Animal Reproduction Science 79 (2003) 265 289
Transgenic animals in biomedicine and agriculture:
outlook for the future
M.B. Wheeler", E.M. Walters, S.G. Clark
366 Animal Sciences Laboratory, Department of Animal Sciences, University of Illinois at Urbana-Champaign,
1207 W. Gregory Dr., Urbana, IL 61801, USA
Received 15 November 2002; received in revised form 14 April 2003; accepted 15 April 2003
Abstract
Transgenic animals are produced by introduction of  foreign deoxyribonucleic acid (DNA)
into preimplantation embryos. The foreign DNA is inserted into the genetic material and may be
expressed in tissues of the resulting individual. This technique is of great importance to many
aspects of biomedical science including gene regulation, the immune system, cancer research,
developmental biology, biomedicine, manufacturing and agriculture. The production of transgenic
animals is one of a number of new and developing technologies that will have a profound impact
on the genetic improvement of livestock. The rate at which these technologies are incorporated into
production schemes will determine the speed at which we will be able to achieve our goal of more
efficiently producing livestock, which meets consumer and market demand.
© 2003 Elsevier B.V. All rights reserved.
Keywords: Transgenic; Biomedicine; Nuclear transfer; Microinjection; Embryonic stem cells; Knock-outs
1. Introduction
The insertion of deoxyribonucleic acid (DNA) into livestock and its stable integration
into the germ line has been a major technical advance. Production of transgenic livestock
provides a method to rapidly introduce  new genes into cattle, swine, sheep and goats
without crossbreeding. It is a more extreme methodology, but in essence, not really different
from crossbreeding or genetic selection in its result. The obvious question is  WHY MAKE
TRANSGENIC ANIMALS? The answer is not so simple; however, some of the reasons
are to: (1) gain new knowledge, (2) decipher the genetic code, (3) study the genetic control
of physiological systems, (4) build genetic disease models, (5) improve animal production
"
Corresponding author. Tel.: [phone]; fax: [phone].
E-mail address: [e-mail] (M.B. Wheeler).
0378-4320/$  see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0378-4320(03)00168-4
266 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
traits, and (6) produce new animal products. This question is sure to be debated, refined and
pondered for a long time.
There are many potential applications of transgenic methodology to develop new and
improved strains of livestock. Practical applications of transgenics in livestock production
include enhanced prolificacy and reproductive performance, increased feed utilization and
growth rate, improved carcass composition, improved milk production and/or composition
and increased disease resistance. Development of transgenic farm animals will allow more
flexibility in direct genetic manipulation of livestock. Gene transfer is a beneficial way of
modifying the genome of domestic livestock. The use of these methodologies will have
a great impact toward improving the efficiency of animal agriculture. There are several
methodologies that can be used for the production of transgenic animals, including: (1)
DNA transfer by retroviruses, (2) microinjection of genes into pronuclei of fertilized ova;
(3) injection of embryonic stem (ES) cells and/or embryonic germ (EG) cells, previously
exposed to foreign DNA, into the cavity of blastocysts; (4) sperm-mediated exogenous
DNA transfer during in vitro fertilization; (5) liposome-mediated DNA transfer into cells
and embryos; (6) electroporation of DNA into sperm, ova or embryos; (7) biolistics, and
(8) nuclear transfer (NT) with somatic cells, ES or EG cells.
Retroviruses are viruses that insert a DNA copy of their genetic material, produced from
RNA as a template, into the host cell DNA following infection. The retrovirus acts as a
naturally occurring delivery system to transfer DNA into various types of mammalian cells
(Varmus, 1998). Preimplantation embryos or oocytes (Jaenisch et al., 1975; Chan et al.,
1998) can be exposed in vitro to concentrated virus solutions or incubated over a single
layer of virus-producing cells. Following exposure to viruses, in vitro infected embryos are
transferred back to recipient females to complete gestation.
Pronuclear injection is another method for introducing foreign genes into animals. Mi-
croinjection of cloned DNA into the pronucleus of a fertilized ovum has been the most
widely used and most successful method for producing transgenic mice (Gordon et al.,
1980; Brinster et al., 1981; Costantini and Lacy, 1981; Wagner et al., 1981a,b). A spe-
cialized microscope equipped with micromanipulators is required for this method of gene
transfer into recently fertilized one-cell embryos. Following the gene transfer, pregnant
recipients will follow normal gestation and deliver young at term.
Two recent developments will profoundly impact the use of transgenic technology in
livestock. These developments are: (1) the ability to isolate and maintain embryonic and
somatic cells directly from embryos, fetuses and adults in vitro and (2) the ability to use these
embryonic and somatic cells as nuclei donors in NT or  cloning strategies. These strategies
have several distinct advantages for use in the production of transgenic livestock that cannot
be attained using pronuclear injection of DNA. ES cells provide the next method to produce
transgenic offspring. This involves injection of embryonic cells into expanded blastocysts
to produce  hybrid embryos composed of two or more distinct cell lines (Robertson et al.,
1986). These embryos are called chimeras. ES cell lines are derived from the inner cell
mass of blastocysts. Once isolated, ES cells can be grown in vitro indefinitely, resulting in
an unlimited number of identical cells each of which has the potential to differentiate into
various tissue types (Wheeler, 1994; Wobus et al., 1984). This has been demonstrated in vivo
through the introduction of ES cells into expanded blastocysts to produce  chimeric animals
that are composed of two or more distinct genotypes. ES cells may also be transformed
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 267
genetically with exogenous DNA before being used to produce chimeric embryos. When
these transformed cells form the gonads and participate in the formation of sperm and eggs,
the offspring that are produced by these chimeric individuals will be transgenic.
Along with ES cells, another type of pluripotent embryonic cells, termed EG cells, have
been derived from the primordial germ cells (PGCs) which are progenitor cells of the sperm
and egg in the adult animal. The EG cells share common characteristics with ES cells both
in vivo and in vitro. EG cell lines have been established in mice (Resnick et al., 1992;
Matsui et al., 1992), pigs (Shim et al., 1997; Piedrahita et al., 1998) and humans (Shamblott
et al., 1998). The protocols for gene transfer using EG cells have been described above in
the section on ES cells.
The next method for introducing exogenous DNA into animals for the purpose of produc-
ing transgenics is sperm-mediated gene transfer. Sperm of many species has been shown to
bind naked DNA (Lavitrano et al., 1989; Horan et al., 1991; Sperandio et al., 1996) as well as
DNA liposome complexes (Bachiller et al., 1991; Rottmann et al., 1992). Generally, sperm
are collected at ejaculation or from the epididymis of the testis and incubated for varying
ć%
lengths of time at 37 39 C in fertilization medium. The transformed sperm may be used for
in vitro fertilization systems (Lavitrano et al., 1989; Sperandio et al., 1996; Maione et al.,
1998) or artificial insemination (Gandolfi et al., 1996; Schellander et al., 1995; Sperandio
et al., 1996); however, the majority of studies have focused on in vitro fertilization systems.
Successful sperm-mediated gene transfer has been reported in the mouse (Lavitrano et al.,
1989; Hochi et al., 1990; Bachiller et al., 1991; Maione et al., 1998), rabbit (Brackett et al.,
1971; Kuznetsov and Kuznetsov, 1995), pig (Gandolfi et al., 1989; Sperandio et al., 1996),
chicken (Fainsold et al., 1990), xenopus (Kroll and Amaya, 1996) and cattle (Perez et al.,
1991; Sperandio et al., 1996).
Liposome/DNA delivery methods are another technique under study for introducing
cloned DNA into cells and embryos. Liposomes are small vesicles consisting of membrane-
like lipid layers that can actually protect foreign DNA from digestion of proteases and
DNAses (Felgner et al., 1987). Cationic liposomes are capable of spontaneously interacting
with DNA molecules, giving rise to lipid DNA complexes (Felgner et al., 1987). Under
appropriate conditions, exogenous DNA can be transferred into cells and a portion of this
DNA becomes localized in the nucleus (Felgner et al., 1987). One-cell embryos may also be
exposed to liposomes carrying cloned genes and potentially incorporate the DNA sequences
into their genome. The methodologies for using liposome carriers with mammalian embryos
are still developing.
Electroporation has been utilized to transfer cloned DNA into cells and embryos (Reiss
et al., 1986; Tur-Kaspa et al., 1986; Inoue et al., 1990; Gagne et al., 1991; Puchalski and
Fahl, 1992; Whitmer and Calarco, 1992). Briefly, the target cells or embryos are placed
in a solution containing the gene of interest. The solution and the cells are exposed to
a very short duration (microseconds) of a high voltage electrical pulse, which allows a
temporary breakdown of the cell membrane. This technique has been used in attempts to
produce transgenic animals by electroporation of DNA into sperm, which then carry the
exogenous DNA to the egg at fertilization (Gagne et al., 1991). As with the liposomes, there
has yet to be a reported instance of germ line transmission utilizing DNA electroporation.
However, electroporation of DNA into mouse ES cell lines (Wurst and Joyner, 1993) and
their subsequent transfer into blastocysts by microinjection have resulted in the production
268 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
of transgenic mice (Camper et al., 1995). This method has great potential either alone or
in combination with others to efficiently transfer genes for the production of transgenic
individuals.
Biolistics or particle bombardment is a physical method that uses accelerated micro-
projectiles to deliver DNA or other molecules into intact tissues and cells (Sanford et al.,
1991). The method was developed initially to transfer genes into plants by Sanford (1988).
The main advantage of biolistics, compared to other transfection methods, is its mechanical
ability to cross biological membranes (Biewenga et al., 1997). Biolistics do not depend
upon the structure and characteristics of target cell membranes (Jiao et al., 1993) nor on the
interaction between DNA molecules and the membranes.
The use of NT (cloning) techniques may have the potential to increase the number of off-
spring from a single female into the thousands and possibly the tens of thousands (Bondioli
et al., 1990). Since the famous cloned sheep  Dolly was born (Wilmut et al., 1997), NT
technology has become another methodology available for the production of transgenic an-
imals. The NT procedure utilizes either in vitro or in vivo matured oocytes as the cytoplasm
donor (cytoplast). The genetic material of the cytoplast is removed (enucleation) leaving
only the cytoplasm. Following enucleation of the oocyte, a donor nucleus (karyoplast) is
injected into the perivitelline space or into the cytoplasm of the enucleated oocyte (Onishi
et al., 2000). The enucleated oocyte and the donor cell are fused by electrofusion. Elec-
trofusion of the cytoplast and the karyoplast is highly species-dependent for the duration,
amplitude of the pulse, fusion medium, and fusion medium equilibration time. After fusion
of the donor nuclei and the enucleated oocyte, the oocyte is activated by either chemical or
mechanical stimulation. Successful activation initiates development to the blastocyst stage,
followed by transfer into a suitable recipient.
The new methods that have recently been reported for the production of genetically
identical individuals from embryonic (Campbell et al., 1996; Wilmut et al., 1997; Onishi
et al., 2000; Polejaeva et al., 2000) and somatic (Wilmut et al., 1997; McCreath et al.,
2000; Polejaeva et al., 2000) cells via NT should allow the rapid development of genetically
identical animals with a targeted gene insertion. These developments will enhance our
ability to produce transgenic animals with genes inserted into specific sites in the genome.
2. Strategies for producing transgenic animals
There are two basic strategies used when producing transgenic animals. These are the
so-called  gain of function or  loss of function transgenics. The basic idea behind the gain
of function paradigm (Table 1) is that by adding a cloned fragment of DNA to an animal s
genome, you can accomplish several objectives. One objective is to get new expression of
a gene product that did not previously exist in that cell or tissue type. An example of this is
the expression of human growth hormone (hGH) in mouse liver (Palmiter et al., 1982). The
same example can be used for adding a gene and getting over expression in the proper tissue
(in this case the mouse pituitary gland) or in a site where expression of this gene product
does not usually occur (i.e., mouse liver, kidney and intestine). Expression of the new gene
product may allow altered regulation of a down stream metabolite or enzyme system in a
metabolic or signal transduction pathway to occur. An increased level of insulin-like growth
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 269
Table 1
Strategies for producing transgenic animals
Gain of function Loss of function
 Knock-in s  Knock-out s
New expression Lost expression
Over expression Over expression
Altered regulation Altered regulation
Antisense RNA Antisense RNA
Insertional mutations Insertional mutations
factor-I (IGF-I) in serum of hGH transgenic mice is an example of this type of situation
(Palmiter et al., 1983). Another potential use of this strategy is the use of DNA constructs,
which encode a messenger RNA in the antisense or reverse orientation with respect to
the promoter. This antisense RNA binds to the sense mRNA strand, transcribed from the
animal s native DNA, forming a heteroduplex which prevents cytoplasmic translation of
the protein (Izant and Weintraub, 1985). If you make an antisense construct to an inhibitory
protein such as growth and differentiation factor-8 (GDF-8, also known as myostatin) you
could potentially increase skeletal muscle growth (McPherron et al., 1997). The final use
of this gain of function strategy is to disrupt an endogenous gene by insertion into the
host s genome. This action induces mutations, which can then be studied. This insertational
mutagenesis is a fortuitous random integration into a functional region of the host genome
and therefore cannot be planned; however, about 5% of the transgenic animals produced
will sustain such mutations. Wilkie and Palmiter, 1987 have shown that a line of transgenic
mice carrying a MT-I-Herpes tk fusion gene were fertile, but the males failed to transmit the
foreign DNA sequence. The conclusion was that the insertion of the transgene inactivated a
host gene such that the sperm inheriting the mutation were destroyed (Wilkie and Palmiter,
1987). They showed that microinjected DNA had the ability to destroy host genes, which
were involved in very subtle and specific developmental processes.
The  loss of function paradigm (Table 1) has many similar applications as the  gain
of function strategy especially when considering over expression, insertional mutations
and antisense situations. It has a major difference in the ability to disrupt genes in a tar-
geted fashion. This strategy relies on the ability of embryonic cells to undergo homolo-
gous recombination. The generation of transgenic animals is dependent on the ability of
the cell to form stable recombinants between the exogenous DNA and the endogenous
chromosomal DNA in the host s genome. Most of these events are non-homologous re-
combination where the introduced DNA inserts randomly in the genome. However, some
cells possess the enzymatic machinery required for recombination between the introduced
DNA sequence and the homologous or identical sequence in the host s genome. This is
called homologous recombination, which is often referred to as  gene targeting . Gene
targeting permits the transfer of genetic alterations created in vitro into precise sites in
the embryonic or cell genome. If the host s cells are totipotent or pluripotent embryonic
cells (i.e., ES, EG cells, PGCs) or re-programmable somatic cells, then these homologous
recombination events can be transferred to the germ line of the offspring. The use of this
strategy has extraordinary potential for making specific genetic changes for use in medicine,
270 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
agriculture and for furthering our understanding of the genetic control of developmental
processes.
3. Applications of transgenic animals
3.1. Biological, biomedical, veterinary and genetic research
The production of transgenic organisms has been a major technical advance in the study of
biology. It is an important method for changing the genetic make-up of an animal providing
a directed, sudden, induced mutation and species crossing technology. Transgenic animals
have been instrumental in providing new insights into the study of the mechanisms of gene
regulation and developmental biology. Additional areas where transgenic technology has
provided significant advances and offers exciting future possibilities are: (1) the action of
genes implicated with having a role in the development of cancer (oncogenes) and oncogenic
viruses; (2) the mechanisms of regulation and cell interaction in the immune system; (3) as
models for human genetic diseases; (4) the mechanisms and control of growth; and (5) the
basic mechanisms of biology and genetics.
Every cell of an individual has all the required information to synthesize proteins that
are characteristic of each and every tissue. However, only a particular cell utilizes a distinct
subset of the information present. The fate of the animal depends on regulation of gene
expression to the appropriate cell types and the orchestration of the expression into the proper
pattern during the development from an embryo to an adult. The utility of transgenic mice
in examining regulation of developmentally controlled and tissue-specific gene expression
has been elegantly illustrated in studies with the -fetoprotein (AFP) gene (Krumlauf et al.,
1985; Godbout et al., 1986; Hammer et al., 1987). -Fetoprotein is a predominant blood
serum protein in the developing mouse fetus which is linked to a related protein, albumin.
These two genes are activated harmonically in the liver, gut and kidney of the developing
mouse fetus. The AFP gene is inactivated shortly after birth, yet the albumin gene continues
to be expressed in the livers of adult animals. Transgenic mice produced by pronuclear
injection have been used to map the regulatory sequences in the AFP gene, which are
essential for its appropriate expression. The particular DNA sequences of interest were those
that restrict AFP expression to a few tissues and initiate its regression after birth. Additional
areas of developmental biology utilizing transgenic animals include nucleus cytoplasm
interactions in cells and the effect of gene location in the chromosome on its expression.
The potential for directing traits in certain cell types by using specific promoters coupled
to foreign genes has prompted attempts to change the physiological function of animals
experimentally (Palmiter and Brinster, 1986). The application of transgenic technology
has been particularly useful for examining the importance of expression of oncogenes or
oncogenic viruses in animal models (Hanahan, 1984, 1985; Palmiter et al., 1985; Van Dyke
et al., 1985, 1987; Hanahan, 1986). Some situations that may be addressed utilizing these
methods are the scope of tissues accessible to the transforming activity of the oncogene, the
relationship between regulation of oncogenes and their ability to cooperate with different
oncogenes in tumor formation, and the role of oncogenes in growth and differentiation.
When oncogenes are incorporated into transgenic mice using various promoters linked
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 271
with the myc or ras oncogenes, tumors form in most cases (Ornitz et al., 1985; Quaife
et al., 1987). However, when the same promoter was used with myc or ras, certain tissues
seemed to be more or less susceptible to tumor generation by either one or the other of
these oncogenes. The pancreas is susceptible to transformation by ras but not by myc,
whereas, the mammary gland formed tumors in response to myc but not to ras. These
results suggest additional cellular events are required to realize oncogene-mediated tumor
development (Land et al., 1983). Relevant transgenic mice have also provided valuable
models for studying the pathology of oncogenic virus-induced diseases such as human
T-cell leukemia.
Transgenic mice are important tools in the study of immunoglobulin genes (Brinster
et al., 1983; Alt et al., 1985; Bucchini et al., 1987; Goodhardt et al., 1987; Storb, 1987).
These genes are responsible for production of antibodies in the immune system. Several
groups have reported that functionally rearranged immunoglobulin genes introduced into
mice could be appropriately activated, resulting in alteration of the mouse s inherent im-
munoglobulin pattern. Further, a functional immune system involves a complex group of
cell-to-cell communications. Transgenic animals will enable the study of immune func-
tion in animals, which are pre-programmed with certain antibodies. This may be of great
value in the study of acquired immunodeficiency syndrome (AIDS) caused by the human
immunodeficiency virus (HIV) (Pezen et al., 1991).
Some genetic diseases which have been studied utilizing transgenic animals are hypogo-
nadism or decreased functional activity of the ovaries or testes, myelination disorders such
as muscular dystrophy and myasthenia gravis (Lou Gehrig s disease), blood disorders such
as thalassemia (Costantini et al., 1986; Ciavatta et al., 1995; Paszty et al., 1995; Yang et al.,
1995), and sickle cell anemia (Paszty et al., 1997; Ryan et al., 1997).
Transgenic mice, rabbits, sheep and pigs have been used as models to examine postnatal
growth of mammals (Brem et al., 1985; Hammer et al., 1985; Pursel et al., 1987, 1989;
Seamark, 1987; Ebert et al., 1988, 1991; Vise et al., 1988; Murray et al., 1989; Polge
et al., 1989; Rexroad et al., 1989, 1991; Wieghart et al., 1990). Growth hormone (GH) and
insulin-like growth factor genes have been incorporated into animals. This has enabled the
study of chronic expression of these hormones independently of their normal regulation
(Brem et al., 1985; Hammer et al., 1985; Pursel et al., 1987, 1989; Seamark, 1987; Ebert
et al., 1988, 1991; Vise et al., 1988; Murray et al., 1989; Polge et al., 1989; Rexroad
et al., 1989, 1991; Wieghart et al., 1990). Results have shown that increasing GH leads to
enhancement of growth and feed efficiency in livestock yet is accompanied by side effects
such as increased incidence of arthritis and thickening of bone (Hammer et al., 1985; Pursel
et al., 1987; Ebert et al., 1988, 1990; Wieghart et al., 1990).
An important application of transgenics is the production of therapeutic proteins for
human clinical use in so-called  bio-reactors . Through genetic engineering it has become
possible to produce any protein from any animal, plant or bacterial species in the milk of
mammals (Bremel et al., 1989). For example, it is possible to express milk proteins and
other proteins of pharmaceutical value in the milk of mice, rabbits, pigs, goats and sheep
(Simons et al., 1987; Buehler et al., 1990; Ebert et al., 1991; Wall et al., 1991; Wright et al.,
1991). Advantages of the mammary synthesis of proteins include the ability of the mammary
secretory cells to properly modify the protein so it is biologically active and then secrete the
protein containing fluid (milk) in large quantities. Smaller quantities of therapeutic proteins
272 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
may also be produced in the eggs of chickens. The albumin fraction can be targeted with
transgene constructs and the resulting proteins easily harvested from the egg white. This
has some advantages over milk as the costs of animal housing, maintenance and harvest
of proteins is much lower. Overall the ability to transgenically produce large quantities of
bioactive proteins and peptides has resulted in the development of a new segment of the
pharmaceutical industry, which has become known as  bio-pharming .
Genes coding for proteins with pharmaceutical value such as human blood clotting factor
IX, which is genetically deficient in hemophiliacs, may be incorporated into transgenic
sheep, goats and cows. This protein can be harvested from the milk, purified and provided
therapeutically to hemophiliacs. Some of the notable examples include -1 antitrypsin for
treatment of emphysema; protein C to limit clot formation (Velander et al., 1992); human
serum albumin for artificial blood substitute; and hepatitis antigens for vaccine production.
In addition to the production of pharmaceutical proteins in milk, such proteins can
also be produced in other biological fluids such as urine, saliva and blood. Utilization
of tissue-specific promoters that target: (1) urine, such as uroplakin; (2) saliva, such as epi-
dermal growth factor (EGF) promoter; and (3) blood, such as hemoglobin or serum albumin
greatly enhance our ability to use these fluids for protein production. Large quantities of
material can be produced in these fluids from livestock species including horses and poultry.
Use of transgenics in  bio-medicine include the production of models to study cellular
prion proteins. One example is the transfer of Creutzfeldt-Jakobs disease (CJD) to trans-
genic mice by introduction of brain extracts of CJD patients. Another application is the
production of transgenic mice containing the human apolipoprotein A-IV that confers re-
sistance to atherosclerosis in a mechanism independent of high-density lipoprotein (HDL)
concentration. The use of transgenics as models in cancer research has and will continue
to be important. An example of how this may be used to treat cancer patients comes from
transgenic mice containing the simian virus T40 antigen. In these mice silencing of the
transgene lead to reversible expression of this gene which led to a reversal of metastatic
hyperplasia in animals up to 4 months of age.
A unique application of transgenic livestock technology is the production of cells, tis-
sues or organs that contain human antigens or proteins for xenotransplantation and other
biomedical uses. Examples using swine include development of: pancreatic -cells for in-
sulin therapy; dopaminergic cells for Parkinson s therapy; human hemoglobin for artificial
blood (Swanson et al., 1992); hepatocytes for artificial livers; hematopoietic stem cells for
leukemia or anemia; and hearts, lungs, kidneys, livers and corneas for organ transplants
(Lin and Platt, 1996). Additionally, specific cell lines derived from transgenic animals may
be useful for other biomedical production or manufacturing applications.
In the United States alone there is a severe shortage of human organs available for organ
transplants. Many patients suffering from organ failure die before even being put on the
waiting list, while others die waiting for an organ. With this shortage of human organs
available for transplant, the idea of using genetically modified animal organs for transplant
into humans was developed. The pig has become the obvious source of these genetically
modified organs because pig s organs are physiologically and anatomically compatible with
humans (Yang et al., 2000). Another advantage of using pigs as organ donors is the ability to
produce large number of the transgenic pigs to be used for xenotransplantation. One of the
major disadvantages to xenotransplantation currently is hyper-acute rejection of the organs.
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 273
Within minutes of receiving the organ, many patients reject the organ because the body
recognizes the organ as foreign tissue. Using transgenics, an organ may be produced that has
the appropriate human proteins expressed on the cell surfaces so the patient s body does not
recognize the organ as foreign tissue. Further, pig organs can be produced that lack proteins
( knock-outs ), such as -1,3-galactosyltransferase (Lai et al., 2002), which are known to be
involved in tissue rejection. Cell therapy may also be an important application of transgenic
technology. The production of transgenic cells that can be used to repair or re-populate
damaged or diseased tissues or organs could potentially alleviate many problems in human
health. An example of this is the use of transgenic pig cells/tissues or even a person s own
cells in NT to make embryos or embryonic cells from which stem cells may be derived.
These stem cells could then be differentiated along specific developmental pathways to
produce healthy replacement cells. These replacement cells could be used to repair damaged
cardiac muscle from heart disease, dopaminergic cells in the brain in Parkinson s patients,
hepatocytes in diseased livers, pancreatic -cells in diabetics, and hematopoetic stems cells
in anemia or leukemia patients. The development of biodegradable matrices has allowed the
production of solid structures such as ears and noses for plastic surgery. These matrices may
be molded into many complex structures, which can then be populated by cells including
stem cells (both transgenic and non-transgenic) to produce replacements for diseased or
missing tissues or organs. Transgenic cells used in these scenarios may also be used to treat
metabolic or enzyme deficiencies. An example of this is the use of bone marrow stem cells
that have had enzyme or genetic defects repaired and then are transferred back to patients.
These are only a few of the exciting possibilities that this technology has to offer.
Transgenic animals may also be very useful for the testing of new drugs or products.
Transgenic rodents that are sensitive to environmental toxins are able to evaluate new drugs,
products or materials for safety much more quickly than their wild-type counterparts. This
type of testing may be done with much fewer animals as the efficiency can be increased
due to the presence of the transgene. Introducing DNA sequences that are sensitive to
breakage may improve our ability to evaluate the mutagenic nature of drugs, products and
environmental conditions. The ability to safely test drugs while at the same time evaluate
potential side effects of those drugs would speed up our ability to bring new safe and
efficacious pharmaceuticals to market more quickly. While at the same time keeping unsafe
and ineffective products away from consumers.
The use of transgenic methodologies has the potential to drastically change the field of
veterinary medicine. The production of animals containing transgenes has been utilized to
make advances in treatment modalities as well as preventative medicine. In order to develop
new therapy for conditions such as cancer, one needs to understand how the gene behaves.
Transgenic technology has allowed scientists in all fields of medicine to study oncogene
expression. Additionally, production traits have been enhanced by the introduction of novel
genes into animals used for food.
The possibilities of this technology are endless for treatment modalities. One aspect of
using transgenes for treatment of disease is the area of oncology. Veterinarians have used
non-pathogenic vectors such as plasmids or viruses that target a particular organ or tissue
and to carry the transgene to the affected cells. Retroviruses and adenoviruses have been
used extensively as direct delivery systems for genes in treating cancer patients. The use of
these organisms in vivo, however, creates several problems in the living patient including
274 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
viral infection, mutation of the virus into a pathogenic form, and induction of cancer. With
these severe side effects being created by the injection of modified viruses, other means of
introducing genes to diseased tissues were needed.
In an effort to alleviate the problems associated with retroviruses and adenoviruses,
cationic liposome delivery systems have been examined. These liposomes have the ability
to transport genes to particular areas of the body so that the DNA could be exposed to
disease cells after endocytosis of the DNA liposome complex, without causing the side
effects seen with the use of viral delivery systems. These liposomes work with the lipid
bilayer of cells to allow incorporation of the DNA into the target cells.
Other prospective uses for genetic transformations of animals are involved with improv-
ing the animal s immune system. The function and expression of a variety of genes can
be assessed using transgenic animals. Transgenic mice have been used to study the func-
tion of major histocompatibility antigens class I and II genes. Another method in which
transgenes can affect the immune system is by producing animals that are disease-resistant.
If a gene could be incorporated into the genome that would prevent a particular organism
from attaching or infecting its target cells, this would lead to more advanced immunization
programs for companion as well as production animals.
There are literally thousands of strains of transgenic animals (primarily rodents) which
have been produced to study some specific aspect of biology, genetics or disease. The
examples described above are only the tip of the iceberg. Only our imagination, creativity
and ingenuity limit the use of transgenic technology in biological, medical and genetic
research.
3.2. Agriculture
There are numerous potential applications of transgenic methodology to develop new
or altered strains of agriculturally important livestock. Practical applications of transgen-
ics in livestock production include improved milk production and composition, increased
growth rate, improved feed utilization, improved carcass composition, increased disease
resistance, enhanced reproductive performance, increased prolificacy, and altered cell and
tissue characteristics for biomedical research (Wheeler and Choi, 1997) and manufacturing.
The production of transgenic swine with GH serves as an excellent example of the value
of this technology (Brem et al., 1985; Hammer et al., 1985). Transgenic alteration of milk
composition has the potential to enhance the production of certain proteins and/or growth
factors deficient in milk (Bremel et al., 1989). The improvement of the nutrient or thera-
peutic value of milk may have a profound impact on survival and growth of newborn in
both humans and animals. Additionally, other animal products, such as eggs and meat could
benefit from the use of transgenesis. Genes could be targeted that could result in continuous
egg production in chickens, and combat reproductive senescence as a result of physiologic
events such as lactation, anorexia, poor nutrition and season of the year (Seidel, 1999).
The application of transgenics is being utilized by commercial aquaculture to enhance
the growth of commercially valuable fish. Fish embryos have been microinjected with a
DNA construct containing either bovine or chinook salmon GH. Another possibility is that
we may be able to improve the nutritional value of fish. Recent studies have shown that
human consumption of fish containing omega-3 fatty acid seems to decrease the occurrence
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 275
of coronary heart disease (Seidel, 1999). Transgenic technology could provide a method to
transfer the nutritionally beneficial traits to other foodstuffs.
3.3. Modification of milk
Advances in recombinant DNA technology have provided the opportunity either to change
the composition of milk or to produce entirely novel proteins in milk. These changes may
add value to, as well as increase the potential uses of milk.
The improvement of livestock growth or survivability through the modification of milk
composition requires production of transgenic animals that: (1) produce a greater quantity
of milk; (2) produce milk of higher nutrient content; or (3) produce milk that contains a
beneficial  nutriceutical protein. The major nutrients in milk are protein, fat and lactose.
By elevating any of these components, we can impact growth and health of the developing
offspring. In many production species such as cattle, sheep and goats, the nutrients available
to the young may not be limiting. However, milk production in the sow limits piglet growth
and therefore pig production (Hartmann et al., 1984). In swine, 44% of the growth rate
of the developing piglets can be attributed to yield and composition of the sow s milk
(Lewis et al., 1978). Methods that increase the growth of piglets during suckling result in
an increase in weaning weights, a decrease in the number of days required to reach market
weight, and thus a decrease in the amount of feed needed for the animals to reach market
weight.
The high percentage in growth rate attributed to milk indicates the potential usefulness
of this technology to the developing piglet. An approach to increase milk production in pigs
may be accomplished by alteration of milk components such as lactose, a major osmole
of milk in mammary gland cells. The over expression of lactose in the milk of pigs will
increase the carbohydrate intake by the developing young, resulting in improvement of
piglet growth (Noble et al., 2002).
Cattle, sheep and goats used for meat production may also benefit from increased milk
yield or composition. In tropical climates, Bos indicus cattle breeds do not produce copious
quantities of milk. Improvement in milk yield by as little as 2 4 l per day may have a
profound affect on weaning weights in cattle such as the Nelore breed in Brazil. Similar
comparisons can be made with improving weaning weights in meat type breeds like the
Texel sheep and Boer goat. This application of transgenic technology could lead to improved
growth and survival of offspring.
A second mechanism by which the alteration of milk composition may affect animal
growth is the addition or supplementation of beneficial hormones, growth factors or bioac-
tive factors to the milk through the use of transgenic animals. It has been suggested that
bioactive substances in milk possess important functions in the neonate with regard to reg-
ulation of growth, development and maturation of the gut, immune system and endocrine
organs (Grosvenor et al., 1993). Transgenic alteration of milk composition has the potential
to enhance the production of certain proteins and/or growth factors that are deficient in milk
(Wall et al., 1991). The over expression of a number of these proteins in milk through the
use of transgenic animals may improve growth, development, health and survivability of
the developing offspring. Some factors that have been suggested to have important biolog-
ical functions in the neonate are obtained through milk included IGF-I, EGF, TGF- and
276 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
lactoferrin (Grosvenor et al., 1993). Recently, it has been suggested that oral administration
of IGF-I may also improve nutrient absorptive function (Alexander and Carey, 1999).
Other properties of milk that bear consideration for modification are those which affect
human and animal health. It has been shown that the preformed specific antibodies can be
produced in transgenic animals (Storb, 1987). It should be possible to produce antibodies
in the mammary gland that are capable of preventing a mastitis infection in cattle, sheep
and goats and MMA (mastitis-metritis-agalactia) in pigs, and/or antibodies that aid in the
prevention of domestic animal or human diseases (Pursel and Rexroad, 1993). Another
example is to increase proteins that have physiological roles within the mammary gland
itself such as lysozyme (Maga et al., 1995) or other anti-microbial peptides.
It is important to consider the use of transgenics to increase specific component(s), which
are already present in milk for manufacturing purposes. An example might be to increase
one of the casein components in milk. This could increase the value of milk in manufacturing
processes such as production of cheese or yogurt. One might also alter the physical properties
of a protein such as increasing the glycosylation of -casein (Choi et al., 1996). This would
result in increased -casein solubility. Increasing the -casein concentration of milk would
reduce the time required for rennet coagulation and whey expulsion. This would produce
firmer curds that are important in cheese making. The deletion of phosphate groups from
-casein would result in softer cheeses. Changes in other physical properties could result
in dairy foods with improved characteristics, such as better tasting low fat cheese (Bleck
et al., 1995). Increasing the -casein content would result in increased thermal stability
of milk that could improve manufacturing properties as well as storage properties of fluid
milk and milk products. It may ultimately be possible to increase the concentration of
milk components while maintaining a constant volume. This could lead to greater product
yield, i.e. more protein, fat or carbohydrate from a liter of milk. This would also aid in
manufacturing processes as well as potentially decreasing transportation costs of the more
concentrated products in fluid milk. The end result would be more saleable product for the
dairy producer.
The overall result of the transgenic modification of milk will be the creation of more uses
of milk and milk products in both agriculture and medicine. This is truly a  value-added
opportunity for animal agriculture by increasing the concentrations of existing proteins or
producing entirely new proteins in milk.
3.4. Modification of growth and carcass composition
The production of transgenic livestock has been instrumental in providing new insights
into the mechanisms of gene action implicated in the control of growth (Brem et al., 1985;
Hammer et al., 1985; Seamark, 1987; Pursel et al., 1989; Vise et al., 1988; Wieghart et al.,
1990). Using transgenic technology it is possible to manipulate known growth factors,
growth factor receptors and growth modulators. Transgenic mice, sheep and pigs have been
used to examine postnatal growth of mammals. GH and IGF genes have been incorporated
and expressed at various levels in transgenic animals (Seamark, 1987). Transgenic livestock
as well as salmon and catfish have been produced which contain an exogenous GH gene.
This type of work enabled the study of chronic expression of these hormones on growth in
mammals (Seamark, 1987) and fish. Results from one study have shown that an increase in
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 277
human GH leads to enhancement of growth and feed efficiency in pigs, yet is accompanied
by side effects such as an increased incidence of arthritis and bone thickening (Pursel
et al., 1990). Similar increases in growth have been shown with the porcine GH (pGH) gene
(Vise et al., 1988). In fish, increased GH levels have lead to dramatic (30 40%) increase in
growth rates in catfish by introducing salmon GH into these animals (Dunham and Devlin,
1999). Introduction of salmonid GH constructs have resulted in a 5 11-fold increase in
weight after 1 year of growth (Du et al., 1992; Devlin et al., 1994, 1995). This illustrates
the point that increased growth rate and ultimately increased protein production per animal
can be achieved via transgenic methodology. Several other genes have also been introduced
into transgenic pigs in an effort to alter growth. An alternative approach was performed
by the introduction of the chicken  ski oncogene, which was previously shown to cause
hypertrophy of numerous muscles, while reducing body fat (Sutrave et al., 1990). This
strategy, however, has resulted in limited success although muscle hypertrophy has been
observed in some transgenic pigs (Pursel et al., 1992) and transgenic cattle (Bowen et al.,
1994).
The Rendement Napole (RN) or Acid-Meat gene has been implicated in lower processing
yields in several lines of Hampshire and Hampshire crossbred pigs. The low pH in the
carcass post-mortem results in differences in pork quality that can be distinguished by
various properties such as water holding capacity, color, marbling, firmness, shear force
and processing yield.  Knocking-out the RN gene (once it is identified and sequenced)
may provide a method to alter the glycolytic potential, post-mortem pH, and, thereby,
meat quality in this species. Other specific loci which may affect growth patterns are the
ryanodine receptor (formerly the halothane sensitivity gene locus, Hal; Fujii et al., 1991),
the myo-D (Harvey, 1991; Sorrentino et al., 1990), GH releasing factor, high affinity IGF
binding proteins (IGFBP-1 to IGFBP-6), the sheep callipyge (Snowder et al., 1994) and the
myostatin (growth/differentiation factor-8, GDF-8; McPherron et al., 1997) genes. Based
on a recent report in the mouse (McPherron and Lawler, 1997) the myostatin gene is an
exceptionally intriguing potential locus for  knock-out using ES cells in meat producing
species. The loss of the myostatin protein results in an increase in lean muscle mass. Mice
lacking this gene have enlarged shoulders and hips. The increased skeletal muscle mass is
widespread throughout the carcass and appears grossly normal. Individual muscle groups
from homozygous knockouts have 2 3 times the weight of control animals. Fat content
was comparable in both the wild type and mutant genotypes (McPherron et al., 1997).
Researchers concluded that a large part of the observed increase in skeletal muscle mass
was due to muscle cell hyperplasia. Certainly, there are numerous potential genes related
to growth, including growth factors, receptors or modulators which have not been used, but
may be of practical importance in producing transgenic livestock with increased growth
rates and/or feed efficiencies.
Another aspect of manipulating carcass composition is that of altering the fat or choles-
terol composition of the carcass. By altering the metabolism or uptake of cholesterol and/or
fatty acids, the content of fat and cholesterol of meats, eggs and cheeses could be low-
ered. There is also the possibility of introducing beneficial fats such as the omega-3 fatty
acids from fish into our livestock. Potential targets are enzymes in the cholesterol and
fat biosynthesis pathways such as cholesterol 7-alpha hydroxlyase (Davis et al., 1998),
hydroxy-methylglutaryl Coenzyme A (HMG-CoA) reductase, fatty acid synthatase and
278 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
lipoprotein lipase. In addition, receptors such as the low-density lipoprotein (LDL) recep-
tor gene and hormones like leptin are potential targets that would decrease fat and cholesterol
in animal products.
The use of transgenic technology to modify feed efficiency and/or appetite could pro-
foundly impact livestock production. Again, increased uptake of nutrients in the digestive
tract, by alteration of the enzyme profiles in the gut, could increase feed efficiency. The
ability to introduce enzymes such as phytase or xylanase into the gut of species where it
is not normally present such as swine or poultry is particularly attractive. The introduction
of phytase would increase the bioavailability of phosphorus from phytic acid in corn and
soy products. Golovan et al. (2001) reported the production of transgenic pigs expressing
salivary phytase as early as 7 days of age. The salivary phytase provided essentially com-
plete digestion of the dietary phytate phosphorus in addition to reducing phosphorus output
by up to 75%. Furthermore, transgenic pigs required almost no inorganic phosphorus sup-
plementation to the diet to achieve normal growth. The use of phytase transgenic pigs in
commercial pork production could result in decreased environmental phosphorus pollution
from livestock operations.
Finally, the introduction of cellulolytic enzymes into the digestive tracts of non-ruminants
could allow for increased digestion of plant cell wall material. This would allow for the
increased utilization of fibrous feedstuffs in the diets of poultry and swine. The ultimate
result would be decreased competition with humans for cereal grains and an increase in the
potential feedstuffs, which could be used for these livestock species.
3.5. Modification of disease resistance
A very interesting aspect of agricultural transgenics is the potential to increase dis-
ease resistance by introducing specific genes into livestock. Identification of single genes
in the major histocompatibility complex (MHC), which influence the immune response,
was instrumental in the recognition of the genetic basis of disease resistance/susceptibility
(Benacerraf and McDevitt, 1972). The application of transgenic methodology to specific
aspects of the immune system should provide opportunities to genetically engineer livestock
with superior disease resistance.
It has only been realized recently that there are many aspects of disease resistance or
susceptibility in livestock that are genetically determined (Lewin, 1989). However, little
information exists regarding the roles of specific genes in the immune or other systems in
the etiology of diseases with economic importance in livestock species (Ebert and Selgrath,
1991). Embryonic cells and/or NT will be very useful for manipulation of genes or large
clusters of genes from the MHC. The use of ES, EG or somatic cells with NT will allow
transfer and integration of much larger (>100 kb) DNA fragments than previously possi-
ble with pronuclear injection. Large yeast artificial chromosome (YAC) vectors containing
extremely large genes (>400 kb) have been transfected into ES cells and produced germ
line transgenics (Choi et al., 1993; Lamb et al., 1993). Manipulation of the MHC in farm
animals through ES cells or NT transgenics could have a major beneficial effect on disease
resistance for livestock producers.
One specific example where transgenesis has been applied to disease resistance in live-
stock is the attempt to produce of pigs that are resistant to influenza. Previously, mice
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 279
and mouse fibroblast cell lines that contain the Mx 1 protein were shown to be resistant
to infection with influenza virus (Haller et al., 1981; Staeheli et al., 1986). Muller et al.
(1992) reported the production of transgenic piglets after introduction of an SV40::Mx con-
struct. However, they also reported that a permanent high level of Mx 1 protein synthesis
might be detrimental to the pigs. Further, all the transgenic piglets born were found to have
rearrangements in the integrated transgenes, which abolished protein synthesis in the live
piglets (Muller et al., 1992). Although, this particular study was not successful in producing
transgenic pigs resistant to influenza, this manipulation is an example of how transgenesis
could be used to increase disease resistance in livestock. It also shows that there needs to
be further research in this important area.
The application of chimeras or NT technology will enable the augmentation of beneficial
alleles and/or the removal (via gene  knock-out ) of undesirable alleles associated with
disease resistance or susceptibility. An example is  knocking-out the intestinal receptor
for the K88 antigen. The absence of the antigen has been shown to confer resistance to both
experimentally and naturally induced infection of K88-positive E. coli (Edfors-Lilia et al.,
1986). Potential areas of investigation include resistance to: (1) parasitic organisms such as
trypanosomes and nematodes, (2) viral or bacterial organisms such as bovine leukemia virus,
pseudorabies virus, foot and mouth virus, clostridium and streptococcus, and (3) genetic
diseases such as deficiency of UMP synthase (DUMPS), mule foot and bovine leukocyte
adhesion deficiency (BLAD).
The opportunity to produce animals that could self-immunize against pathogens is an
exciting application of transgenic technology. The design of transgenes that would be ex-
pressed in response to specific stimuli or physiological state could produce antigens that
result in immunization of the transgenic animal to that particular disease. Transgenes will
be designed which respond to specific stimuli like feeding zinc or a specific antibiotic to
produce antigens that could raise protective antibody titers.
In the future we may be able to produce prion-free, scrapie-free and BSE-free livestock
using the genetics from naturally resistant animals in cloning schemes. An example of this
is the production of fetuses that are resistant to Brucellosis (Shin et al., 1999). This is only
a partial list of organisms or genetic diseases that decrease production efficiency and may
also be targets for manipulation via transgenic methodologies.
3.6. Modification of reproductive performance and prolificacy
Several potential genes have recently been identified which may profoundly affect re-
productive performance and prolificacy. These include the estrogen receptor (ESR) and the
Boroola fecundity (FECB) genes. Rothschild et al. (1994) have reported an association of a
polymorphism in the ESR gene with litter size in pigs. They found a difference of 1.4 more
pigs born per litter between the two homozygous genotypes. Introduction of a mutated or
polymorphic ESR gene could increase litter size in a number of diverse breeds of pigs.
A single major autosomal gene for fecundity, the FECB gene, which allows for increased
ovulation rate, has been identified in Merino sheep (Piper et al., 1985). Each copy of the
gene has been shown to increase ovulation rate by approximately 1.5 ova, although the
increase in litter size is not completely additive (Piper et al., 1985). Production of trans-
genic sheep containing the appropriate FECB allele could increase fecundity in a number
280 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
of diverse breeds. Identification of additional genes involved in prolificacy and fecundity
from hyperprolific breeds/strains of swine (Meishan); sheep (Finnish Landrace) and cattle
(high twinning) will provide additional opportunities to increase reproductive performance.
Other transgenic applications in reproduction could be a visual indicator of estrus in farm
animals. In the baboon, estrogen levels increase at the time of estrus making their posterior
bright red. Transgenic pigs could be made to have bright red posteriors at the time of estrus
(Seidel, 1999). This type of transgenic pigs could save swine producers time, money and
possibly increase conception rates.
The use of the so called  suicide genes to specifically kill cells could be a great benefit
to livestock production. These genes, once incorporated into a cell, can be stimulated or
induced to initiate apoptosis or programmed cell death. The incorporation of such strate-
gies into the production of transgenic animals could allow for precise control over the
reproduction of certain strains and even specific sex distributions of livestock. An example
would be using a testis specific promoter that when expressed would kill  Y chromosome
bearing sperm in the case of sex selection and would kill all sperm in the case of con-
trol of reproduction. Similar strategies could be envisioned for female transgenics. This
type of manipulation could produce sterile individuals that could only be used for food
production without fear of an accidental release of a reproductively competent transgenic
animal into the environment. A good application would be the use of these strategies in
the production of transgenic salmon, trout and catfish. The use of suicide genes could also
limit the breeding of valuable transgenic livestock by inappropriate parties. This would
offer some protection to the large investment usually required to develop and produce such
animals.
The manipulation of reproductive processes using transgenic methodologies is only be-
ginning and should be a very rich area for investigation in the future.
3.7. Modification of fiber and hair
The control of the quality, color, yield and even ease of harvest of hair, wool and fiber
for fabric and yarn production has been an area of focus for transgenic manipulation in
livestock. The manipulation of the quality, length, fineness and crimp of the wool and hair
fiber from sheep and goats has been examined using transgenic methods (Hollis et al.,
1983; Powell et al., 1994). Other aspects that transgenic methods will allow modification
of are increasing the elasticity of the fiber and increasing the fiber strength (Bawden et al.,
1998). In the future transgenic manipulation of wool will focus on the surface of the fibers.
Decreasing the surface interaction could decrease shrinkage of garments made from such
fibers (Bawden et al., 1999).
Another application of this technology is the efforts to induce sheep to shed their wool
at specific times to alleviate the need for hand shearing of fiber producing animals (Hollis
et al., 1983; Powell et al., 1994). Genes such as EGF with inducible promoters have been
introduced into sheep (Hollis et al., 1983). The idea is that when EGF expression is induced,
a weak spot is produced in the wool fiber that allows the fleece to be removed with hand
pressure. The fleece can literally be peeled off without the need for metal shears or clippers.
This would greatly reduce the expense of wool harvest. Similar strategies could be developed
for mohair goats, alpacas, camels and other fiber-producing animals.
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 281
A very novel approach to produce useful fiber has been recently accomplished us-
ing the milk of transgenic goats (Karatzas et al., 1999). Spiders that produce orb-webs
synthesize as many as seven different types of silk used in making these webs. Each
of these silks has very specialized mechanical properties that makes them distinct from
other synthetic and natural fibers (Karatzas et al., 1999). One of the most durable vari-
eties is dragline silk. This material can be elongated up to 35% and has tensile properties
close to those of the synthetic fiber Kevlar. This silk has the energy absorbing capabili-
ties before snapping; which exceeds those of steel. The protein monomers that assemble
to produce these spider silk fibers have been produced in the milk of transgenic goats
using the BELE® (Breed Early Lactate Early) goat system. The protein monomers can
be assembled in the laboratory or factory to produce fibers with properties approaching
those seen in the natural spider silk. The numerous potential applications of these fibers
include medical devices, suture, ballistic protection, aircraft, automotive composites and
clothing to name a few. The use of the mammary gland to produce protein components of
fibers has a great deal of utility in producing new products or value-added products from
livestock.
In summary, the potential applications of transgenic technology in livestock production
are tremendous. The utility of this technology is limited only by our ability to identify
appropriate genes and gene functions to manipulate in our production livestock species.
3.8. Pitfalls with transgenic production
As one can imagine, problems occur. These problems can be: (1) unregulated expression
of genes resulting in over or under production of gene products; (2) too high a copy number
resulting in over expression of products; (3) possible side effects, GH transgenic swine had
arthritis, altered skeletal growth, cardiomegaly, dermatitis, gastric ulcers and renal disease;
(4) insertional mutations which result in some essential biological processes being altered;
(5) mosaicism in the founders which results in transmission of the transgene to only some
of the offspring; and (6) transgene integration on the  Y chromosome which results in only
males carrying the transgene. Many, if not all, of these problems are related to the transgene
itself, integration site, copy number and transgene expression.
3.9. Ethics and animal welfare issues associated with transgenic production
The technology involved in production of transgenic animals holds great promise for both
agriculture and biomedicine. This as with other areas of biological research has both benefits
and potential risks. The public s perception of biotechnology is different depending on its
uses. Development of new vaccines to treat infectious diseases may be widely accepted
whereas production of transgenic livestock that grow at a faster rate, for consumption as
food for humans, may not. There is no doubt that this type of research will experience ever
growing public scrutiny. It is also evident that not only self-regulation but also increasing
governmental regulation is imminent. Mechanisms are already in place in a number of
European countries to evaluate the ethics and animal welfare of proposed manipulation
of animal genomes. The production of transgenic livestock, at present, is inefficient and
financially costly. To date, improvement in productivity traits of these animals have been
282 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
modest. One aspect that must be determined is whether, in the long-term, these costs are
proportional to the increased productivity (Mench, 1999) or increased consumer benefit.
It is clear that for the long-term benefit of society and the area of transgenic technology,
the impacts on the environment, farmers, consumers and especially the animals must be
carefully evaluated. Issues that need further discussion and evaluation are animal welfare,
behavioral freedom, food safety, product labeling, freedom to adopt or decline technology
and biodiversity to name a few (Mench, 1999). It is important for scientists using this tech-
nology to become engaged and be willing participants in the discussion and consideration
of ethical issues and concerns surrounding the implementation of this work.
It is worth pointing out here that the goal of using this technology is for the benefit not the
detriment of mankind. As previously stated use of this technology is not simple, efficient or
inexpensive. Scientists using this technology are trying to develop models to study diseases,
produce bio-pharmaceuticals and produce more wholesome, healthy and economical food.
These studies are difficult and great care must be taken before such investigations begin.
Such considerations are critical due to the time, cost, welfare, ethics, concerns, risks and
benefits involved in these kinds of investigations. Realize none of these groups, farmers,
consumers or scientists, are motivated to produce inappropriate medical models, ineffective
or dangerous pharmaceuticals, or unsafe food. None of these groups would be around very
long if that were the case. This does not mean that animal care, concern, animal welfare,
ethics, societal benefit and vigilance should be ignored. On the contrary, they should be
embraced when designing and conducting such investigations. Consideration of these as
well as scientific issues will lead us forward to reaping the benefits from this important
technology.
3.10. Perspectives
The overall goal of producing transgenic animals is to (1) increase our knowledge of
biology and biomedical science and (2) increase our ability to efficiently produce milk, meat
and fiber. To successfully obtain these improvements, we will test our ability to quantify
desirable traits, to identify genes responsible for these traits and to introduce those genes
into laboratory animals as well as production livestock. We must select and/or  re-design
populations of superior individuals which can be propagated. The incorporation of cell, NT
and recombinant DNA technologies into these strategies will continue to be an important
aspect of future advances. However, we must remember that the production capability
of genetically selected and/or genetically engineered animals will only be realized when
their true genetic potential is attained through appropriate environmental and management
considerations.
4. Conclusion
The ultimate utility and value of transgenic technology will be limited by our ability
to: (1) identify genes and appropriate regulatory sequences for the production of traits we
wish to study, improve or include in development of transgenic animals; and (2) incor-
porate these desired genes in an appropriately expressed and regulated manner into our
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 283
domestic livestock. The establishment of ES, EG and somatic cell lines and NT methods in
livestock will be useful for the production of transgenic livestock as well as for studies of
cell differentiation, development and gene regulation in farm animals. The use of ES/EG
cell-mediated gene transfer has not been employed in domestic livestock mainly due to the
lack of established, stable ES/EG cell lines. There are two barriers to overcome to produce
transgenic livestock with this technology: (1) establishment of undifferentiated ES/EG cell
lines and (2) the successful transformation of the ES/EG cells with the  foreign gene(s).
Great progress has been made towards overcoming the first of these barriers (Gerfen and
Wheeler, 1995; Wheeler et al., 1995; Rund et al., 1996; Shim et al., 1997; Piedrahita et al.,
1998), that is, the isolation of undifferentiated ES or EG cell lines. However, a significant
amount of work still remains to enable the routine production of transgenic livestock from
ES or EG cells.
Finally, there are a number of new and developing technologies that will have a profound
impact on the genetic improvement of livestock. The rate at which these technologies are
incorporated into production schemes will determine the speed at which we will be able to
achieve our goal of more efficiently producing livestock, which meets consumer and market
demand.
References
Alexander, A.N., Carey, H.V., 1999. Oral IGF-I enhances nutrient and electrolytes absorption in neonatal piglet
intestine. Am. J. Physiol. 277, G619 G625.
Alt, F.W., Blackwell, T.K., Yancopoulos, G.D., 1985. Immunoglobulin genes in transgenic mice. TIG 1, 231 236.
Bachiller, D., Schellander, K., Peli, K., Ruther, U., 1991. Liposome-mediated DNA uptake by sperm cells. Mol.
Reprod. Dev. 30, 194 200.
Bawden, C.S., Powell, B.C., Walker, S.K., Rogers, G.E., 1998. Expression of a wool intermediate filament keratin
transgene in sheep fiber alters structure. Transgenic Res. 7, 273 287.
Bawden, C.S., Dunn, S.M., McLaughlan, C.J., Nesci, A., Powell, B.C., Walker, S.K., Rogers, G.E., 1999.
Transgenesis with ovine keratin genes: expression in the sheep wool follicle for fibres with new properties.
Transgenic Res. 8, 474 (abstract).
Benacerraf, B., McDevitt, H.O., 1972. Histocompatibility linked immune response genes. A new class of genes
that controls the formation of species immune response has been identified. Science 175, 273 279.
Biewenga, J.E., Destre, O.H.J., Schrama, L.H., 1997. Plasmid-mediated gene transfer in neurons using the biolistics
technique. J. Neuro. Meth. 71, 67 75.
Bleck, G.T., Jiminez-Flores, R., Wheeler, M.B., 1995. Production of transgenic animals with altered milk as a tool
to modify milk composition, increase animal growth and improve reproductive performance. In: Greppi, G.F.,
Enne, G. (Eds.), Animal Production & Biotechnology. Elsevier, Amsterdam, pp. 1 19.
Bondioli, K.R., Westhusin, M.E., Loony, C.R., 1990. Production of identical bovine offspring by nuclear transfer.
Theriogenology 33, 165 174.
Bowen, R.A., Reed, M., Schnieke, A., Seidel, G.E., Stacey, A., Thomas, W.K., Kaijkawa, O., 1994. Transgenic
cattle resulting from biopsied embryos: expression of c-ski in a transgenic calf. Biol. Reprod. 50, 664 668.
Brackett, B.G., Boranska, W., Sawicki, W., Koprowski, H., 1971. Uptake of heterologous genome by mammalian
spermatozoa and its transfer to ova through fertilization. Proc. Natl. Acad. Sci. USA 68, 353 357.
Brem, G., Brenig, B., Goodman, H.M., Selden, R.C., Graf, F., Kruff, B., Springman, K., Hondele, J., Meyer, J.,
Winnacker, E.-L., Kraublich, H., 1985. Production of transgenic mice, rabbits and pigs by microinjection into
pronuclei. Zuchthygiene 20, 241 245.
Bremel, R.D., Yom, H.-C., Bleck, G.T., 1989. Alteration of milk composition using molecular genetics. J. Dairy
Sci. 72, 2826 2833.
Brinster, R.L., Chen, H.Y., Trumbauer, M.E., Senear, A.W., Warren, R., Palmiter, R.D., 1981. Somatic expression
of herpes thymidine kinase in mice following injection of a foreign gene into eggs. Cell 27, 223 231.
284 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
Brinster, R.L., Ritchie, K.A., Hammer, R.E., O Brien, R.L., Arp, B., Storb, U., 1983. Expression of a microinjected
immunoglobulin gene in the spleen of transgenic mice. Nature 306, 332 336.
Bucchini, D., Reynaud, C.-A., Ripoche, M.-A., Grimal, H., Jami, J., Weill, J.-C., 1987. Rearrangement of a chicken
immunoglobulin gene occurs in the lymphoid lineage of transgenic mice. Nature 326, 409 411.
Buehler, T.A., Bruyere, T., Went, D.F., Stranzinger, G., Buerki, K., 1990. Rabbit -casein promoter directs secretion
of human interleukin-2 into the milk of transgenic rabbits. Biotechnology 8, 140 143.
Campbell, K.H.S., McWhir, J., Ritchie, W.A., Wilmut, I., 1996. Sheep cloned by nuclear transfer from a cultured
cell line. Nature 380, 64 66.
Camper, S.A., Saunders, T.L., Kendall, S.K., Keri, R.A., Seasholtz, A.F., Gordon, D.F., Birkmeier, T.S., Keegan,
C.E., Karolyi, I.J., Roller, M.L., Burrows, H.L., Samuelson, L.C., 1995. Implementing transgenic and embryonic
stem cell technology to study gene expression, cell cell interactions and gene function. Biol. Reprod. 52, 246
257.
Chan, A.W.S., Homan, E.J., Ballou, L.U., Burns, J.C., Bremel, R.D., 1998. Transgenic cattle produced by
reverse-transcribed gene transfer in oocytes. Proc. Natl. Acad. Sci. USA 95, 14028 14033.
Choi, T.K., Hollenbach, P.W., Pearson, B.E., Udea, R.M., Weddell, G.N., Kurahara, C.G., Woodhouse, C.S., Kay,
R.M., Loring, J., 1993. Transgenic mice containing a human heavy chain immunoglobulin gene fragment
cloned in a yeast artificial chromosome. Nat. Genet. 4, 117 123.
Choi, B.K., Bleck, G.T., Wheeler, M.B., Jiminez-Flores, R., 1996. Genetic modification of bovine -casein and
its expression in the milk of transgenic mice. J. Agric. Food Chem. 44, 953 960.
Ciavatta, D.J., Ryan, T.M., Farmer, S.C., Townes, T.M., 1995. Mouse model of human beta zero thalassemia:
targeted deletion of the mouse beta maj- and beta min-globin genes in embryonic stem cells. Proc. Natl. Acad.
Sci. USA 92, 9259 9263.
Costantini, F., Lacy, E., 1981. Introduction of a rabbit -globin gene into the mouse germ line. Nature 294, 92 94.
Costantini, F., Chada, K., Magram, J., 1986. Correction of murine -thalassemia by gene transfer into the germ
line. Science 233, 1192 1194.
Davis, A.M., Pond, W.G., Wheeler, M.B., Ishimura-Oka, K., Su, D.R., Li, C.M., Mersmann, H.J., 1998. Distribution
of alleles of the cholesterol 7 alpha-hydroxylase (CYP7) gene in pigs selected for high or low plasma total
cholesterol. Proc. Soc. Exp. Biol. Med. 217, 466 470.
Devlin, R.H., Yesaki, T.Y., Donaldson, E.M., Du, S.-J., Hew, C.L., 1994. Extraordinary salmon growth. Nature
371, 209 210.
Devlin, R.H., Yesaki, T.Y., Donaldson, E.M., Du, S.-J., Hew, C.L., 1995. Production of germline transgenic Pacific
salmonids with dramatically increased growth performance. Can. J. Fish Aquat. Sci. 52, 1376 1384.
Du, S.J., Gong, Z., Flecther, G.L., Schears, King, M.A., King, M.J., Idler, D.R., Hew, C.L., 1992. Growth
enhancement in transgenic salmon by the use of an  all fish chimeric growth hormone gene construct.
Biotechnology 10, 176 181.
Dunham, R.A., Devlin, R.H., 1999. Comparison of traditional breeding and transgenesis in farmed fish with
implications for growth enhancement and fitness. In: Murray, J.D., Anderson, G.B., Oberbauer, A.M., Mc
Gloughlin, M.M. (Eds.), Transgenic Animals in Agriculture. CABI Publishing, New York, pp. 209 230.
Ebert, K.M., Selgrath, J.P., 1991. Changes in domestic livestock through genetic engineering. In: Pedersen, R.A.,
McLaren, A., First, N.L. (Eds.), Current Communication in Cell and Molecular Biology: Animal Applications
of Research in Mammalian Development, pp. 233 266.
Ebert, K., Low, M., Overstrom, E., Buonoma, F., Roberts, T.M., Lee, A., Mandel, G., Goodman, R.A., 1988.
Moloney MLV-RAT somatotropin fusion gene produces biologically active somatotropin in a transgenic pig.
Mol. Endocrinol. 2, 277 283.
Ebert, K.M., Smith, T.E., Buonoma, F.C., Overstrom, E.W., Low, M.J., 1990. Porcine growth hormone gene
expression from viral promoters in transgenic swine. Anim. Biotechnol. 1, 145 159.
Ebert, K.M., Selgrath, J.P., Ditullio, P., Denman, J., Smith, T.E., Memon, M.A., Schindler, J.E., Monastersky,
G.M., Vitale, J.A., Gordon, K., 1991. Transgenic production of a variant of human tissue-type plasminogen
activator in goat milk: generation of transgenic goats and analysis of expression. Biotechnology 9, 835 840.
Edfors-Lilia, I., Petersson, H., Gahne, B., 1986. Performance of pigs with or without the intestinal receptor for
Escherichia Coli K88. Anim. Prod. 42, 381 387.
Fainsold, A., Frumpkin, A., Rangini, Z., Revel, E., Yarus, S., Ben-Yehuda, A., Gruembaum, Y., 1990. Chicken
homeogenes expressed during gastrulation and the generation of transgenic chicken. In: Proceedings of the
EMBO-EMBL Symposium, Mol. Biol. Vertebrate Dev. 31.
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 285
Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., Danielsen,
M., 1987. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci.
USA 84, 7413 7417.
Fujii, J., Otsu, K., Zorzto, F., De Leon, S., Khanna, V.K., Weiler, J.E., O Brian, P.J., MacLennan, D.H., 1991.
Identification of a mutation in the porcine ryanodine receptor associated with malignant hyperthermia. Science
253, 448 451.
Gagne, M.B., Pothier, F., Sirad, M.-A., 1991. Electroporation of bovine spermatozoa to carry foreign DNA in
oocytes. Mol. Reprod. Dev. 29, 6 15.
Gandolfi, F., Lavitrano, M., Camaioni, A., Spadafora, C., Siracusa, G., Lauria, A., 1989. The use of sperm-mediated
gene transfer for the generation of transgenic pigs, J. Reprod. Fertil. 4 (10) (abstract).
Gandolfi, F., Terqui, M., Modina, S., Brevini, T.A.L., Ajmone-Marsan, P., Foulon-Gauze, F., Courot, M., 1996.
Failure to produce transgenic offspring by intra-tubal insemination of gilts with DNA treated sperm. Reprod.
Fertil. Dev. 8, 1055 1060.
Gerfen, R.W., Wheeler, M.B., 1995. Isolation of pluripotent embryonic cell lines from porcine blastocysts. Anim.
Biotechol. 6, 1 14.
Godbout, R., Ingram, R., Tilghman, S.M., 1986. Multiple regulatory elements in the intergenic region between
the -fetoprotein and albumin genes. Mol. Cell Biol. 6, 477 487.
Golovan, S.P., Meidinger, R.G., Ajakaiye, A., Cottrill, M., Wiederkehr, M.Z., Barney, D.J., Plante, C., Pollard,
J.W., Fan, M.Z., Hayes, M.A., Laursen, J., Hjorth, J.P., Hacker, R.R., Phillips, J.P., Forsberg, C.W., 2001. Pigs
expressing salivary phytase produce low-phosphorus manure. Nature Biotechnol. 19, 741 745.
Goodhardt, R., Cavelier, F., Akimenko, M.A., Lutfalla, G., Babinet, C., Rougeon, F., 1987. Rearrangement and
expression of rabbit immunoglobulin kappa light chain gene in transgenic mice. Proc. Natl. Acad. Sci. USA
84, 4229 4233.
Gordon, M.F., Scangos, G.A., Plotkin, D.J., Barbosa, J.A., Ruddle, F.H., 1980. Genetic transformation of mouse
embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. USA 77, 7380 7384.
Grosvenor, C.E., Picciano, M.F., Baumrucker, C.R., 1993. Hormones and growth factors in milk. Endocrinol Rev.
14 (6), 710 728.
Haller, O., Arnheiter, H., Gresser, I., Lindenmann, J., 1981. Virus-specific interferon action/protection of newborn
Mx carries against lethal infection with influenza virus. J. Exp. Med. 154, 199 203.
Hammer, R.E., Pursel, V.G., Rexroad Jr., C.E., Wall, R.J., Bolt, D.J., Ebert, K.M., Palmiter, R.D., Brinster, R.L.,
1985. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315, 680 683.
Hammer, R.E., Krumlauf, R., Camper, S.A., Brinster, R.L., Tilghman, S.M., 1987. Diversity of alpha fetoprotein
gene expression in mice is generated by a combination of separate enhancer elements. Science 235, 53 58.
Hanahan, D., 1984. Oncogenes in transgenic mice. Nature 312, 503 504.
Hanahan, D., 1985. Heritable formation of pancreatic -cell tumors in transgenic mice expression recombinant
insulin/simian virus 40 oncogenes. Nature 315, 115 122.
Hanahan, D., 1986. Oncogenesis in transgenic mice. In: Kahn, P., Graf, T. (Eds.), Oncogenes and Growth Control.
Springer, Heidelberg, Germany, pp. 349 363.
Hartmann, P.E., McCauley, I., Gooneratne, A.D., Whitely, J.L., 1984. Inadequacies of sow lactation: survival of
the fittest. Symp. Zool. Soc. Lond. 51, 301 326.
Harvey, R.P., 1991. Widespread expression of MyoD genes in Xenopus embryos is amplified in presumptive
muscle as a delayed response to mesoderm induction. Proc. Natl. Acad. Sci. USA 88, 9198 9202.
Hochi, S., Ninomiya, T., Mizuna, A., Honma, M., Yuki, A., 1990. Fate of exogenous DNA carried into mouse
eggs by spermatozoa. Anim. Biotechnol. 1, 25 30.
Hollis, D.E., Chapman, R.E., Panaretto, B.A., Moore, G.P., 1983. Morphological changes in the skin and wool
fibers of Merino sheep infused with mouse epidermal growth factor. Aust. J. Biol. Sci. 36 (4), 419 434.
Horan, R., Powell, R., McQuaid, S., Gannon, F., Houghton, J.A., 1991. Association of foreign DNA with porcine
spermatozoa. Arch. Androl. 26, 83 92.
Inoue, K., Yamashita, S., Hata, J., Kabeno, S., Asada, S., Nagahisa, E., Fujita, T., 1990. Electroporation as a new
technique for producing transgenic fish. Cell Diff. Dev. 29, 123 128.
Izant, J.G., Weintraub, H., 1985. Constitutive and conditional suppression of exogenous and endogenous genes
by anti-sense RNA. Science 229, 345 352.
Jaenisch, R., Fan, H., Croker, B., 1975. Infection of preimplantation mouse embryos and of newborn mice with
leukaemia virus: tissue distribution of viral DNA and RNA leukemogenesis in adult animal. Proc. Natl. Acad.
Sci. USA 72, 4008 4012.
286 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
Jiao, S., Cheng, L., Wolff, J., Yang, N., 1993. Particle bombardment-mediated gene transfer and expression in rat
brain tissues. Biotechnology 11, 497 502.
Karatzas, C.N., Zhou, J.F., Huang, Y., Duguay, F., Chretien, N., Bhatia, B., Bilodeau, A., Keyston, R., Tao, T.,
Keefer, C.L., Wang, B., Baldassare, H., Lazaris, A., 1999. Production of recombinant spider silk (BiosteelTM)
in the milk of transgenic animals. Transgenic Res. 8, 476 477.
Kroll, K., Amaya, E., 1996. Transgenic Xenopus embryos from sperm nuclear transplantation reveal FGF signaling
requirements during gastrulation. Development 122, 3173 3183.
Krumlauf, R., Hammer, R.E., Tilghman, S.M., Brinster, R.L., 1985. Developmental regulation of -fetoprotein
genes in transgenic mice. Mol. Cell Biol. 5, 1639 1648.
Kuznetsov, A.V., Kuznetsov, I.V., 1995. The binding of exogenous DNA pRK3lacZ by rabbit spermatozoa, its
transfer to oocytes and expression in preimplantation embryos. Ontogenez 26, 300 309.
Lai, L., Kolber-Simonds, D., Park, K.W., Cheong, H.T., Greenstein, J.L., Im, G.S., Samuel, M., Bonk,
A., Rieke, A., Day, B.N., Murphy, C.N., Carter, D.B., Hawley, R.J., Prather, R.S., 2002. Production of
alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295, 1089 1092.
Lamb, B.T., Sisodia, S.S., Lawler, A.M., Slunt, H.H., Kitt, C.A., Kearns, W.G., Pearson, P.L., Price, D.L., Gearhart,
J.D., 1993. Introduction and expression of the 400 kilobase precursor amyloid protein gene in transgenic mice.
Nat. Genet. 5, 22 30.
Land, H., Parada, L.F., Weinberg, R.A., 1983. Tumorigenic conversion of primary embryo fibroblasts requires at
least two cooperating oncogenes. Nature 304, 596 602.
Lavitrano, M., Camaioni, A., Fazio, V.M., Dolci, S., Farace, M.G., Spadafora, C., 1989. Sperm cells as vectors
for introducing foreign DNA into eggs: genetic transformation of mice. Cell 57, 717 723.
Lewin, H.A., 1989. Disease resistance and immune response genes in cattle: strategies for their detection and
evidence of their existence. J. Dairy Sci. 72, 1334 1348.
Lewis, A.J., Speer, V.C., Haught, D.G., 1978. Relationship between yield and composition of sow s milk and
weight gains of nursing pigs. J. Anim. Sci. 47, 634 638.
Lin, S.S., Platt, J.L., 1996. Immunological advances towards clinical xenotransplantation. In: Tumbleson, M.E.,
Schook, L.B. (Eds.), Advances in Swine in Biomedical Research. Plenum Press, New York, pp. 147 162.
Maga, E.A., Anderson, G.B., Murray, J.D., 1995. The effect of mammary gland expression of human lysozyme
on the properties of milk from transgenic mice. J. Dairy Sci. 78, 2645 2652.
Maione, B., Lavitrano, M., Spadafora, C., Kiessling, A., 1998. Sperm-mediated gene transfer in mice. Mol. Repod.
Dev. 50, 406 409.
Matsui, Y., Zsebo, K., Hogan, B.L.M., 1992. Derivation of pluripotent embryonic stem cells from murine primordial
germ cells in culture. Cell 70, 841 847.
McCreath, K.J., Howcroft, J., Campbell, K.S., Coleman, A., Schnieke, A.E., Kind, A.J., 2000. Production of
gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405, 1066 1069.
McPherron, A.C., Lawler, A.M., Le, S.-J., 1997. Regulation of skeletal muscle mass in mice by a new TGF-beta
superfamily member. Nature 387, 83 90.
Mench, J.A., 1999. Ethics, animal welfare and transgenic farm animals. In: Murray, J.D., Anderson, G.B.,
Oberbauer, A.M., McGloghlin, M.M. (Eds.), Transgenic Animals in Agriculture. CABI International, pp.
25 268.
Muller, M., Brenig, B., Winnacker, E.L., Brem, G., 1992. Transgenic pigs carrying cDNA copies encoding the
murine Mx1 protein which confers resistance to influenza virus infection. Gene 121, 263 270.
Murray, J.D., Nancarrow, C.D., Marshall, J.T., Hazelton, I.G., Ward, K.A., 1989. The production of transgenic
Merino sheep by microinjection of ovine metallothionein-growth hormone fusion genes. Reprod. Fertil. Dev.
1, 147 155.
Noble, M.S., Rodriguez-Zas, S., Bleck, G.T., Cook, J.S., Hurley, W.L., Wheeler, M.B., 2002. Lactational
performance of first parity transgenic gilts expressing bovine -lactalbumin in their milk. J. Anim. Sci. 80,
1090 1096.
Onishi, A., Iwamoto, M., Akita, T., Mikawa, S., Takeda, K., Awata, T., Hanada, H., Perry, A.C.F., 2000. Pig cloning
by microinjection of fetal fibroblast nuclei. Science 289, 1188 1190.
Ornitz, D.M., Palmiter, R.D., Messing, A., Hammer, R.E., Pinkert, C.A., Brinster, R.L., 1985. Elastase promoter
directs expression of human growth hormone and T-antigen genes to pancreatic acinar cells in transgenic mice.
In: Proceedings of the Cold Spring Harbor Symposium on Quant Biology, USA, vol. 50, pp. 399 409.
Palmiter, R.D., Brinster, R.L., 1986. Germ-line transformation of mice. Ann. Rev. Genet. 20, 465 499.
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 287
Palmiter, R.D., Brinster, R.L., Hammer, R.E., Trumbauer, M.E., Rosenfeld, M.G., Birnberg, N.C., Evans, R.M.,
1982. Dramatic growth of mice that develop from egg microinjected with metallothionein-growth hormone
fusion genes. Nature 300, 611 615.
Palmiter, R.D., Norstedt, G., Gelinas, R.E., Hammer, R.E., Brinster, R.L., 1983. Metallothionein-human GH fusion
genes stimulate growth of mice. Science 222, 809 814.
Palmiter, R.D., Chen, H.Y., Messing, A., Brinster, R.L., 1985. SV40 enhancer and large T-antigen are instrumental
in development of choroid plexux tumors in transgenic mice. Nature 316, 457 463.
Paszty, C., Mohandas, N., Stevens, M.E., Loring, J.F., Liebhaber, S.A., Brion, C.M., Rubin, E.M., 1995. Lethal
alpha-thalassaemia created by gene targeting in mice and its genetic rescue. Nat. Genet. 11, 33 39.
Paszty, C., Brion, C.M., Manci, E., Witkowska, H.E., Stevens, M.E., Mohandas, N., Rubin, E.M., 1997. Transgenic
knockout mice with exclusively human sickle hemoglobin and sickle cell disease. Science 278, 876 878.
Perez, A., Solano, R., Castro, R., Lleonart, R., de Armas, R., Martinez, R., Aguilar, A., Herrera, L., de la Fuente,
J., 1991. Sperm cells mediated gene transfer in cattle. Biotechnol. Aplicada 8, 90 94.
Pezen, D.S., Leonard, J.M., Abramczuk, J.W., Martin, M.A., 1991. Transgenic mice carrying HIV proviral DNA.
In: First, N.L., Haseltine, F.P. (Eds.), Transgenic Animals. Butterworth, Boston, Heinemann, pp. 213 226.
Piedrahita, J.A., Moore, K., Oetama, B., Lee, C.K., Scales, N., Ramsoondar, J., Bazer, F.W., Ott, T., 1998.
Generation of transgenic porcine chimera using primordial germ cell-derived colonies. Biol. Reprod. 58, 1321
1329.
Piper, L.E., Bindon, B.M., Davis, G.H., 1985. The single inheritance of the high litter size of the Borolla Merino.
In: Land, R.B., Robinson, D.W. (Eds.), Genetics of Reproduction in Sheep. Butterworths, London, pp. 115 125.
Polejaeva, I.A., Chen, S.H., Vaught, T.D., Page, R.L., Mullins, J., Ball, S., Dai, Y., Boone, J., Walker, S., Ayares,
D., Coleman, A., Campbell, K.H.S., 2000. Cloned pigs produced by nuclear transfer from adult somatic cells.
Nature 407, 505 509.
Polge, E.J.C., Barton, S.C., Surani, M.H.A., Miller, J.R., Wagner, T., Elsome, K., Davis, A.J., Goode, J.A.,
Foxroft, G.R., Heap, R.B., 1989. Induced expression of a bovine growth hormone construct in transgenic pigs.
In: Heap, R.B., Prosser, C.G., Lamming, G.E. (Eds.), Biotechnology of Growth Regulation. Butterworths,
London, pp. 189 199.
Powell, B.C., Walker, S.K., Bawden, C.S., Sivaprasad, A.V., Rogers, G.E., 1994. Transgenic sheep and wool
growth: possibilities and current status. Reprod. Fertil. Dev. 6 (5), 615 623.
Puchalski, R.B., Fahl, W.E., 1992. Gene transfer by electroporation, lipofection, and DEAE-dextran transfection:
compatibility with cell-sorting by flow cytometry. Cytometry 13, 23 30.
Pursel, V.G., Rexroad Jr., C.E., 1993. Status of research with transgenic farm animals. J. Anim. Sci. 71 (Suppl.
3), 10 19.
Pursel, V.G., Rexroad Jr., C.E., Bolt, D.J., Miller, K.F., Wall, R.J., Hammer, R.E., Pinkert, C.A., Palmiter, R.D.,
Brinster, R.L., 1987. Progress on gene transfer in farm animals. Vet. Immunol. Immunopathol. 17, 303 312.
Pursel, V.G., Pinkert, C.A., Miller, K.F., Bolt, D.J., Cambell, R.G., Palmiter, R.D., Brinster, R.L., Hammer, R.E.,
1989. Genetic engineering of livestock. Science 244, 1281 1288.
Pursel, V.G., Hammer, R.E., Bolt, D.J., Palmiter, R.D., Brinster, R.L., 1990. Integration, expression and germ-line
transmission of growth-related genes in pigs. J. Reprod. Fertil. 41 (Suppl.), 77 87.
Pursel, V.G., Sutrave, P., Wall, R.J., Kelly, A.M., Hughes, S.H., 1992. Transfer of c-ski gene into swine to enhance
muscle development. Theriogenology 37, 278 (abstract).
Quaife, C.J., Pinkert, C.A., Ornitz, D.M., Palmiter, R.D., Brinster, R.L., 1987. Pancreatic neoplasia induced by
ras expression in acinar cells of transgenic mice. Cell 48, 1023 1034.
Reiss, M., Jastreboff, M.M., Bertino, J.R., Narayanan, R., 1986. DNA-mediated gene transfer into epidermal cells
using electroporation. Biochem. Biophys. Res. Commun. 137, 244 249.
Resnick, J.L., Bixler, L.S., Cheng, L., Donovan, P.J., 1992. Long-term proliferation of mouse primordial germ
cells in culture. Nature 359, 550 551.
Rexroad Jr., C.E., Hammer, R.E., Bolt, D.J., Mayo, K.M., Frohman, L.A., Palmiter, R.D., Brinster, R.L., 1989.
Production of transgenic sheep with growth regulating genes. Mol. Reprod. Dev. 1, 164 169.
Rexroad Jr., C.E., Mayo, K.M., Bolt, D.J., Elsasser, T.H., Miller, K.F., Behringer, R.R., Palmiter, R.D., Brinster,
R.L., 1991. Transferrin- and albumin-derected expression of growth-related peptides in transgenic sheep. J.
Anim. Sci. 69, 2995 3004.
Robertson, E., Bradley, A., Kuehn, M., Evans, M., 1986. Germ-line transmission of genes introduced into cultured
pluripotential cells by retroviral vector. Nature 323, 445 448.
288 M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289
Rothschild, M.F., Jacobson, C., Vaske, D.A., Tuggle, C.K., Short, T.H., Sasaki, S., Eckardt, G.R., McLaren, D.G.,
1994. A major gene for litter size in pigs. In: Proceedings of the Fifth World Congress Genet Applied to
Livestock Production, Guelph, Canada, vol. 21, pp. 225 228.
Rottmann, O.J., Antes, R., Hoefer, P., Maierhofer, G., 1992. Liposome mediated gene transfer via spermatozoa
into avian egg cells. J. Anim. Breed Genet. 109, 64 70.
Rund, L.A., Bleck, G.T., Izard, M.M., Grum, L.R., Gerfen. R.W., White, B.R., Wheeler, M.B., 1996. Development
of swine embryonic stem (ES) cells. In: Tumbleson, M.E., Schook, L.B. (Eds.), Advances in Swine in
Biomedical Research. Plenum Press, New York, pp. 207 222.
Ryan, T.M., Ciavatta, D.J., Townes, T.M., 1997. Knockout-transgenic mouse model of sickle cell disease. Science
278, 873 876.
Sanford, J.C., 1988. The biolistic process. TIBTECH 6, 299 302.
Sanford, J.C., DeVit, M.J., Russel, J.A., Smith, F.D., Harpending, P.R., Roy, M.K., Johnston, S.A., 1991. An
improved, helium-driven biolistic device. Tech. J. Meth. Cell Mol. Biol. 3, 3 16.
Schellander, K., Peli, J., Schmall, F., Brem, G., 1995. Artificial insemination in cattle with DNA-treated sperm.
Anim. Biotechnol. 6, 41 50.
Seamark, R.E., 1987. Potential of transgenic pigs and related technology for the pig industry. In: Barnett, J.L.,
Batterham, E.S., Cronin, G.M., Hansen, C., Hemsworth, P.H., Hennessy, D.P., Hughes, P.E., Johnston, N.E.,
King, R.H. (Eds.), Manipulating Pig Production. VIP Printing Pty. Ltd., Australia, pp. 165 170.
Seidel, G.E., 1999. The future of transgenic farm animals. In: Murray, J.D., Anderson, G.B., Oberbauer, A.M.,
Mc Gloughlin, M.M. (Eds.), Transgenic Animals in Agriculture. CABI Publishing, New York, pp. 269 283.
Shamblott, M.J., Axelman, J., Wang, S., Bugg, E.M., Littlefield, J.W., Donovan, P.J., Blumenthal, P.D., Huggins,
G.R., Gearhart, J.D., 1998. Derivation of pluripotent stem cells from cultured human primordial germ cells.
Proc. Natl. Acad. Sci. USA 95, 13726 13731.
Shim, H., Gutierrez-Adan, A., Chen, L.R., BonDurant, R.H., Behboodi, E., Anderson, G.B., 1997. Isolation of
pluripotent stem cells from cultured porcine primordial germ cells. Biol. Reprod. 57, 1089 1095.
Shin, T., Adams, L.G., Templeton, J.W., Westhusin, M.E., 1999. Nuclear transfer using somatic cell line derived
from a bull genetically resistance to brucellosis. Transgenic Res. 8, 488 (abstract).
Simons, J.P., McClenaghan, M., Clark, A.J., 1987. Alteration of the quality of milk by expression of sheep
beta-lactoglobulin in transgenic mice. Nature 328, 530 532.
Snowder, G.D., Busboom, J.R., Cockett, N.E., Hendrix, F., Mendenhall, V.T., 1994. Effect of the Callipyge gene
on lamb growth and carcass characteristics. In: Proceedings of the Fifth World Congress Genet Applied to
Livestock Production, Guelph, Canada, vol. 18, pp. 51 54.
Sorrentino, V., Pepperkok, R., Davis, R.L., Ansorge, W., Phillipson, L., 1990. Cell proliferation inhibited by
MyoD1 independently of myogenic differentiation. Nature 345, 813 814.
Sperandio, S., Lulli, V., Bacci, M.L., Forni, M., Maidone, B., Spadafora, C., Lavitrano, M., 1996. Sperm-mediated
DNA transfer in bovine and swine species. Anim. Biotechnol. 7, 59 77.
Staeheli, P., Haller, O., Boll, W., Lindemann, N.J., Weismann, C., 1986. Mx protein: constitutive expression in
3T3 cells transformed with cloned MxcDNA confers selective resistance to influenza virus. Cell 44, 147 158.
Storb, U., 1987. Transgenic mice with immunoglobin genes. Annu. Rev. Immunol. 5, 151 174.
Sutrave, P., Kelly, A.M., Hughes, S.H., 1990. ski can cause selective growth of skeletal muscle in transgenic mice.
Genes Dev. 4, 1462 1472.
Swanson, M.E., Martin, M.J., O Donnell, J.K., Hoover, K., Lago, W., Huntress, V., Parsons, C.T., Pinkert, C.A.,
Pilder, S., Logan, J.S., 1992. Production of functional human hemoglobin in transgenic swine. Biotechnology
10, 557 559.
Tur-Kaspa, R., Teicher, L., Levine, B.J., Skoultchi, A.I., Shafritz, D.A., 1986. Use of electroporation to introduce
biologically active foreign genes into primary rat hepatocytes. Mol. Cell Biol. 6, 716 718.
Van Dyke, T., Finlay, C., Levine, A.J., 1985. A comparison of several lines of transgenic mice containing the
SV40 early genes. In: Proceedings of the Cold Spring Harbor Symposium on Quant Biology, USA, vol. 50,
pp. 671 678.
Van Dyke, T., Finlay, C., Miller, D., Marks, J., Lozano, G., Levine, A.J., 1987. Relationship between simian virus
40 large tumor antigen expression and tumor formation in transgenic mice. J. Virol. 61, 2029 2032.
Varmus, H., 1998. Retroviruses. Science 240, 1427 1435.
Velander, W.H., Johnson, J.L., Page, R.L., Russell, C.G., Subramanian, A., Wilkins, T.D., Gwazdauskas, F.C.,
Pittius, C., Drohan, W.N., 1992. High-level of expression of a heterologous protein in the milk of transgenic
swine using the cDNA encoding human protein C. Proc. Natl. Acad. Sci. USA 89, 12003 12007.
M.B. Wheeler et al. / Animal Reproduction Science 79 (2003) 265 289 289
Vise, P.D., Michalska, A.E., Ashuman, R., Lloyd, B., Stone, A.B., Quinn, P., Wells, J.R.E., Seamark, R.R., 1988.
Introduction of a porcine growth hormone fusion gene into transgenic pigs promotes growth. J. Cell Sci. 90,
295 300.
Wagner, T.E., Hoppe, P.C., Jollick, J.D., Scholl, D.R., Hondinka, R.L., Gault, J.B., 1981a. Microinjection of a
rabbit -globin gene in zygotes and its subsequent expression in adult mice and their offspring. Proc. Natl.
Acad. Sci. USA 78, 6376 6380.
Wagner, E.F., Stewart, T.A., Mintz, B., 1981b. The human -globin gene and a functional thymidine kinase gene
in developing mice. Proc. Natl. Acad. Sci. USA 78, 5016 5020.
Wall, R.J., Pursel, V.G., Shamay, A., McKnight, R.A., Pittius, C.W., Henninhausen, L., 1991. High-level synthesis
of a heterologous milk protein in the mammary glands of transgenic swine. Proc. Natl. Acad. Sci. USA 88,
1696 1700.
Wheeler, M.B., 1994. Development and validation of swine embryonic stem cells: a review. Reprod. Fertil. Dev.
6, 563 568.
Wheeler, M.B., Choi, S.J., 1997. Embryonic stem cells and transgenics: recent advances. Arch. Fac. Vet. UFRGS
25, 64 83.
Wheeler, M.B., Rund, L.A., Bleck, G.T., Izard, M.M., Grum, L.R., Davis, A.M., Hunt, E.D., White, B.R., Barnes,
J., 1995. Production of chimeric swine by embryonic stem (ES) cells. Biol. Reprod. 52 (Suppl 1), 319 (abstract).
Whitmer, K.J., Calarco, P.G., 1992. HIV-1 expression during early mammalian development. AIDS 6, 1133 1138.
Wieghart, M., Hoover, J.L., McGrane, M.M., Hanson, R.W., Rottman, F.M., Holtzman, S.H., Wagne, T.E., Pinkert,
C.A., 1990. Production of transgenic pigs harbouring a rat phosphoenolpyruvate carboxykinase-bovine growth
hormone fusion gene. J. Reprod. Fertil. 41 (Suppl.), 89 96.
Wilkie, T.M., Palmiter, R.D., 1987. Analysis of the integrant in Myk-103 transgenic mico in which males fail to
transmit the integrant. Mol. Cell. Biol. 7, 1646 1655.
Wilmut, I., Schneieke, A.E., McWhir, J.M., Kind, A.J., Campbell, K.H.S., 1997. Viable offspring from fetal and
adult mammalian cells. Nature 385, 810 813.
Wobus, A.M., Holzhausen, H., Jakel, P., Schoneich, J., 1984. Characterization of a pluripotent stem cell line
derived from a mouse embryo. Exp. Cell Res. 152, 212 219.
Wright, G., Carver, A., Cottom, D., Reeves, D., Scott, A., Simons, P., Wilmut, I., Garner, I., Colman, A., 1991. High
level of expression of active alpha-1-antitrypsin in the milk of transgenic sheep. Biotechnology 9, 830 834.
Wurst, W., Joyner, A.L., 1993. Production of targeted embryonic stem cells clones. In: Joyner, A.L. (Ed.), Gene
Targeting: A Practical Approach. Oxford University Press, Oxford, pp. 33 61.
Yang, B., Kirby, S., Lewis, J., Detloff, P.J., Maeda, N., Smithies, O., 1995. A mouse model for beta 0-thalassemia.
Proc. Natl. Acad. Sci. USA 92, 11608 11612.
Yang, X., Tian, X.C., Dai, Y., Wang, B., 2000. Transgenic farm animals: applications in agriculture and biomedicine.
Biotechnol. Annu. Rev. 5, 269 292.