness, kidney
disease, and pneumonia. Therefore these pigs were eutha- than $600 million (USD), and the resultant drug may not be
nized, and biologists imposed a voluntary moratorium on affordable to most patients. It is difficult for the therapeutic-
performing further studies on mammals involving alteration protein manufacturing industry to keep pace with the rapid
in the expression of growth hormone. increase in drug discovery and development, resulting in
unmet patient needs and dramatically rising medicine costs.
Superfish Genetically engineered animals may provide an important
source of these protein/peptide drugs in the future because
The creation of “Superfish” was another attempt at creating the production of recombinant proteins in the animal secre-
a more efficient food source. Tilapia, a species of engineered tive products (e.g. the milk, blood, or eggs of transgenic
fish, was created at the Centro de Ingenieria Genética y Bio- animals) presents a much less expensive approach than pro-
tecnologia in Havana, Cuba, to overexpress its own growth ducing therapeutic proteins in animal cells. The first human
hormone, which was microinjected. This animal was not therapeutic protein, Antithrombin III (ATryn, GTC Biother-
transgenic, as it was not created by using another animal’s apeutics, Framingham, MA), was derived in 2006 from the
gene, but it was genetically engineered. Superfish showed milk of genetically engineered goats and was approved by
increased growth, but it reached an adult size that was not the European Commission for the treatment of patients with
larger than normal tilapia (Martinez et al., 1996). Similarly, hereditary antithrombin deficiency (Avidan et al., 2005).
a transgenic salmon was engineered that produced growth Serum biopharmaceutical products are also obtained from
hormone continuously, irrespective of the season (turning transgenic animals, such as antibodies that can be used for
it off depending on the season). In this experiment the eggs the treatment of infectious diseases, cancer, organ transplant
of a species of usually slow-growing trout were used for rejection, and autoimmune diseases such as rheumatoid
microinjection with the gene of a salmon that grew very fast arthritis (Patel et al., 2007; Dunn et al., 2005). Presently, the
after many generations of selective breeding (Devlin et al., main production system for such blood products is donated
2001). There was concern that these fish might escape into human blood, a system that is limited by disease concerns
the environment, and therefore tight control was kept over (e.g. HIV/AIDS), lack of qualified donors, and regulatory
transgenic fish farms (Stokstad, 2002). The biggest fear issues. Genetically engineered animals, such as cattle carry-
is that these fish will breed with native wild-type fish and ing human antibody genes, provide a steady supply of poly-
outcompete them for food, though one of the companies clonal antibodies for the treatment of a variety of infectious
involved in the farming of transgenic salmon claimed that and other diseases. Much information about human dis-
the salmon were raised on fish pellets and therefore would eases has also been obtained from transgenic mice models,
not know how to forage for themselves in the wild natural which have become important in biological and biomedical
environment. There is still considerable opposition to the research.
generation and farming of these “superfish,” though this A transgenic sheep has been produced for production of
transgenic fish looks like a much more likely source of food α1-proteinase inhibitor (α1-PI) protein that when released
than any transgenic animal species. in blood serum binds to the elastase secreted by neutrophils
in response to certain spores, bacteria, and other antigens.
TRANSGENIC ANIMALS FOR DRUG Elastase released in large amounts can damage elastin in the
walls of lung alveoli, leading to severe emphysema. People
AND INDUSTRIAL PRODUCTION who have dysfunctional gene controls for α1-antitrypsin
Transgenic animals may better serve as important models in production (emphysema or cystic fibrosis) can be sup-
translating basic science breakthroughs into potential clini- ported in two ways: by gene therapy using a functional
cal applications. Moreover, the use of transgenic animals as α1-antitrypsin gene or by administration of high doses of
bioreactors in the pharmaceutical industry has far-reaching α1-PI as an aerosol. Gene therapy is still debated; therefore,
implications, from protein production in various end organs the only way of producing α1-PI in large quantities is by
(e.g. milk, blood, urine, and other tissues) to the modifi- creating a transgenic animal with an α1-PI-producing gene.
cation of tissues and organs for transplantation. Thus, the Pharmaceutical Protein Ltd. of Midlothian, Scotland, tried
applications of transgenic technologies, although poten- to produce this enzyme in sheep’s milk. There are potential
tially of great significance, have yet to be fully recognized. advantages of using sheep; being mammals they will pro-
Transgenic animal research has been used mostly in duce the same kind of α1-PI as in humans. Sheep are less
the field of human medicine. Various therapeutic proteins expensive than cows and they mature more quickly. The
or peptides for the treatment of human diseases require enzyme is formed only in the milk, which can be collected
animal cell-specific modifications to be effective, and are easily, and the sheep remain fit and healthy for a long time.
generally produced in mammalian cell-based bioreactors. Further, large quantities of enzyme can be produced because
To start a new cell culture-based manufacturing facility flocks of these sheep can be easily bred and the purified
for a single therapeutic protein or peptide may cost more enzyme from milk will therefore be inexpensive. Once the
purified enzyme is produced, it must undergo clinical trials

and receive approval from the regulatory authorities before TABLE 22.1 Pharmaceutically Related Products Derived
it becomes available on the market. from Transgenic Animals
Several biomedical research models are being pro- Transgenic Company/
duced from transgenic animals, including livestock spe- Product Use Animal Organization
cies produced specifically for various human afflictions
ABX-EGF Cancer mouse Abgenix–
such as Alzheimer’s disease and ophthalmic disease, and
Amgen
for the possible xenotransplantation of cells, tissues, and
organs (Forsberg, 2005). Transgenic animals are also help- Antithrombin 3 Thrombosis goat GTC
ful in studying animal diseases such as “mad cow” disease (ATIII)
(bovine spongiform encephalopathy) and infection of the Butyrylcholines- Biodefense goat Nexia
udder (mastitis) (Maga et al., 2006). Although scientists terase
have now developed transgenic livestock for agricultural CFTR Cystic fibrosis sheep, mouse PPL
purposes, including some with enhanced production traits
Collagen I Tissue repair cow Pharming
(Brophy et al., 2003), environmental benefits (Golovan
et al., 2001), and disease resistance traits (Maga et al., Collagen II Rheumatoid cow Pharming
2006), no company other than Aqua Bounty (Boston, CTLA4Ig Rheumatoid goat BMS-GTC
MA), which produced the growth-enhanced salmon, has arthritis
announced its intent to pursue the commercialization of
Factor IX Hemophilia pig, cow Pharming PPL
these agricultural applications. Economic profits are higher sheep
with the production of genetically engineered/transgenic
animals for human medicine than for agricultural appli- Factor VIII Hemophilia pig sheep Pharming PPL
cations. Commercialization of agricultural applications Fibrinogen Wound cow sheep Pharming PPL
of transgenic animals is hindered by concerns about the healing
cost and timelines associated with the regulatory process, Glutamic acid Type 1 mouse, goat GTC
and by consumer acceptance issues. Also, potential inves- decarboxylase diabetes
tors are reluctant because public acceptance of agricultural Human Osteoporosis rabbit PPL
applications of genetic engineering has generally been calcitonin
lower than acceptance of medical applications (e.g. recom-
Human Thrombosis pig, sheep PPL
binant insulin). Several chimeric and humanized antibodies
protein C
have been marketed, and other therapeutically useful pro-
teins are in development (Table 22.1). Msp-1 Malaria mouse GTC
Pro542 HIV mouse, goat GTC

TRANSGENIC ANIMALS’ IMPACT ON THE Tissue plasmin- Thrombosis mouse, goat PPL
ENVIRONMENT ogen activator
(tPA)
The possible environmental impact of transgenic animals
α-1 Anti-trypsin Hereditary sheep PPL
will initially depend on their phenotype rather than on (AAT) emphysema,
the fact that they are transgenic per se (Kapuscinski and Cystic fibrosis
Hallerman, 1990). Thus, if the animal in question has a phe-
α-Fetoprotein Myasthenia goat GTC
notype giving it absolutely no chance of even short-term (rhAFP) gravis
survival in the wild, then the fact that it is transgenic is Multiple
of little to no concern as an environmental issue. If, con- sclerosis
versely, the animal’s phenotype does not limit, or perhaps Rheumatoid
increases, its chances for survival and dispersal in the wild, arthritis
and if this animal is also capable of breeding in the wild, α-Glucosidase Pompe rabbit Pharming
then this raises serious environmental concerns. The only disease
way to ensure that transgenic animals can have no envi- α-Lactalbumin Anti-infection cow PPL
ronmental impact is to make their escape or intentional
(Modified from Breekveldt and Jongerden, 1998.)
release into the wild impossible, something that can never
be assured. Although the long-term environmental impact
of transgenic animals is hard to predict with certainty, it is
generally accepted that if such impacts arise, they will be
difficult or impossible to reverse.
The production and evaluation of transgenic salmon transgenic salmon should reduce the amount of fishmeal
currently emphasize accelerating growth rates through required per unit body mass produced. Although not yet
engineered changes in growth hormone expression (Du realized, the engineering of transgenic fish that can use
et al., 1992). The rapid growth of these fish clearly sug- plant meal as a protein source would have tremendous
gests a competitive advantage over wild fish should trans- environmental benefit in this regard. Engineering disease-
genic individuals escape. Given that absolute body size is and parasite-resistant fish could potentially reduce the use
often a deciding factor in the outcome of competition for of antibiotics and pesticides in aquaculture. Finally, if the
access to resources and/or mates, any animal that more rap- use of transgenic fish makes on-shore, fully contained fish
idly attains a certain size is likely to displace or prey upon farming an economically viable enterprise, then this will
smaller, slower-growing individuals of the same species. allow the concentration and better management of fish
Such animals may also exhibit interspecific competitive/ waste products. These include organic wastes in the form of
predatory advantages due to changes in spatial and tempo- uneaten food and feces, as well as inorganic wastes such as
ral distributions as a result of their faster growth. Similar phosphates and nitrates. The accumulation of these wastes
outcomes are possible from fish engineered for traits such from aquaculture can cause increased biochemical oxygen
as freeze resistance, salinity tolerance, disease resistance, or demand, eutrophication, and sedimentation problems, all
other economically valuable characteristics (Maclean and of which can be eliminated in on-shore systems with re-
Penman, 1990). Based on what is currently known about circulated water supplies.
the phenotype of growth hormone-transgenic salmon, it is
impossible to adequately predict the environmental out-
comes should these fish escape or be released into the wild.
PATENTING TRANSGENIC ANIMALS
Beyond these near-term ecological effects, the greatest con- In general, consumers have expressed support for the prin-
cerns with transgenic fish relate to the genetic effects of ciple of patenting, including the patenting of genes and
interbreeding with wild populations. Even if they are not gene sequences (Pollara and Earnscliffe Strategy Group,
well adapted for survival in the wild, transgenic animals 2000). When the Canadian Court of Appeals agreed (deci-
may have detrimental impacts on the genetic structure of sion overturned by the Supreme Court of Canada) with
wild populations by allowing the introgression of “exotic” the patentability of the Harvard OncoMouse, only about
genes into natural gene pools. Changes in the genetic make- 50% of Canadians said they were not comfortable with
up of a well-adapted wild population may ultimately affect the Appeals Court decision. When asked – “Is it okay for
the animals’ abilities to withstand environmental change. Of someone to have a patent on a new plant modified through
particular concern in this regard is the so-called Trojan gene the use of transgenesis?” – 66% disagreed. However, only
effect, whereby transgenic animals that are poorly adapted 30% agreed that “granting a patent on an animal modi-
for survival in the wild but exhibit mating advantages (e.g. fied through the use of transgenesis is no different than
through faster growth and/or larger size) drive populations granting a patent on a consumer product.” Furthermore,
to extinction by successfully breeding with wild individu- 66% agreed that “we should not grant patents on a new
als, thereby reducing the fitness of their progeny (Muir species of guinea pig that includes human genes,” and the
and Howard, 1999). Non-transgenic farmed salmon exhibit same majority were in agreement that patents should not
characteristics that predispose them to such Trojan gene be granted on “a new species of chimpanzee that includes
effects as reduced survival of progeny from mating between human genes” (Environics, 1998).
farmed and wild salmon (Fleming et al., 2000). Harvard’s OncoMouse raised general ethical issues
An additional environmental concern with transgenic regarding transgenic technology. It also raised two key
fish is their capacity for transferring diseases and/or parasites issues for the patent system: (1) Should patents be granted
to wild populations. This is a general concern with aqua- at all for animals or animal varieties, particularly for higher-
culture (Saunders, 1991), not so much because farmed fish order animals such as mammals, even if they do otherwise
are an initial source of pathogens or parasites, but because meet patentability criteria (e.g. novelty, industrial applica-
their confinement allows the amplification of pathogen and bility/usefulness, inventive step)? (2) How should moral
parasite loads for subsequent transmission back to wild implications be addressed in relation to specific cases (e.g.
populations. Transgenic fish that are engineered for disease the question of suffering caused to the transgenic animal)?
resistance would potentially increase the risk of becoming These issues have been resolved in different ways by the
vectors for transferring pathogens or parasites to wild popu- patent authorities of different countries, as the following
lations either through direct contact or through the release examples illustrate.
of their feces or contaminated rearing water. The United States Patent Office in 1988 granted Patent
The use of transgenic salmon also offers several possi- #4,736,866 to Harvard College, claiming the creation of
ble environmental benefits to aquaculture. For instance, the “a transgenic non-human mammal whose germ cells and
improved food conversion efficiency of growth hormone somatic cells contain a recombinant activated oncogene
sequence introduced into said mammal.” The claim explic- are not unique to genetic engineering, and traditional breed-
itly excluded humans, apparently reflecting moral and legal ing and selection practices can also change animals in
issues regarding patents on human beings or modification similar ways. Cows from the Belgian Blue cattle breed, for
of the human genome. example, require cesarean delivery of their calves because
The European Patent Office (EPO) considered the Onco- they have been selected for increased birth weight resulting
Mouse case at length and at several levels. The EPO applies from the naturally occurring “double-muscle” trait and the
the patent standards of the European Patent Convention, narrowness of the cow’s pelvic passageway (Vandenheede
which contains two key relevant provisions: Article 53(a) et al., 2001).
excludes patents for inventions “the publication or exploita- The success rate in creating transgenic animals is still
tion of which would be contrary to ordre public or moral- low. There are more failures than successes, and the general
ity,” and Article 53(b) excludes patents on “animal varieties assumption is that the higher the species of transgenic ani-
or essentially biological processes for the production of mal, the greater the cloning failure. The failures in transgen-
animals.” The EPO applied the utilitarian test and decided esis are animals that die before they are born or animals that
that the prohibition on patenting animal varieties did not are born without the transgene and are of no use for fulfill-
constitute a ban on patenting animals as such. It concluded ing the desired purpose. However, the number of failures is
further that the OncoMouse was not an animal variety and decreasing as cloning techniques improve. Comparing the
so did not fall within that exclusion. prenatal deaths of a few animals against saving the lives of
In order to address the ordre public or morality excep- potentially thousands of humans suggests that the reward
tion, the EPO developed a utilitarian balancing test. This is very high, and improvements in the process are continu-
aimed to assess the potential benefits of a claimed invention ously decreasing the costs.
against negative aspects, in this case weighing the suffer- A very good example of a transgenic animal that does
ing of the OncoMouse against the expected medical ben- not suffer is Alzheimer’s mouse. Alzheimer’s mouse does
efits to humanity. Other considerations could also be taken poorly on maze tests, but does not feel any pain related to
into account in the balancing test, such as environmental its condition by any standards used in laboratories. Because
risks (neutral in this case), or public unease (there was no Alzheimer’s mouse spends its days in a laboratory setting,
evidence in European culture for moral disapproval of the any survival skills that would be hampered in the wild by its
use of mice in cancer research, thus no moral disapproval diminished memory do not come into play. Also, Alzheim-
of the proposed exploitation of the invention in this case). er’s mouse continues to provide significant information on
The EPO concluded that the usefulness of the OncoMouse Alzheimer’s disease that could lead to a cure. The Alzheim-
in furthering cancer research satisfied the likelihood of er’s mouse that was created in part at Worcester Polytechnic
substantial medical benefit and outweighed moral con- Institute (Worcester, MA) (Games et al., 1995) was used to
cerns about suffering caused to the animal. In the original develop a vaccine that lowers senile plaque burden in mice
application, the claims referred to animals in general, but in (Schenk et al., 1999); it is now in Phase II human clini-
the course of the proceedings, the patent was amended and cal trials by Elan Pharmaceuticals (Dublin, Ireland). The
finally maintained with claims limited to mice. benefits are tremendous, and the animal suffering is almost
nonexistent. Thus, creation of Alzheimer’s mouse was a
brilliant idea.
ETHICAL ISSUES The OncoMouse is more complicated ethically. In 2008,
Production of transgenic animals remains a hot topic of an estimated 7.5 million people died of cancer world-
debate as some people are ethically uncomfortable with the wide (American Cancer Society, 2008). Any transgenic
idea of genetically engineering animals. Two central ethi- or genetically engineered animal/mouse that can help
cal concerns are associated with the creation of transgenic increase knowledge about this deadly disease would have
animals. The first relates to breaching species barriers or enormous medical benefits. However, the problem with the
“playing God.” According to this view, life should not be OncoMouse is that as it grows, it begins to suffer from the
regarded solely as if it were a chemical product subject to tumors just as humans do with this disease. Therefore, in
willful genetic changes and patentable for economic benefit this complex case, though the animal has enormous medical
or commercialization. The second major ethical issue is the benefits, its use is associated with strong ethical contraindi-
belief that the transgenesis of animals interferes with the cations. However, because most of the work on OncoMouse
integrity or telos of the animal. “Telos” can be defined as focuses on early development of the tumors, the mouse can
“the set of needs and interests which are genetically based, be euthanized before its suffering increases unbearably. If
and environmentally expressed, and which collectively con- advanced oncogenesis needs to be studied, university/insti-
stitute or define the form of life or way of living exhibited tutional animal care committees could require that painkill-
by that animal, and whose fulfillment or thwarting matter to ers be used. Therefore, it is difficult to say that the creation
that animal” (Holland and Johnson, 1998). Such concerns of the OncoMouse was completely a good idea, because the
mice do suffer like humans and die in pain. The need to fertilized egg or embryo that will then eventually grow into
prevent cancer and palliate its symptoms is overwhelming, a transgenic animal. Similarly, a particular disease condition
however, and to save millions of human lives most scientists can be generated in transgenic animals to study the effect
believe that OncoMouse is the best hope. of a specific drug that cannot be directly applied to human
beings. No doubt animals do feel pain and do suffer during
laboratory experiments like human beings, but this suffer-
FDA GUIDELINES ON GENETICALLY
ing is still less if compared to the relief of pain or cure from
ENGINEERED ANIMALS disease for millions of people worldwide. At the same time,
The U.S. Food and Drug Administration is the lead agency it is important to consider the relevance of the end results,
responsible for the control and regulation of genetically engi- and any kind of haphazard science must not be practiced on
neered animals to be used for food, and it plans to regulate transgenic animals. Thus, one should not blindly follow the
transgenic animals under the “new animal drug” provisions of misconceptions of allowing or denying use of transgenic ani-
the Food, Drug, and Cosmetic Act (FDCA). The new animal mals, and should instead become aware of the actual benefits
drug provisions ask: (1) Is the new drug safe for the animal? and ethical issues associated with these animals.
(2) Is the new drug effective? (3) If the drug is for an animal
that is used for food, is the resulting food safe to eat? Although
premarket regulatory review of genetically engineered animals WORLD WIDE WEB RESOURCES
is mandatory, the FDA has not yet issued general guidelines The World Wide Web has become a common source of
explaining what information will be required for this regula- information about any subject, including transgenic ani-
tory review. Also, the regulatory path to commercialization mals and various topics related to transgenics. One of the
of genetically engineered animals remains ill defined (Pew most important resources where one can get scientifically
Initiative on Food and Biotechnology, 2005). However, trans- validated information is PubMed (www.ncbi.nlm.nih.
genic animal research is subject to existing FDA regulations gov/pubmed). Another good resource for this type of infor-
governing animal research. All organizations or institutions mation is the National Institutes of Health (http://www.
receiving or applying for federal funding to carry out research nih.gov). Apart from these two major sources, the research
using animals are required by the federal Animal Welfare Act departments at a number of educational institutions provide
(AWA) of 1966 to have the IACUC review research protocols good web sites with information regarding this subject, such
involving dogs, cats, rabbits, guinea pigs, hamsters, gerbils, as the following: http://www.harvard.edu (Harvard Univer-
nonhuman primates, marine mammals, captive wildlife, and sity), http://www.stanford.edu (Stanford University), http://
domestic livestock species used in nonagricultural research www.mit.edu (Massachusetts Institute of Technology), and
and learning. The AWA also requires the following: (1) http://www.wustl.edu (Washington University St. Louis).
research institutions must have a veterinary care program in Apart from the web resources created by academic institu-
place; (2) all personnel using or caring for live animals must tions, several government organizations, like the Food and
be well qualified to do so; and (3) a mechanism must be in Drug Administration (FDA, http://www.fda.gov) and the
place at the organization for reporting of issues regarding ani- U.S. Patent and Trademark Office (http://www.uspto.gov)
mal care and unethical use. The AWA is administered through also provide information about transgenic animals.
the U.S. Department of Agriculture (USDA) and is enforced Although this is not an exhaustive list of resources on the
through unannounced and random inspections by a veterinary subject, and new sites are constantly being created and updated
medical officer designated by the USDA. On an international on a regular basis, the following are particularly useful:
level, the Association for Assessment and Accreditation of
Laboratory Animal Care (AAALAC) oversees the voluntary http://www.transgenelifesciences.com
accreditation and assessment of research organizations com- http://www.med.umich.edu
mitted to responsible animal care and use. http://www.hhmi.org
http://www.rnainterference.org
http://www.medicalnewstoday.com
TRANSLATIONAL SIGNIFICANCE http://www.who.int
Transgenic animals are an important part of today’s consumer http://www.wisc.edu
health world, and we may expect even more dependency in http://www.dnalc.org
the near future as most of the recombinant proteins or mono-
clonal antibodies are derived from these animals. Thus, it
becomes relevant to know how transgenic animals are pro- ACKNOWLEDGMENTS
duced and can be used for fulfilling our needs. For example, The authors wish to acknowledge the United States Public Health Ser-
it is not possible to use human subjects to study the role of vice/National Institutes of Health grants AI 093172-01 to ZKK and AI
a particular gene that can be easily studied if inserted into a 077414 to PJ.
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ABBREVIATIONS SHORT ANSWER QUESTIONS

AAALAC Association for Assessment and Accreditation of 1. What was the first animal for which a patent was issued?
Laboratory Animal Care 2. What is the “Trojan gene” effect?
AWA Animal Welfare Act 3. What is RNAi?
EPO European Patent Office 4. What is the difference between cloning and transgenesis?
ES Cell Embryonic Stem Cell
5. Name the agency responsible for regulation of animal
FDCA Food, Drug, and Cosmetic Act
experimentation in various organizations.
GFP Green Fluorescence Protein
PCR Polymerase Chain Reaction
RISC RNA-Inducing Silencing Complex ANSWERS TO SHORT ANSWER
RNAi RNA Interference
shRNA Small Hairpin RNA
QUESTIONS
siRNA Short Interfering RNA 1. OncoMouse was the first animal to be patented in 1988.
2. Transgenically produced animals (e.g. transgenic
salmon over-expressing growth hormone gene), if they
LONG ANSWER QUESTIONS escaped into the environment, could wipe out their wild-
1. How are transgenic animals useful for medical research? type counterparts by competitively growing faster.
Describe with examples. 3. RNAi (RNA interference) is a technique used to inhibit
2. What are the different methods of creating transgenic the expression of protein by degrading its mRNA. This
animals? Explain in detail. technique was first discovered in plants.
3. How can transgenic animals affect the natural environ- 4. Cloning is the reproduction of an exact copy of a living
ment? Explain with suitable examples. organism using the DNA (without manipulation) of that
4. How can the use of transgenic animals over cell-based organism, whereas transgenesis (genetic engineering)
bioreactors be justified? Do you think transgenic ani- refers to the human manipulation of genetic material in
mals can meet world food demands? a manner that does not occur in nature.
5. Are there any controls on the creation of transgenic 5. Institutional Animal Care and Use Committee (IACUC)
animals? What is your opinion about patenting of trans- is the agency responsible for regulation of animal exper-
genic animals? imentation in most of the institutions and organizations.

Therapeutically Important Proteins - Transgenic Animals

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Chapter 22

Transgenic Animals and their Applications

SUMMARY transplant. These proteins have significant therapeutic

Animal Biotechnology. http://dx.doi.org/10.1016/B978-0-12-416002-6.00022-5

FLOW CHART 22.1

the result of complementary sequence homology (Fire

Efforts at gain-of-function and loss-of-function modeling

TRANSGENIC ANIMALS AS TRANSGENIC ANIMALS AS FOOD SOURCES

purified enzyme is produced, it must undergo clinical trials

Pro542 HIV mouse, goat GTC

ABBREVIATIONS SHORT ANSWER QUESTIONS

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