transgenic animals: how they are made and their role in animal production and research

REVIEW

Transgenic animals: how they are made and their role in animal production and research

M DZIADEK Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3052

Introduction Transgenesis is the use of genetic engineering technology to intro-

duce foreign DNA into the genetic make-up (genome) of an animal so that all the cells, including the germ line (sperm and ova), are genetically altered. Transgenic technology was pioneered in laboratory mice (Gordon and Ruddle 1981) and has more recently been applied to rats (Paul era1 1994), rabbits (Hammer etal 1985a), sheep (Hammer etal 1985a), pigs (Hammer ef a1 1985a; Purse1 et a1 1989) and birds (Shuman 1991). The possibility of altering the genome by introducing specific gene sequences stimulated a wealth of research into understanding gene regulation and gene function in the whole animal. The substantial knowledge gained from these studies has been used to generate animal models of human genetic disease and to genetically modify farm animals for agricultural and biotechnological benefits. Transgenic technology has already had a major impact on advances in biomedical research, and further improvements in this approach will undoubtedly see its greater and more successful utilisation in farming and pharmaceutical production. It is thus timely to review the technical advances in transgenic animal production, the various roles of transgenic animals and the ethical issues associated with their production.

Construction of Transgenes Gene Structure

A gene consists of different segments of DNA (Figure 1). Some segments directly code for the specific protein that will be produced from the gene, while other segments have a function in the regulation of expression of the gene. The structural, or coding, part of the gene is formed from a series of DNA segments called exons (expressed sequences) that are separated by non-coding DNA segments called introns or intervening sequences that in most cases have no known function (Figure 1). The exons and introns in each gene are transcribed to give an intermediary product called messenger RNA (mRNA). The introns are then spliced out by specific enzyme com- plexes and only the exon sequences are present in the mature mRNA that is the template for protein synthesis (Figure 1). Not all exons are expressed as protein. Some genes contain only a few exons while others may have more than a hundred. A major regulatory element for each gene is the promoter, which is situated in front of (proximal to) the first exon and controls the initiation of transcription and specifies the rate of transcription (see legend to Figure 1 for defini- tions). The promoter also plays a role in determining the cell types in which the gene will be transcribed and thus the tissue-specificity of expression. Additional elements also regulate gene transcription, and these can be situated upstream or downstream of the gene or within the introns of the gene itself (Figure 1). Individual regulatory elements can confer a positive influence on gene expression and are thus called ‘enhancers’, or can be negative regulators, in which case they are called ‘silencers’. The combination of enhancers and silencers, together with the promoter, specify the rate or level of gene transcription and the tissue-specificity of expression. The number of regulatory elements, their position and relative importance varies from gene to gene. However, while these elements are essential in the control of ‘when’, ‘where’ and ‘how much’ a particular gene is

transcribed, additional regulatory steps determine how much functional protein is produced from a specific gene in a particular tissue. These steps include mRNA splicing, transport and stability of mRNA in the cytoplasm, efficiency of translation into protein, stability of the protein and post-translational modifications necessary for correct protein function. The regulation of gene expression is a highly complex process and the relative importance of each step in this process can be quite different for each gene.

Genetic Engineering Genetic engineering is, in simple terms, the ‘cutting’ and ‘pasting’

of different DNA segments to produce new combinations of regulatory and structural sequences. Molecular biology techniques are used to cut genes into pieces using restriction enzymes and to reassemble DNA segments using enzymes called ligases. Specific sequences of DNA can be artificially synthesised and specific mutations can be introduced. The technology now exists to create novel gene struc- tures from any animal species and to generate many types of mutations. It is these novel DNA constructs that are introduced into embryos to generate new or aberrant pathways of protein synthesis in specific cell types. The types of genetic constructs engineered for transgenesis include:

Attachment of a stronger promoter to the structural gene to increase the level of transcription. Replacing the normal tissue-specific regulatory sequences with ones that redirect gene expression to a different tissue (for example, targetting gene expression to mammary glands so that the protein product of the gene can be obtained by milking the animal) . Introduction of specific mutations (for example, deletions, single base changes) within the structural part of the gene to produce aberrant protein (for example, animal models of human genetic disease). Introduction of DNA constructs with end regions that are identi- cal (homologous) to intron segments of the gene of interest but in which new DNA sequences are inserted into an exon or exons are replaced by different gene sequences. Recombination between the construct and the homologous regions of the endogenous gene results in the endogenous gene being replaced by the gene construct (called a targetting construct) and produces a functional ‘knockout’ of the gene. This generates a ‘null’ mutation in which no functional protein for the gene of interest is produced. Such transgenic animals are used to study the functional importance of individual genes in tissue development, function and pathology, and to generate models of human genetic disease.

Production of Transgenic Animals Pronuclear Injection

The most common procedure for producing transgenic animals is by microinjection of foreign DNA (the transgene) into one of the pronuclei of a recently fertilised egg (Gordon 1993). A very small quantity of a solution containing the purified DNA (lo-’ mL) is injected into each egg using a fine glass pipette attached to a micro-

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upstream regulatory region structural regionlcoding region downstream regulatory region I I

Figure 1 . Diagrammatic representation of the different elements present within a gene. The structural part of the gene that codes for a specific protein product contains exons (open boxes, 1,2 ,3) that are separated by intervening sequences or introns. Expression of the gene is regulated by the promoter (black box) that is situated in front of the first exon. Other regulatory elements are the enhancers or silencers (shaded boxes, US) that can be situated upstream of the structural part of the gene (US1, E/S2), downstream of the gene (E/S4) or within an intron (E/S3). The promoter and enhancer/silencer elements control the level of gene expression (how many copies of mRNA are made), when the gene is expressed and the cell types in which the gene is expressed. Transcription refers to the process by which messenger RNA (mRNA) copies are made from the DNA sequence of the gene. Only exons and introns are transcribed into mRNA. The introns are then spliced out by specific enzymes to generate a mature mRNA that contains only exon sequences. These mature mRNA copies are used as a template for the synthesis of the protein that is coded for by the gene. Translation refers to the process by which the RNA sequence is read to produce an amino acid sequence that is specific for each protein.

manipulator. Injected eggs are transferred to a foster mother to allow theembryos to develop to term. All the animals born are tested using DNA diagnostic assays to determine whether they have the transgene incorporated within their genome. The chromosomal site of integration of the transgene is random, and between one and several hundred copies of the transgene can become integrated at one or several sites. Integration of the transgene relies on normal chromosomal breakage and repair and most frequently occurs in the fertilised egg before any cell division takes place. Once integrated into the animal’s genome the transgene is very stable and is replicated and inherited by all cells of the animal in the same way as the normal endogenous genes. The transgene is also present in cells of the germ line (sperm or oocytes) and can thus be stably transmitted to subsequent generations. I f integration of the transgene occurs after the fertilised egg has divided a few times only some parts of the embryo will be transgenic.

The success rate of producing trangenic animals by microinjection is not very high and depends largely on the technical skills of the experimenter (Mann and McMahon 1993). Most mouse studies report transgenesis rates of 10 to 15% of live-born animals, but rates of 25 to 40% are possible (Mann and McMahon 1993). Fewer studies have been done using other species and the success rates are generally much lower. However, in practical terms, the rate of successful transgenic animal production is even lower, since only about 50% of transgenes are actually functional after integration (see later). For all species a large number of fertilised eggs and surrogate females need to be used to generate a few founder transgenic animals for breeding. The time and cost of generating transgenic farm animals in particular is substantial.

Use of Embryonic Stem Cells Embryonic stem (ES) cells are permanent cell lines that are equiva-

lent to undifferentiated cells present in the very early embryo. To date, only mouse ES cells have been produced routinely in a number of laboratories. ES cells are isolated from the inner cell mass (ICM, those cells that form the foetus proper) of the preimplantation embryo at the blastocyst stage of development and can be maintained as a stable cell line in culture (Abbondanzo ef af 1993). Millions of these

cells can be generated in a matter of weeks. ES cells are the precursor (progenitor) cells of all the different cell types that form during development of the foetus, including the gametes. Transgenes can be introduced into ES cells in culture by a number of techniques, including electroporation and the use of viral vectors. Selection procedures are then used to isolate ES cells that have incorporated the transgene into their genome (about 1 in lo4 cells) and stable cell lines can be established from single, genetically modified (transgenic) ES cells. Small numbers of these ES cells are microinjected into blastocyts and these are transferred to foster mice to develop to term. If the injected ES cells become incorporated into the ICM the animals generated by this procedure are chimeric (mosaic), being composed of cells derived from the host blastocyst and from the injected ES cells. If some of the injected cells become incorporated to form those cells in the gonads that produce the gametes, the chimera is called germ-line. When germ-line chimeric animals are mated, any gametes derived from the transgenic ES cells will produce offspring that are entirely transgenic (Stewart 1993). The advantages of this technique over pronuclear injection are that only genetically modified ES cells are used for injection into host blasto- cysts and all ES-derived mice will be transgenic. This technique thus utilises fewer animals to generate transgenic lines, although an additional generation is required to produce animals that are entirely transgenic. This procedure has been used extensively to generate transgenic mice, but has not been used in other species as yet. Recent isolation of rat ES cells (Iannaccone ef a1 1994) will no doubt result in similar applications. However, there have not yet been any sub- stantiated reports of ES cell lines from larger animals (pigs, sheep, cows), even though a substantial effort has been made by many research groups to generate such cell lines. The reasons why ES cell lines are difficult to generate from animals other than rodents is not at all clear, but are likely to be due to species differences in additives required in cell culture to allow cell division and survival of cells isolated from early embryos.

ES cell lines are not only a useful alternative to pronuclear microinjection for genetic modification by random integration of transgenes but are essential for targeted integration of foreign DNA by

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homologous recombination. This procedure involves the construction of a transgene that contains sequences homologous to the specific endogenous gene that is being targeted for mutation (Capec- chi 1989). A recombination event that causes the endogenous gene to be replaced by the transgene occurs at low frequency (1 in I 0’ ), but can be selected for in ES cells by placing selectable marker genes within the transgene construct (for example, genes that confer resistance to antibiotics such as neomycin). This technology has so far only been used in mice and several hundred specific null mutations have now been generated.

Retroviral infection Transgenes can be incorporated into the genome of retroviruses that

are used to infect embryonic cells. Stable integration of retroviral DNA into the mouse genome was in fact one of the first examples of heritable gene modification by exogenous gene insertion (Jaenisch 1976). Gene transfer by retroviral vectors is particularly useful when many cells are to be transfected and it has been used successfully to generate transgenic birds (Shuman 1991). The microinjection technique is not suitable for avian embryos since the fertilised egg has divided to generate 60 000 cells by the time the egg is laid. Retroviral vectors that are able to replicate can produce a high rate of transgenesis since the virus replicates after integration into the genome of individual cells and new viral copies can spread and infect neigh- bowing cells. However, since the transmission of infection between cells and to other animals can have significant pathological consequences, replication-defective retroviruses are generally used for transgenesis. These viruses are unable to replicate after insertion into the genome but remain stably incorporated and the viral and transgene DNA replicate only during the normal processes of DNA replication and cell division in tissues of the transgenic animal.

Purposes for Transgenic Animal Production Agriculture

Farm animals have been subject to genetic modifications for many decades through natural and artificial selection of desirable traits in body growth, wool growth and milk yield. However, any significant progress in genetic gain by selection is very slow. In addition, any advantageous genes in one animal species cannot be transferred to another because of the biological barriers against inter-species breeding. Transgenic technology makes it possible to introduce specific genetic modifications without relying on conventional selection and breeding programmes. The goals in this area are to introduce eco- nomically significant genetic traits into farm animals, such as a more efficient utilisation of feed, leaner meat, faster growth to a marketable size, and immunity to livestock diseases (Ward and Nancarrow 1991). The range of transgenic animals already produced (sheep, goats, pigs, cows) indicates that most major domestic animal species can be genetically modified by transgenesis.

Transgenic pigs have been generated that overexpress bovine growth hormone (GH) or growth hormone releasing factor (GHRF) in order to increase their growth rate. Overproduction of GH in pigs significantly increased their rate of growth and reduced the amount of feed required per unit weight gain (Pursel et a1 1989). These animals had greatly reduced levels of body fat, with significant reduction in lipids and cholesterol, and lowered saturated and un- saturated fatty acids (Solomon et a1 1994). However, these highly positive traits were offset by negative effects that included reduced reproductive performance, arthritis, gastric ulcers, dermatitis, renal disease and premature death (Pursel er a1 1989). Similar effects were seen in transgenic sheep. While sheep had the desired trait of producing lean meat, the undesirable traits included diabetes, impaired renal function, abnormalities in bone growth, large internal organs, and premature death (Rexroad et a1 1990; Nancarrow et a1 1991). Recent studies have shown that long-term elevation of GH in trans-

genic mice also results in a reduced life-span and pathological changes in the liver and kidneys (Wolf el a1 1993).

Transgenesis has also been considered as a potential strategy for introducing changes to milk composition, but little progress has been made in this area. Milk contains a mixture of proteins and lipid in ratios that vary between different breeds of dairy cattle (Gibson et a1 1991). The even more significant differences in milk composition between species reinforces the fact that genetic differences influence milk composition. A higher protein : lipid ratio in milk would be advantageous to diet-conscious consumers and could be achieved by increasing the relative expression of milk proteins such as casein in transgenic animals. Researchers at the Victorian Institute of Animal Science in Melbourne are investigating this possibility in collabora- tion with scientists at the Monash University and the University of New South Wales, with support from the Australian Dairy Industry. An alternative strategy could be the partial inhibition of lipid biosynthesis in mammary glands by the manipulation of genes in this pathway. However, changes in fat composition that may improve the quality of milk for direct consumption may be detrimental to the quality of milk products such as cheese (Gibson 1991). I t may be necessary to generate specific transgenic dairy cattle strains that produce milk to be used for specific milk products and therefore the feasibility of this within the dairy cattle industry would need to be evaluated.

A number of studies are being performed to genetically modify production of textile fibres such as wool, cashmere and angora. Wool growth is dependent on the levels of cysteine, an amino acid that is not normally synthesised by animal cells, but must be obtained from the diet. The transgenic introduction of bacterial genes that allow cysteine to be synthesised from dietary serine in sheep would potentially have a significant positive effect on wool growth (Ward and Nancarrow 1991; Bawden et a1 1999, and this work is ongoing at the CSIRO Division of Animal Production in Blacktown, New South Wales, and the University of Adelaide, South Australia. Two bacterial genes encoding enzymes important for cysteine biosynthesis, cysE and cysK, have been transferred to mice and shown to be expressed in the intestinal tract (Ward et a1 1994). This introduced biosynthetic pathway was fully functional, and able to prevent hair loss in mice on a diet deficient in sulphur-containing amino acids. A similar approach has been tried in sheep but no transgenic animals that express these enzymes in the intestine have yet been produced (Bawden ef a1 1995; CD Nancarrow personal communication). An- other transgenic approach that has been considered is the introduction of genes to increase glucose supply or metabolism within the wool follicles, to promote the synthetic properties of these follicles and hence their growth. Further possibilities include the genetic modification of wool structural proteins (keratins) to alter wool quality, to engineer proteins with increased dye-retention and to introduce proteins into wool that are toxic to moths (Ward and Nancarrow 199 I). Significant economic benefits to the wool industry are likely if these genetic modifications are successful.

One of the most beneficial applications of transgenesis in agriculture would be the introduction of genes to protect livestock from disease. Differential genetic disease-resistance has been recognised to exist in farm animals, and specific retroviral gene products have been identified that confer disease-resistance in mice and chickens (Ikeda and Sugimura 1980; Crittenden and Salter 1990). The Mxl gene (myxovirus-resistant) in mice confers selective resistance to influenza viruses and Mx homologues have now been described in other animals, including humans, cattle, pigs and rats (Muller and Brein 1991). Introduction of the mouse Mxl gene into the pig genome by transgenesis does appear to protect animals against virus infection if a high level of gene expression is achieved (Muller and Breim 1991). Significant advances in this area of transgenic work are expected in the coming years.

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Biotechnology Many human therapeutic proteins are currently being produced in

microorganisms, such as bacteria, with the aid of recombinant DNA technology. However, while these microbial production systems are very cost-effective there are limitations on the types of proteins that can be produced. Bacteria can synthesise the correct protein sequences from human genes, but do not have the necessary machinery to carry out the post-translational modifications that are critical for correct biological activity of the protein. Large-scale animal cell culture systems have been used for this purpose but are very costly to maintain. Transgenic animals are considered to be a viable alternative for large-scale production of bioactive pharmaceutical proteins by targetting transgene expression to the mammary gland such that the protein is secreted into milk. Genes can be targetted to the mammary gland by placing them under the control of regulatory elements from genes that encode milk proteins (for example, p-casein, P-lactoglobulin, whey acidic protein) (Wilmutetal1991; Janne et a1 1994). Human blood clotting factor IX (Clark et a1 1989) and al-antitrypsin (Wright e ta l 1991) have been produced at high levels in sheep milk. In one animal 50% of milk protein was biologically active al-antitrypsin (Wright e ta l 1991). Biologically active human plasminogen activator has been successfully produced in goats milk (Ebert et a1 199 l ) , and pigs have been used to produce protein C with anticoagulant activity (Velander ef a1 1992). While pigs are not traditionally used for milk production they are used for transgenic studies because of their large litter size. However, while several successful transgenic studies have been reported in sheep, goats and pigs, that prove the feasibility of ‘molecular farming’, the level of protein production is generally very low and highly variable. Studies on the expression of a P-lactalbumin transgene in the mouse mammary gland have shown that there can be as much as a 10-fold variation in protein production between mice from the same transgenic line (that is, offspring from one founder animal) and even a 3-fold variation in samples taken from a single lactation in one animal (Bleck and Bremel 1994). Further research is clearly needed to understand the basis for this variation before the successful utilisation of farm animals as ‘bioreactors’ becomes a viable proposition for biotechnological industries. The rapid advances being made in this technology suggest that this scenario may be reasonably close.

Biomedical Research One of the most important applications of transgenic technology in

biomedical research has been the production of animal models of human genetic disease (Smithies 1993; Clarke 1994). Such transgenic animals allow detailed study of the precise molecular mechanisms that contribute to the disease pathology and are also useful for the testing of therapeutic agents that may prevent the onset of the disease, slow its progression, or reduce its symptoms.

Mouse models have now been generated for a large number of human disorders for which the genetic basis is known, including cystic fibrosis (Colledge 1994), osteogenesis imperfecta (Stacey ef a1 1988), sickle cell disorder (Ryan et a1 1990; Greaves ef a1 1990) and P-thalassemia (Shehee et a1 1993). Animal models for genetic disorders such as cystic fibrosis are now proving to be extremely valuable for testing gene therapy techniques to introduce normal copies of the cystic fibrosis gene into affected cell types (Colledge 1994). Transgenic studies have also been important in determining the

roles of candidate genes in human diseases. Studies on transgenic rats have implicated a role for overexpression of components of the renin-angiotensin system in the development of hypertension (Mullins et a1 1990; Ohkubo ef a1 1990; Paul ef a1 1994), while transgenic overexpression of atrial natriuretic peptide causes hypo- tension in mice (Steinhelper et a1 1990). Long-term changes to cardiovascular homeostasis by altered expression of cardiovascular hormones in transgenic animals will provide opportunities for the

early diagnosis, prevention and future therapy of cardiovascular disease.

Neurodegenerative disease is another important group of human disorders that are now starting to be understood as a result of transgenic studies. Overexpression of specific genes such as inter- leukin-6 (IL-6) in the central nervous system is sufficient to produce neurodegeneration, the severity of which correlates directly with levels of IL-6 in the brains of transgenic animals (Campbell et a1 1993). IL-6 levels are elevated in a number of inflammatory, autoim- mune, infection- or trauma-induced disorders of the central nervous system, and the transgenic studies demonstrate that this cytokine can have a direct role in the pathology of neurodegenerative changes in the brain. Overexpression of a mutant B-amyloid peptide in transgenic mice has been shown to cause neurodegenerative changes, and may prove to be a valuable model for Alzheimer’s disease (Games et a1 1995). Another human neurodegenerative disease, Gerstmann- Straussler-Scheinker syndrome, is linked to mutations in the prion protein, a protein implicated in scrapie (Prusiner 1994). Introduction of mutations into the prion protein in mice by transgenesis has demonstrated a direct role for this mutant protein in neurodegeneration (Hsiao er a1 1994).

Perhaps the widest application of transgenic technology has been in the field of cancer research. A variety of oncogenes (cancer-caus- ing genes) from viral and cellular sources have been shown to cause cancer in transgenic mice. These include gastric cancers, leukaemia, neurofibromatosis, mammary cancers, brain tumours, liver tumours and lymphomas (Clarke 1994). These studies demonstrate the causal relationship between genetic mutations and cancer. Cancer arises by a multi-step sequence of genetic and cellular changes, requiring activation of a number of different oncogenes or inactivation of tumour-suppressor genes. Production of doubly transgenic mice for distinct oncogenes, such as ras and myc, has facilitated studies of the cooperation between these genes in tumour progression (Pattengale et a1 1989; Sandgren ef a1 1989). Animals doubly transgenic for an activating oncogene and a null mutation of the p53 tumour-suppressor gene form more aggressive tumours than when each transgene is present alone (Symonds et a1 1994). The treatment of transgenic animals with various chemical carcinogens has been used to determine whether these carcinogens cooperate or interact with particular oncogenic transgenes. For example, overexpression of the N-ras oncogene in transgenic mice predisposes these animals to tu- morigenesis and cooperates with carcinogens such as N-methyl- nitrosourea to generate mammary tumours and lymphomas (Mangues et a1 1994). The impact of nutrition, factors that regulate tissue growth and repair, and age-related factors on tumour forma- tion can also be assessed in animals that carry a transgene that predisposes them to cancer. Transgenic animals thus provide an invaluable resource for elucidation of genetic, physiological and environmental influences on cancer development.

Specific transgenic animal models have also been created for measuring the frequency of genetic mutation that occurs either spontaneously or during specific treatment regimens. Vectors that serve as targets for mutation are introduced into mice by transgenesis. These vectors are constructed so that they can be easily isolated from the host genome (shuttle vectors), and mutations within them are then detected by various assays to measure gene function (Dy- caico e/ a1 1994). These animals are particularly useful for rapidly measuring tissue-specific mutation rates following treatment of animals with mutagens.

Difficulties in obtaining human organs for transplantation has generated interest in the possible use of animal organs (xenografts) for human transplantation. Of critical importance in organ transplantation is the need to overcome hyperacute tissue rejection which results from complement activation on vascular endothelial cells that leads to the occlusion of blood vessels. Several approaches are being undertaken in different laboratories to produce xenografts that have

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been genetically modified to prevent rejection. One approach is to provide donor tissue that expresses membrane-bound complement inhibitor that would protect cells from the effects of complement activation after transplantation. Transgenic mice and pigs have been engineered to highly express human complement inhibitor on the surface of large vessel and capillary endothelia (Fodor et a1 1994). Organs from these animals were shown to be resistant to human complement, and are thus potentially useful for transplantation to humans. The use of organs from transgenic pigs, together with more advanced immunosuppressive treatment, may thus allow successful xenotransplantation into humans.

Basic Research Transgenic animals provide important model systems for elucidat-

ing the mechanisms that regulate gene expression during foetal development and in adult tissues, and have generated significant new information in areas that have been difficult to study using normal animals. Several thousand reports are published annually by researchers worldwide that describe the generation, characterisation and use of transgenic animals.

Transgenic studies have made an important contribution to our understanding of the regulation of gene expression, as has been described above. For example, the presence of regulatory elements within introns was discovered by transgenic studies (Brinster et a1 1988; Palmiter et a1 1991). The promoter, enhancer and silencer elements within the regulatory regions of many genes have now been identified and characterised using transgenic technology. A range of promoters has been characterised that control tissue-specificity of gene expression (for example, in kidney, liver, brain, blood, mammary gland), and these promoters are now widely used in transgenic studies to direct the synthesis of other proteins to a tissue of interest (Koretsky 1992).

One of the most extensive uses of transgenic technology has been in studies of embryonic development to analyse the genetic processes that control the conversion of a single fertilised egg to a complex multicellular organism. Transgenic studies have helped to construct a detailed map of developmental genes that has had a major impact in developmental biology. Mutations generated by transgenesis have demonstrated the roles of specific genes in embryo survival, growth, and the development of malformations. Transgenic experiments have shown the role of specific genes in diverse developmental processes such as sex determination (Koopman et a1 1991), lung development and maturation (Glasser et a1 1994), differentiation of hair follicles (Rogers and Powell 1993) and brain development (Takahashi et a1 1994). A combination of transgenic approaches to overexpress, eliminate expression, or aberrantly express growth factors have identified the roles of individual growth-controlling genes such as growth hormone (GH) and insulin-like growth factors in foetal and neonatal development (Palmiter et a1 1982; Hammer et a1 1985b; De Chiara et a1 1990; Carson et a1 1993; Liu et a1 1993; Wolf e f a1 1993, 1994).

Undesired Effects of Transgenesis Unexpected, or undesired, effects of transgenesis in laboratory or

farm animals are due to (a) an incomplete understanding of regulatory elements that are required for normal patterns of expression, (b) effects on transgene expression that depend on the site of transgene integration, and (c) incomplete knowledge of all the physiological functions of specific gene products. Further research in all of these areas is important if genetic modification of livestock is to be feasible in both economic and ethical respects.

Transgenic mouse studies have demonstrated that the site of transgene insertion has a major influence on transgene expression. If a transgene inserts into a region of the genome where it comes under the control of very strong enhancer or silencer elements from an endogenous gene, these regulatory elements will override those situated within the transgene itself and will have a significant effect

on the levels of transgene expression. These strong endogenous elements may also drive expression of the transgene in an inappro- priate cell type which may have developmental or pathological consequences. In about 10% of transgenic mice the transgene is integrated within an endogenous gene (Gridley et a1 1987). Altered expression of the endogenous gene, by this ‘insertion mutation’ can cause indirect phenotypic effects that are independent of transgene expression. In other cases the transgene can remain totally silent by having been inserted into a region of the genome that is transcrip- tionally inactive. These regions influence the conformation of the transgene such that it also becomes inactivate. Production of animals by pronuclear injection or transfection of ES cells is a random process in which neither the site of transgene integration nor the number of transgene copies can be controlled by the experimenter. Improvements in transgenic technology need to be made to improve the efficiency of transgenesis and ensure that single copies of the transgene integrate into sites that will allow the correct level of tissue-specific expression of the transgene. Progress has recently been made in targeting of transgenes by using site-specific recombination systems (Chambers 1994), which may overcome some of the problems described above. In addition, the introduction of transgenes into animal cells using very large DNA constructs, such as in yeast artificial chromosomes (Peterson et a1 1993; Jakobovits et a1 1993), may insulate the transgene from any effects of the chromatin at the insertion site and should allow correct transgene expression. Further knowledge of the regulation of transgenes in transgenic animals is clearly necessary to achieve maximal control over the outcome of genetic manipulation.

The results from transgenic studies to improve traits in livestock (for example, GH transgenics to improve growth) demonstrate that significant improvements in productivity are frequently associated with detrimental effects that lead to lowered overall performance. Further research is required to understand what levels of transgene product will not disturb physiological properties that are normally delicately balanced in the animal. The combined efforts of physiolo- gists and molecular biologists are required to understand what modifications to an animal’s metabolism will not compromise its health. The long-term benefits and risks of transgenesis should be very carefully evaluated. The impact of any negative effects on the welfare of animals needs to be carefully balanced against the potential positive benefits (for example, medical advances, economic benefits) and clear guidelines need to be established concerning the acceptability of maintaining and breeding such animals.

Care of Transgenic Animals The difference between transgenic animals and other genetic mu-

tations is that transgenic animals are deliberately created by insertion of known DNA sequences that may be from the same species or a different species, while other mutations arise spontaneously or occur after chemical or radiation mutagenesis. These latter mutations are random deletions, single base changes, or rearrangements of the endogenous genome. In principle, transgenic animals can be viewed as a specific class of ‘man-made’ mutations. They are distinguished from other mutations only by the nature of their creation and the variety of possible genetic changes that are introduced. However, the range of possible phenotypic effects is as extensive in transgenic animals as in non-transgenic mutations. From an animal welfare perspective transgenic animals do not require any special care that does not also apply to other genetic strains that have their health compromised as a result of a specific mutation. In some instances the transgenic insert or the gene knockout causes immunodeficiency, and these animals require housing in a barrier facility to prevent losses due to infection. Transgenic animals created to simulate disease states or malformations must be monitored closely and not be allowed to suffer undue pain or stress. Considerations of animal ethics are particularly pertinent, especially since the phenotypic effects of many transgenic experiments cannot be predicted.

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A considerable amount of time, money and number of animals are used to produce transgenic animals and each transgenic founder animal is extremely valuable. These animals therefore require the level of care that is appropriate for any valuable genetic strain that is hard to obtain. Particular care should be taken to protect these strains from losses due to disease, reproductive failure or any accidental losses. Techniques for embryo cryopreservation have been devel- oped for mice and several farm animals (cows, sheep, goats) and are a useful way to preserve transgenic strains.

Widespread community concern exists about the desirability, safety and long-term utility of transgenic animals, particularly those generated for agricultural and biotechnological purposes to provide products for eventual human consumption or direct clinical usage. Transgenic laboratory and farm animals are required to be kept in secure environments, as recommended by the Genetic Manipulation Advisory Committee, a committee established by the Federal Gov- ernment in 1987 to review and oversee all research and development using genetic manipulation techniques. This committee reviews all applications of transgenic animal technology, whether basic research, medical research, agriculture or biotechnology.

Communication between the public and scientists involved in transgenic technology is important to provide the community with information about developments in this area and to discuss the particular concerns that genetic manipulation raises. These discus- sions are critical if transgenic technology is to receive general acceptance within the community, particularly by those many per- sons who stand to gain from advances in medical research, agriculture and biotechnology that such studies are providing.

Acknowledgments I am very grateful to Ian Lewis and David Hopkins for their

comments on this paper from a veterinary perspective, and to Drs Chris Langford (Victorian Institute of Animal Production) and Colin Nancarrow (CSIRO Division of Animal Production, Blacktown, NSW) for discussing their current unpublished research.

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transgenic animals: how they are made and their role in animal production and research

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