Transgenic animals in biomedicine and agriculture: outlook for the future

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.: +1-217-333-2239; fax: +1-217-333-8286. E-mail address: [email protected] (M.B. Wheeler).

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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. Microinjection 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 specialized 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

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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 producing 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 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 membranelike 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

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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 microprojectiles 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 offspring 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 animals. 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. Electrofusion 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

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Table 1 Strategies for producing transgenic animals Gain of function

Loss of function

“Knock-in’s” New expression Over expression Altered regulation Antisense RNA Insertional mutations

“Knock-out’s” Lost expression Over expression Altered regulation Antisense RNA 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 targeted fashion. This strategy relies on the ability of embryonic cells to undergo homologous 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 recombination 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,

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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

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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 immunoglobulin pattern. Further, a functional immune system involves a complex group of cell-to-cell communications. Transgenic animals will enable the study of immune function 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 hypogonadism 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

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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 epidermal 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 transgenic 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 resistance 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, tissues or organs that contain human antigens or proteins for xenotransplantation and other biomedical uses. Examples using swine include development of: pancreatic ␤-cells for insulin 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.

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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

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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 improving 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 function 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 transgenics 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 therapeutic 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

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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 bioactive 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 regulation 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 biological functions in the neonate are obtained through milk included IGF-I, EGF, TGF-␣ and

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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

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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 cholesterol 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 lowered. 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

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lipoprotein lipase. In addition, receptors such as the low-density lipoprotein (LDL) receptor 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 profoundly 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 complete digestion of the dietary phytate phosphorus in addition to reducing phosphorus output by up to 75%. Furthermore, transgenic pigs required almost no inorganic phosphorus supplementation 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 disease 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 possible 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 livestock is the attempt to produce of pigs that are resistant to influenza. Previously, mice

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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 construct. 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 expressed 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 reproductive 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 transgenic sheep containing the appropriate FECB allele could increase fecundity in a number

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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 strategies 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 control 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 beginning 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.

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A very novel approach to produce useful fiber has been recently accomplished using 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 varieties 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 capabilities 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

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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 technology 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) incorporate these desired genes in an appropriately expressed and regulated manner into our

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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.

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