Transgenesis may affect farm animal welfare: A case for systematic risk assessment C. G. Van Reenen1, T. H. E. Meuwissen, H. Hopster, K. Oldenbroek, Th. A. M. Kruip, and H. J. Blokhuis Institute for Animal Science and Health (ID-Lelystad), Division of Animal Sciences, 8200 AB Lelystad, The Netherlands
ABSTRACT: This paper considers (potentially) harmful consequences of transgenesis for farm animal welfare and examines the strategy of studying health and welfare of transgenic farm animals. Evidence is discussed showing that treatments imposed in the context of farm animal transgenesis are by no means biologically neutral and may compromise animal health and welfare. Factors posing a risk for the welfare of transgenic farm animals include integration of a transgene within an endogenous gene with possible loss of host gene function (insertional mutations), inappropriate transgene expression and exposure of the host to biologically active transgene-derived proteins, and in vitro reproductive technologies employed in the process of generating transgenic farm animals that may result in an increased incidence of difficult parturition and fetal and neonatal losses and the development of unusually large or otherwise abnormal offspring (large offspring syndrome). Critical components of a scheme for evaluating welfare of transgenic farm animals are identified, related to specific characteristics of transgenic animals and to factors that may interact with the effects of transgenesis. The feasibility of an
evaluation of welfare of transgenic farm animals in practice is addressed against the background of the objectives and conditions of three successive stages in a long-term transgenic program. Concrete steps with regard to breeding and testing of transgenic farm animals are presented, considering three technologies to generate transgenic founders: microinjection, electroporation and nuclear transfer, and gene targeting including gene knockout. The proposed steps allow for unbiased estimations of the essential treatment effects, including hemi- and homozygous transgene effects as well as effects of in vitro reproductive technologies. It is suggested that the implementation of appropriate breeding and testing procedures should be accompanied by the use of a comprehensive welfare protocol, specifying which parameters to monitor, at which stages of the life of a farm animal, and in how many animals. Some prerequisites and ideas for such a protocol are given. It is anticipated that systematic research into the welfare of farm animals involved in transgenesis will facilitate the use of the safest experimental protocols as well as the selection and propagation of the healthiest animals and, thereby, enable technological progress that could be ethically justified.
Key Words: Animal Welfare, Farming, Risk Assessment, Transgenic Animals 2001 American Society of Animal Science. All rights reserved.
Introduction A transgenic (or genetically modified) animal is one whose genome contains DNA of exogenous origin that has been introduced through experimental manipulation (Jacenko, 1997). Since the early 1980s, when for the first time transgenic mice were generated, research with transgenic animals has evolved into a major scientific field. During the last 15 yr transgenesis has been
Correspondence: P.O.Box 65 (phone: +31 320 23 82 03; fax: +31 320 23 80 94; E-mail:
[email protected]). Received October 2, 2000. Accepted March 26, 2001. 1
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extended to livestock, with the aim of benefiting human health, but also to improve animal production (Pinkert and Murray, 1999). At the same time there is growing concern about health and welfare of the animals involved in transgenesis research (e.g., Masood, 1997; Mench, 1999). However, few studies have been conducted to evaluate the welfare of transgenic farm animals. Moreover, although scientists or companies directly involved in the production of transgenic farm animals have recognized the importance of animal welfare (e.g., Pinkert and Murray, 1999; Pharming, 2000), they generally fail to specify concrete steps to monitor and prevent possibly adverse effects of transgenesis on animal health and welfare (see also Klotzko, 1998). We argue that opinions on welfare of transgenic farm animals should be based on relevant biological data
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obtained in scientifically valid experimental setups. In this paper we will discuss (potential) welfare problems in farm animals associated with transgenic technologies, and we will examine the strategy of studying health and welfare of transgenic farm animals. We first briefly describe the experimental methods that are currently employed to create transgenic farm animals and summarize the most important applications of transgenesis in farm animals.
tions in livestock which may, eventually, be as powerful as ES cell-mediated gene transfer in mice (Wilmut et al., 1999; Piedrahita, 2000). One important characteristic shared by various technologies is the fact that transgenic animals resulting from the actual manipulation of early embryos (socalled founders or zero-generation transgenic animals) generally carry exogenous DNA at a single site in one of the host chromosomes, giving so-called hemizygous (TO) transgenic individuals (Wells and Wall, 1999).
Technology to Generate Transgenic Livestock Applications of Transgenesis in Farm Animals For the production of transgenic livestock, the following two technologies are most relevant.
Current applications of transgenesis in farm animals fall into three categories.
Pronuclear Microinjection With pronuclear microinjection, multiple copies of a foreign gene (transgene or DNA construct) are introduced into the pronucleus of fertilized eggs (Jacenko, 1997). Thus far, this method has created the majority of transgenic farm animals. Generating transgenic cattle and sheep with microinjection typically involves a series of successive in vitro manipulations, including collection and in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) prior to microinjection, in vitro embryo culture (IVC) following microinjection, and transfer of embryos (ET) to recipients (e.g., Krimpenfort et al., 1991; Eyestone, 1999).
Nuclear Transfer Nuclear transfer is the most recently developed technology to generate transgenic farm animals. So far, transgenic sheep (Schnieke et al., 1997), calves (Cibelli et al., 1998; Brink et al., 2000), and goats (Keefer et al., 2001) have been produced using nuclear transfer. The starting point is cultured cells, derived from fetal fibroblasts, that are genetically modified in vitro by electroporation. A nucleus from a modified fetal cell is introduced into an enucleated oocyte and fused to the recipient cytoplasm. The reconstructed oocyte obtains early embryonic properties and is transferred to a recipient after a period of IVC. The latest breakthrough in nuclear transfer-mediated genetic modification is the first-time generation of gene-targeted transgenic sheep, carrying a transgene construct at the same predetermined site of the genome (McCreath et al., 2000). Transgene insertion was achieved via homologous recombination in fetal fibroblasts. Homologous recombination is used to manipulate chromosomal DNA in mouse embryonic stem (ES) cells and allows for the introduction of precise changes into specific sites of the genome, including selective deletion of genes (gene knockout), as opposed to all other technologies, which result in random integration of transgenes (Jasin et al., 1996; Pati et al., 1998). Homologous recombination in fetal cells in combination with nuclear transfer is believed to provide a route for precise genetic modifica-
Transgenic Animals as Bioreactors This application of farm animal transgenesis aims at the production of heterologous biomedical proteins in the mammary gland (Wall, 1999). By using transgenes that contain regulatory sequences of milk protein genes, tissue-specific expression of the transgene in mammary tissue should be obtained, with the foreign proteins entering the milk. Harvesting the desired protein involves the collection and subsequent purification of the milk. Up to the point where expression of the transgene in the mammary gland has been achieved, this approach has been successfully applied in rabbits (Bijvoet et al., 1999), pigs (Bleck et al., 1998), sheep (Carver et al., 1993), goats (Ebert et al., 1994) and dairy cows (Eyestone, 1999; Brink et al., 2000). Moreover, transgene-derived products obtained in this manner have recently entered clinical trials in humans (see Pharming, 2000; PPL Therapeutics, 2000). In another approach, the desired therapeutical protein is produced in bodily fluids other than milk, for example blood or urine (Lubon, 1998). This approach has been followed in pigs for the production of human hemoglobin (Sharma et al., 1994).
Transgenic Animals as Donors of Xenografts This application of transgenesis technology has the objective of generating transgenic animals genetically modified in such a manner that their organs (so-called xenografts, e.g., heart or kidneys) can be used for transplantation to humans (White and Langford, 1998). One of the key elements in the suggested strategy is the prevention, in human recipients, of rejection of the xenograft resulting from the activation of complement factors belonging to the human immune system. Transgenic pigs have been produced expressing genes that encode for human complementary inhibitory factors (e.g., Rosengard et al., 1995). Other potential strategies that still await practical implementation involve the generation of transgenic pigs lacking specific genes that are responsible for the presence of antigenic epitopes.
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Table 1. Factors that may affect the welfare of transgenic farm animals Factor
Actual or potential welfare hazard
Insertional mutations
Random transgene insertion Loss of host gene function
Transgene expression
Lack of control of transgene expression (level, site, developmental control, temporal control) Exposure of host to biologically active transgene-derived proteins Perturbation of homeostasis
In vitro reproductive biotechnologies
Disturbance of regulation of early gene expression Large offspring syndrome (LOS)
Transgenic Animals with Improved Production Traits The most important livestock production traits that are currently subject to manipulation by transgenesis technology are 1) growth and body composition, 2) quality of wool production, and 3) disease resistance. Improving growth and body composition has been attempted by the introduction into the genome of pigs and sheep of fusion genes that encode for various growthpromoting substances such as growth hormone or IGF-I (Pursel and Rexroad, 1993; Pursel et al., 1999). Several lines of transgenic sheep harboring different transgenes have been produced with the aim of improving wool quality. Genes of particular interest encode for biochemical substrates critical in the synthesis of wool, including wool keratin and enzymes required for the synthesis of cysteine and IGF-I (Powell et al., 1994; Damak et al., 1996; Ward et al., 1999). Transgenic approaches to increase disease resistance of farm animals are still at a preliminary stage (Mu¨ller and Brem, 1998). Suggested strategies are based on the introduction into the germline of genes that encode for various factors modulating the immune response (e.g., cytokines, in particular interferons), congenital immunization by transgenic expression of genes encoding for specific immunoglobulins or for other (cell-bound) proteins that are able to interfere with pathogen replication, and the introduction of specific disease resistance genes.
Consequences of Transgenesis for Animal Welfare On both empirical and theoretical grounds, three separate sets of factors can be distinguished, that, potentially, may affect the welfare of transgenic farm animals (see Table 1).
Insertional Mutations Microinjection of DNA constructs can result in the integration of foreign DNA within or close to an endogenous gene, thereby creating a new, so-called insertional mutation, with a resultant loss of host gene function. Depending on the function of the disturbed gene, this may harm animal welfare. Estimates of the frequency of insertional mutations among transgenic mice range
from 7 to 20% (Gordon, 1989; Meisler, 1992; Jacenko, 1997). Examples of harmful insertional mutations in transgenic mice have been reported by several authors, some showing that a particular defect was present in all transgenic mice, which points to a dominant mutation (e.g., Weiher et al., 1990; Ting et al., 1994), and others demonstrating that, in certain transgenic mouse lines, only homozygous descendants (TT) of hemizygous founders exhibited abnormalities, which is consistent with a recessive trait (e.g., McNeish et al., 1988; Woychik et al., 1988; Jones et al., 1993).
Transgene Expression With endogenous genes, strict regulation of gene expression is mediated by regulatory sequences associated with the active gene, in terms of site of expression (cell-type, tissue), developmental and temporal control, and levels of expression. In animal transgenesis, including similar regulatory elements within a gene construct is an attempt to obtain a pattern of expression that mimics that of the endogenous gene (Wells and Wall, 1999). However, many transgenic animal models fail in one or more of the conditions conferring proper transgene expression. For example, transgenes containing milk protein gene-specific promotor sequences and designed to express in the mammary gland of lactating females only have frequently been shown to exhibit expression in inappropriate tissues (so-called ectopic expression), including brain, heart, spleen, kidney, and salivary gland (e.g., Carver et al., 1992; Niemann et al., 1999). In the case of potentially deleterious transgene-derived proteins such as erythropoeitin (EPO) or growth hormone, ectopic expression outside mammary tissue was accompanied by various pathologies consistent with systemic actions of the gene-product (e.g., Limonta et al., 1995; Massoud et al., 1996). Similarly, transgenic pigs and sheep harboring transgenes encoding for biologically active growth-promoting factors suffered from a range of serious, often lethal, pathological conditions caused by chronically elevated levels of circulating recombinant protein produced in many different organs (e.g., Pursel et al., 1989; Rexroad et al., 1990; Pinkert et al., 1994). Studies in mice clearly demonstrate that, in the absence of a rigorous regulation of transgene expression, the production
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of transgene-derived factors modulating the immune response, such as cytokines, may result in profoundly deleterious side effects (Lewis et al., 1993; Fattori et al., 1994). An important reason behind insufficient transgene regulation is related to the methodology most frequently used so far to generate transgenic farm animals (i.e., microinjection). The integration site of microinjected DNA is essentially random. Depending on the genomic region of insertion, regulatory elements of neighboring endogenous genes may override those situated within the transgene itself, which may result in aberrant expression patterns (so-called position effects, or integration site effects), including no expression, an enhanced expression, or ectopic expression in an inappropriate cell-type or tissue. In addition, control elements of the transgene may lack essential sequences necessary for appropriate transgene regulation and expression (Niemann et al., 1999; Wells and Wall, 1999). Gene targeting via homologous recombination is considered a promising strategy to avoid unwanted position effects and insertional mutations (e.g., Wilmut et al., 1999; Robl et al., 1999; Polejaeva and Campbell, 2000), although this has yet to be demonstrated in livestock. Similar to endogenous milk proteins, foreign proteins secreted into milk may also enter the general circulation via leakage from the milk to blood (Carver et al., 1992; Lubon, 1998). Mechanisms underlying this phenomenon may involve paracellular transport across leaked tight junctions or intracellular transport of newly synthesized proteins to the base of mammary epithelial cells and secretion through the basal membrane (McFadden et al., 1987; Devinoy et al, 1995). Thus, transgenic dairy animals as bioreactors may be systemically exposed to transgene-derived proteins irrespective of proper transgene regulation. Particularly when high transgene expression levels in milk are obtained (multiple mg/mL), relatively high plasma levels (multiple ng/mL) are feasible. Depending on the biological properties and function of the protein, such levels may well be outside a safe and physiologically acceptable range in terms of animal welfare. A more fundamental criticism of transgenesis in farm animals not only considers potentially deleterious effects of individual gene-products as such (e.g., growth hormones or cytokines), but also questions the basic assumptions taken, particularly the idea that sophisticated biological functions such as growth or immunity can be improved by manipulating single loci in isolation (e.g., Regal, 1994; Gordon, 1999). Such an approach may not sufficiently take into consideration that many (if not all) biologically important processes rely on many different components, mediated by a multitude of genes, and operating in a complex regulatory setting designed to maintain homeostasis based on intricate cross-relations within and between different physiological systems, including the endocrine system, the immune system, and the nervous system (see, for example, Elsasser et al., 1997; Johnson et al., 1997; Davis, 1998,
for discussions on neuroendocrine-immune interactions modulating disease resistance, metabolism, and growth). Therefore, as pointed out by several authors (e.g., Gordon, 1999; Seidel, 1999; Ward et al., 1999), genetic modification per se may place an animal at risk because of an alteration of an existing delicate balance that has been established through many generations of selection, and that represents a wide range of optimized gene combinations that may be difficult to manipulate without causing unexpected deleterious effects on the phenotype. The available evidence, for example on growth hormone and cytokine transgenes, clearly supports this suggestion. Notably, after thoroughly reviewing numerous studies on transgenic mice expressing growth hormone transgenes, Bartke et al. (1994) concluded that the consequences of prolonged exposure to supraphysiological levels of growth hormone cannot always be predicted from the known or the presumed physiological actions of this hormone. Lubon (1998) argued that unexpected effects, including, for example, premature shut-down of milk production and impaired mammary development after transgenic expression of putatively nondetrimental proteins in milk (e.g., Shamay et al., 1992; Ebert et al., 1994), may be associated with as-yet unknown biological functions of transgenederived proteins, which may become apparent especially when a transgene is expressed in, or the geneproduct is exposed to, tissues other than the tissue of origin.
In Vitro Reproductive Biotechnologies To date, there is a large body of evidence showing that in vitro technologies employed in the process of generating transgenic cattle and sheep (e.g., in vitro fertilization, in vitro embryo culture, and nuclear transfer), relative to in vivo procedures (e.g., artificial insemination and in vivo embryo culture), may result in a host of deleterious side effects commonly known as the large offspring syndrome (LOS), including a high incidence of abortion throughout pregnancy, more congenital malformations, an increased birth weight, a longer gestation period, and a high perinatal mortality rate (e.g., Walker et al., 1996; Kruip and den Daas, 1997; Van Wagtendonk-de Leeuw et al., 2000). This syndrome, however, does not represent a predictable or uniform set of phenomena, and not all of the symptoms described have been observed universally (Young et al., 1998). Postmortem examinations performed on lambs and calves, either stillborn or animals that died postnatally, derived after nuclear transfer or after in vitro fertilization followed by in vitro embryo culture, have revealed a wide range of abnormalities, including incomplete development of the vascular system and the urinogenital tract (Campbell et al., 1996), thymic atrophy and lymphoid hypoplasia, preceded clinically by severe anemia (Renard et al., 1999), and brain lesions (Schmidt et al., 1996). Sinclar et al. (1999) demonstrated significantly increased allometric growth coefficients (i.e., rel-
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ative to body mass) for liver and heart in sheep fetuses derived from in vitro-cultured embryos relative to in vivo controls, evidencing major shifts in patterns of fetal organ and tissue development due to in vitro culture. In a survey by Garry et al. (1996), monitoring postnatal characteristics in a group of 40 live-born calves generated by nuclear transfer and born after Caesarian section, the majority of calves were characterized neonatally as slow or weak and in need of some (clinical) intervention (e.g., medical treatment, respiratory support, or assistance to nurse). Reported data on actual transgenesis experiments fully substantiate the association between in vitro technologies and LOS and have shown high perinatal mortality rates (up to more than 50%), increased birth weights, and severe pre- and postmortem pathologies among transgenic lambs and calves generated by nuclear transfer (Schnieke et al., 1997; Hill et al., 1999; McCreath et al., 2000) and a high incidence of congenital malformations, an increased birth weight, and a high perinatal mortality rate among calves originating from in vitro-produced embryos subjected to microinjection (Van Reenen and Blokhuis, 1997). Mechanisms underlying these effects have not yet been identified, although a plausible, and generally accepted, hypothesis has been raised. It has been postulated that LOS symptoms result from environmentally induced disturbances in the regulation of early gene expression (Kono, 1998; Niemann and Wrenzycki, 2000; Young and Fairburn, 2000). A specific category of genes, the so-called imprinted genes, is assumed to be crucial in this respect, for a number of reasons. First, imprinted genes are critically involved in the control of embryonic, placental, fetal, and neonatal growth. Second, unlike classic genes, imprinted genes are expressed from one of the parental alleles only. The process of differentiating between the active and silent allele, so-called imprinting, is associated with extrinsic, or so-called epigenetic, modifications that alter gene expression in an allele-specific manner, without changing the DNA sequence. The epigenetic modification of imprinted genes most likely involves methylation of DNA (i.e., the attachment of methyl groups), which may either silence or allow gene expression in a tissue-specific manner. Many imprinted genes undergo allele-specific changes in their DNA methylation (i.e., are imprinted) during early embryogenesis, a developmental stage that, in case of the generation of transgenic farm animals, coincides with various in vitro manipulations. Thus, it is suggested that embryo manipulations may interfere with epigenetic modifications of imprinted genes, for example by interacting with or removal of methyl groups, leading to inappropriate gene expression and developmental abnormalities. Such abnormalities are also believed to be implicated in a disturbed communication between manipulated embryos or fetuses and their recipients during pregnancy, and an inappropriate signaling in preparation for birth (Walker et al., 1996; Wells et al., 1999).
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One likely environmental factor that may target imprinted genes during early embryonic development is the medium that is used to culture reconstructed or microinjected embryos before transfer (e.g., Kruip et al., 2000). Nuclear transfer constitutes additional factors that are assumed to be able to detrimentally affect the regulation of imprinted genes. First, donor nuclei transferred to enucleated oocytes have to go through the process of genetic reprogramming, which is the transformation from the pattern of gene expression that is characteristic of the donor cell to one that is appropriate for early embryonic development. This process in itself may be incomplete and result in inappropriate patterns of gene expression. Second, nuclear transfer involves the exposure of reconstructed oocytes to various environmental stimuli intended to facilitate fusion between nucleus and recipient cytoplasm and to promote oocyte activation, for example electric shock or treatment with protein inhibitors (Brower, 1998; Colman, 1999/2000). Such stimuli may also disrupt epigenetic modifications of imprinted genes. The relative contributions of in vitro embryo culture or nuclear transfer to the induction of abnormality remain to be determined (Young and Fairburn, 2000). Theoretically, any procedure at any stage of the sequential process of in vitro embryo production and manipulation may influence embryo development and characteristics of offspring (Van Wagtendonk-de Leeuw et al., 2000). Some essential questions arise in appreciating the ultimate possible consequences of in vitro embryo manipulations for animal welfare. For example, to what extent do phenotypic abnormalities induced by in vitro embryo manipulations persist postnatally, or become apparent later in life? Are such abnormalities heritable in that they may be transmitted to offspring conceived after normal mating? Although it has been suggested that long-term effects of reproductive technologies on farm animal health and welfare may exist (e.g., Wilmut et al., 1998; Van der Lende et al., 2000), there are few published reports on this matter from studies in livestock. Preliminary results on the adult performance of a limited number of selected dairy cattle produced by in vitro reproductive technologies seem to indicate that in vitro embryo production does not negatively affect semen production and semen quality of bulls or reproduction results of heifers (Van Wagtendonk-de Leeuw et al., 2000). However, as mentioned by the authors, these results should be interpreted with caution given the biased nature of the observed sample. It has also been shown that, in addition to a higher weight at birth, calves derived from in vitro-manipulated embryos exhibited normal growth rates and slaughter weights at the age of 13 mo (Wilson et al., 1995; McEvoy et al., 1998) but had significantly enlarged hearts at slaughter (McEvoy et al., 1998) compared with animals derived from embryos produced in vivo. So far, studies in mice have provided the clearest evidence that in vitro embryo manipulations may result in long-lasting phenotypic changes. In a carefully designed experiment by Duli-
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oust et al. (1995), a comparison was made between mice derived from cryopreserved embryos (i.e., freezethawed during preimplantation stages) and control mice in several morphophysiological, sensimotor, and behavioral traits, from birth to senescence. Embryo cryopreservation did not induce major anomalies, but significant differences between mice that developed from cryopreserved embryos and control mice were found in a number of developmental and behavioral features, some of which appeared during adulthood and in senescence. Epigenetic changes due to cryopreservation were proposed as a possible explanation for these results. Reik et al. (1993) produced hybrid mice using a technology similar to nuclear transfer in livestock, in that the female pronucleus of a fertilized oocyte was removed and transferred into a recipient egg from a different genotype. Compared to non-hybrid controls, mice born from reconstructed oocytes unvariably showed an aberrant phenotype with growth deficiency resulting in reduced adult body weight, as well as increased gene methylation and repressed expression in major urinary protein genes in the liver. These latter findings also strongly support the putative link between embryo manipulation, altered epigenetic modification of (an) imprinted gene(s), and an aberrant phenotype. Most importantly, a follow-up study by Roemer et al. (1997) showed that the typical phenotype observed in hybrid mice created by pronuclear transfer, involving growth retardation and altered DNA methylation patterns, was present in the majority of offspring from normal matings between hybrid males and normal females, demonstrating that an aberrant epigenetic modification associated with defective gene regulation was transmitted through the germline. This particular result may have far-reaching implications for the progression and implementation of farm animal biotechnology including transgenesis, because it would suggest that, contrary to established theory that epigenetic modifications are erased during gametogenesis (e.g., Latham, 1999), environmentally induced epigenetic changes, including possibly detrimental ones associated with LOS symptoms, may be transmitted from parent to offspring.
Welfare Research in Transgenic Farm Animals As discussed in previous sections of this paper, there are convincing arguments to support the idea that treatments imposed in the context of farm animal transgenesis are by no means biologically neutral in their effects on animal health and welfare. On the contrary, several treatments seem to directly threaten the preand postnatal survival of transgenic farm animals, and there is every reason to assume that overt pathogenicity and lethality merely represent the very extremes of a wide range of possible detrimental effects of experimental manipulations and phenotypic changes related to transgenesis on animal health and welfare. Therefore, we believe that a substantial effort into the study of welfare of transgenic farm animals is appropriate.
Some methodological and strategical issues with regard to welfare research in transgenic farm animals will be considered below.
Characteristics of Transgenic Animals There are a number of characteristics of transgenic animals that are essential in determining the parameters of a useful scheme for evaluating welfare of transgenic farm animals. These characteristics are the following.
Predictability of Phenotypic Properties of Transgenic Animals is Technology-Dependent. The predictability of phenotypic properties of transgenic animals depends on the technology that is used to create them. Microinjected DNA is integrated in a random fashion with respect to both the number of copies and the site of integration. Therefore, each transgenic founder generated after microinjection is strictly unique in its genetic makeup and in the expression pattern of the transgene (see, for example, comprehensive reports by Pursel et al., 1989 on transgenic pigs or by Carver et al., 1993 on transgenic sheep, evidencing major genetic and phenotypic differences between different founders, and between offspring of different founders, harboring the same gene-construct). Unless targeted insertion of a transgene is accomplished, the same is basically true for founders created by independent cycles of nuclear transfer-mediated transgenesis, because electroporation does not resolve random insertion and its associated random position effects (Wells and Wall, 1999). The crucial methodological implications of random transgene insertion are that each of the resulting transgenic founders is unique in terms of the nature of any defects that may arise from transgenesis and that, therefore, possible deleterious transgenic effects must be evaluated for each transgenic founder separately, case by case (see Table 2). Consequently, the outcome of an evaluation of the welfare of one founder produced by random transgene insertion cannot be used to predict the outcome of the evalution of the welfare of another founder, even if they carry the same transgene. Predictability of founder animal phenotype could be much higher when gene targeting via holomologous recombination would be used to generate transgenic farm animals. Gene targeting as such would not eliminate position effects but is assumed to result in similar position effects and, hence, similar transgene expression patterns among independently generated transgenic founders and their offspring carrying the same transgene on the same site of the genome (e.g., Robl et al., 1999; Polejaeva and Campbell, 2000). Here, the implication would be that an assessment of the welfare of a founder generated by targeted transgenesis may have predictive value for an assesement of the welfare of another, independently generated founder carrying the same targeted gene-construct. Transgenic Status Interacts with Other Factors. The effect of the transgenic status of an animal may signifi-
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Table 2. Essential properties of transgenic founder animals when evaluating effects of transgenesis on animal welfare Technology
Transgene insertion
Phenotypic properties
Microinjection
Random
Unique in each founder
Case by case for each unique founder
Electroporation and nuclear transfer
Random
Each cycle of nuclear transfer-mediated transgenesis creates unique line of founders
Case by case for each unique line of founders
Gene targeting
Specific site
Each cycle of gene targeting creates similar line of founders
In both sexes and in different genetic backgrounds
cantly interact with other sources of variation, including sex and genetic background. For measures related to growth and body composition, significant interactions between transgenic status and sex have been identified in progeny of transgenic founder pigs and sheep carrying growth-promoting transgenes (Ward and Brown, 1998; Pursel et al., 1999). Substantial genotypedependent differences in transgene expression levels and in transgene-associated pathologies have been demonstrated between lines of transgenic mice resulting from crosses of the same transgenic founder or strain with different nontransgenic strains (Keller et al., 1994; Doetschman, 1999). Likewise, phenotypical consequences of identically targeted transgenes or gene knockouts in mice may differ according to the ES cell type used (Donehower et al., 1995; Wells and Wall, 1999). These findings suggest that even with gene targeting, predicting the phenotype of a transgenic animal can still be uncertain, and that effects of transgenes must be evaluated in different genetic backgrounds (see Table 2). Importantly, effects of in vitro embryo manipulations not related to transgene insertion and expression also seem to interact with sex and genotype. Data from several nuclear transfer studies using similar cell lines derived from different bovine embryos as nuclear donors suggest that genotype affects developmental rate to the blastocyst stage and later in vivo development (Stice, 1998a). The effects of mouse embryo cryopreservation on postnatal developmental and behavioral characteristics were influenced both by strain and by sex (Dulioust et al., 1995).
Transgenic Program A program aiming at the generation of transgenic farm animals represents a long-term, step-by-step process, starting with the creation of transgenic founders and ending with the production of a herd of transgenic animals exhibiting the desired trait (e.g., Brink et al., 2000). Each stage in this sequential process poses its own set of conditions and resources available for research into animal welfare, and, correspondingly, its own scope of answers that can be provided. We will summarize some of the prevailing objectives, limita-
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tions, and opportunities of three successive, and partially overlapping, stages in a transgenic program (see Table 3). First-Time Generation of Transgenic Founders. The main objectives at this stage are, first, to innovate and validate experimental procedures and, second, to demonstrate that the production of an improved or novel type of transgenic farm animal is technically feasible. At this stage, the number of transgenic founders generated in the same microinjection or nuclear transfer trial is generally low (e.g., Schnieke et al., 1997; Brink et al., 2000). Unless extreme or clearly anomalous features are consistently found in different founders across different experiments (as in the case of, for example, LOS symptoms such as perinatal death and high birth weights), observations on such animals do not allow for a reliable estimation of effects of transgenesis on animal health and welfare (see also Van Reenen and Blokhuis, 1993). Initial studies may provide valuable information on various molecular biological determinants of transgenesis, including site of integration, integrity of the transgene, transgene expression (level, temporal pattern and tissue-specificity), and characteristics of the transgene product (Kuipers et al., 1997; Brink et al., 2000). These mostly descriptive data, in combination with anecdotal information on animal health and abnormalities, may also serve as an initial screen in the selection or culling of founders.
Increasing the Number of Transgenic Farm Animals. This is the stage at which a dependable protocol to generate transgenic farm animals has been established and increasing numbers of transgenic founders become available. With the objective to more reliably determine transgene expression and phenotypical characteristics of transgenic animals, groups of identical founders (i.e., clones) are generated after nuclear transfer (e.g., Brink et al., 2000), or TO transgenic offspring are produced by mating founders with nontransgenic animals (e.g., Carver et al, 1993; Damak et al., 1996). With gene targeting, this would be the stage to select the most favorable chromosomal site of insertion, conferring the highest level of expression or otherwise desired expression pattern in transgenic founders and their offspring. This is also the appropriate stage to commence comprehensive and quantitative research into transgenic ani-
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mal health and welfare, investigating lines of transgenic clones or progeny. Based on a sufficient number of observations, accurate comparisons can be made between different types of transgenes, between different founders, and perhaps between different experimental protocols that have been used so far to generate transgenic animals (for example, with regard to the prevalence of LOS symptoms). Results of welfare research should support and guide the important decisions on the continuation of the program, for example on the selection or culling of (lines of) founders, or on the definition of a safe level of expression of the transgene.
Establishment of Production Herds, or Dissemination of Transgenes into the Population. Until now, completion of the final stage of a transgenic program has rarely been achieved. The establishment of secluded production herds has been documented for transgenic programs aiming at the production of biomedical proteins in the milk of transgenic sheep (PPL Therapeutics, 2000) or rabbits (Pharming, 2000). So far, the introduction of transgenes into the commercial population is not precedented. Necessary steps in this respect have been examined from an animal breeding point of view with emphasis on population genetic considerations (e.g., Smith et al., 1987; Cundiff et al., 1993). Regardless of the actual strategy in terms of breeding and multiplication of transgenic animals that would be applied at this stage, it is anticipated that large numbers of transgenic farm animals will be produced, including multiple generations of TO or TT transgenic progeny, with different genetic backgrounds and carrying different types of transgenes. Provided that a systematic recording of health- and welfare-related parameters is sustained throughout a transgenic program, the final stage would permit a more complete evaluation of transgene effects, involving the accurate estimation of relatively small effects as well as interactions of transgene effects with genetic background or sex. In addition, using an epidemiological approach, insight should be gained into long-term effects of various reproductive technologies on farm animal health and welfare (Van der Lende et al., 2000). This latter approach should entail not only the (meta-)analyses of data from all stages of the same transgenic program, but also
the use of similar information from other transgenic programs. The final assessment within the context of a specific transgenic program would concern the ultimate choice to maintain and propagate certain lines or breeds of transgenic animals carrying certain types of transgenes. In a more general sense, the knowledge obtained in the course of a transgenic program should allow for evaluating, at least preliminarily, which traits to genetically manipulate, which types of transgenes to insert, and which experimental methods and protocols to use for the generation of transgenic farm animals. Finally, suggestions for changing or improving the approach that was taken should be converted into clear hypotheses that could then be tested in follow-up studies or could give rise to a new program.
Breeding and Testing The strategy of selecting, breeding, and disseminating transgenic livestock has been theoretically explored in detail, on the assumptions that animals carry transgenes intended to genetically improve economic merit and that founders were generated by microinjection (e.g., Smith et al., 1987; Cundiff et al., 1993). Many of the suggested procedures also directly apply to the strategy of evaluating health and welfare of transgenic farm animals. We will discuss concrete steps with regard to breeding and testing of transgenic farm animals and consider the following three situations: a situation in which founders are generated by microinjection, a situation in which founders are created by electroporation and nuclear transfer, and a situation in which founders are created by gene targeting, including gene knockout. Basic assumptions are that all testing should involve the most efficient comparisons between treatment and control groups and that effects of transgenes should be distinguished or separated as much as possible from effects of reproductive technologies used in the process of generating transgenic animals (e.g., nuclear transferor in vitro embryo production). Eventually, the proposed steps should be logically (not necessarily chronologically) incorporated into a transgenic program as discussed earlier. Microinjection. Steps in the breeding and testing of transgenic farm animals generated by microinjection
Table 3. Welfare evaluation in three successive stages of a transgenic program Stage
Objectives
Transgenic animals
Basis for welfare evaluation
1. First-time generation of transgenic founders
Innovate experimental procedures and test feasibility
Low numbers of (unique) founders
Descriptive data and anecdotal information
2. Increasing numbers of animals
Determination of transgene expression and phenotypic properties
Groups of clones or transgenic offspring
Quantitative research and accurate comparisons
3. Establishment of production herds
Utilization of desired animals or harvesting desired (gene) product
Many animals
Epidemiological surveillance and meta-analyses
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Table 4. Steps in the breeding and testing of transgenic farm animals produced by microinjection Creation of founders and breedinga
Testing
G0
Founders T1O, T2O, . . . , TkO Mating TiO × OO
Identify unique characteristics in each individual founder TiO
2
G1
50% TiO and 50% OO Mating TiO × OO
Comparison between TO and OO
3
G2
50% TiO and 50% OO Interbreeding TiO × TiO
Comparison between TO and OO
4
G3
25% TiTi, 50% TiO , 25% OO
Comparisons between TT and TO and between TT and OO
Step
Generation
1
a A hemizygous transgenic founder animal is indicated by TiO, with Ti representing the transgene inserted at a unique and single site in one of the host chromosomes. Nontransgenic animals are indicated by OO. Mating the transgenic founder TiO with nontransgenic animals produces nontransgenic (OO) and hemizygous (TiO) offspring in equal proportions. Homozygous transgenic animals, harboring the same transgene at the same site on both chromosomes within a pair, are indicated by TiTi and are produced by intercrossing of hemizygous transgenic animals.
are outlined in Table 4. With microinjection, unique hemizygous founders (T1O to TkO) are produced at generation 0 (G0). Each individual founder, denoted TiO, is mated with nontransgenic (OO) animals, to produce first-generation (G1) nontransgenic (OO) and hemizygous (TiO) half-sibs in equal proportions, on the assumption of a Mendelian inheritance of the transgene. A first test on the fitness of the transgene (or gametes/ embryos carrying the transgene) is performed by testing whether the ratio of TiO/OO offspring is 50/50. Direct comparison of hemizygous transgenic half-sibs, each carrying the same genetic modification on the same site of the genome, with their half-sib nontransgenic counterparts provides an unbiased estimate of the effect of the transgene. If a founder is female, multiple ovulation and embryo transfer (MOET) could be used to provide multiple offspring, or a transgenic G1 son could be used to give many grandprogeny (Smith et al., 1987). The number of transgenic animals can be increased by the production of subsequent generations (G2, G3, etc.) of TO transgenic offspring, and TT transgenic animals can be produced by intercrossing of hemizygous individuals. By several generations of systematic backcrossing of hemizygous animals to nontransgenic animals (for example, for two generations, see Table 4), before the actual intercrossing, the level of inbreeding in homozygous transgenic animals can be reduced (Gama et al., 1992). For example, an intercross of G2 or G3 parents results in offspring with inbreeding coefficients of 3.1 or 1.5%, respectively. At the intercross stage, the progeny are expected to be 25% TT, 50% TO, and 25% OO. Testing the realized numbers of offspring per genotype against these expected proportions yields a test for the fitness of the transgene in homozygous form. Comparisons between homozygous and hemizygous animals and between homozygous and nontransgenic animals provide the estimation of the homozygous transgene effects. Especially, testing homozygotes allows for the detection of deleterious recessive
insertional mutations. When the production of purebreeding homozygous transgenic stock is an important objective of the breeding program, early detection of reduced viability or lethality among homozygous transgenic animals could be useful, because it would avoid the wasting of evaluation and breeding effort during the backcrossing and subsequent intercrossing phases (Smith et al., 1987; Gama et al., 1992). This could be accomplished by interbreeding already at G1, with matings between hemizygous transgenic half-sibs or between founder and hemizygous transgenic G1 offspring. Interactions of the transgene with genetic background could be examined by introducing different OO lines or breeds into the breeding program. As to the assessment of effects on animal health and welfare of in vitro reproductive technologies, a precise comparison should be made between animals produced according to exactly the same protocol as applied during microinjection trials, but without the actual microinjection (i.e., in vitro oocyte maturation, in vitro fertilization, or in vitro embryo culture and embryo transfer), and the appropriate control animals, produced either after in vivo embryo culture or after AI. Such an experiment could also be part of a more comprehensive series of studies looking into several experimental conditions and factors such as the presence or absence of a specific component(s) of the culture medium (e.g., Walker et al., 1996; Van Wagtendonk-de Leeuw et al., 2000). Electroporation and Nuclear Transfer. Table 5 shows steps in the testing of transgenic farm animals produced by electroporation and nuclear transfer. It is assumed that several independent electroporation-nuclear transfer trials are performed (Trial 1 to Trial k), with the same nuclear donor cells and the same geneconstruct. Within the same nuclear transfer trial, a unique group of several identical hemizygous transgenic founders (clones) of the same sex is produced (n1T1O in Trial 1 to nkTkO in Trial k, with n1 to nk
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Table 5. Steps in the testing of transgenic farm animals produced by electroporation and nuclear transfer Creation of clonesa
Step
Generation
Testing
1
G0
Trial 1: founders n1T1O Trial 2: founders n2T2O ... Trial k: founders nkTkO
Identify unique characteristics in each group of identical founders (clones) niTiO
2
G0
Create control clones: njOO
Comparison between niTiO and njOO
a A group of identical hemizygous transgenic founder animals, created in the same nuclear transfer trial, is indicated by niTiO, with ni the number of individuals (clones).
the respective numbers of identical clones generated in each independent trial). If few identical clones are obtained per trial, or if a similar breeding strategy is aimed at as previously described, including the production of homozygous transgenic animals, the breeding and testing strategy is similar to the one presented in Table 4. In that case, every independently generated group of identical clones (niTiO) has the same status from a methodological point of view as each individual founder after microinjection (TiO). If the key objective of a transgenic program is the creation of an instant herd or flock of identical hemizygous transgenic animals, for example, a herd of only females producing biomedical proteins in their milk (e.g., Brink et al., 2000), and if large numbers of clones are obtained per trial, then steps mentioned in Table 5 would be appropriate. To test for transgene effects, the most efficient comparison would be between each group of identical founders (niTiO) and a control group of identical, but nontransgenic, clones (njOO, with nj the number of clones; see Table 5, step 2) created according to the same nuclear transfer protocol using unmodified cells as nuclear donors. To investigate the effect of nuclear transfer on animal health and welfare, nontransgenic clones should be compared with in vivo-produced control animals. However, comparisons between a group of identical clones (njOO), derived from the same nuclear donor cell line, and any in vivo control group are biased because genetic background is confounded with nuclear transfer. Unbiased comparisons can only be made when many independent groups (i.e., lines) of clones are generated, with each line derived from a different nuclear donor cell line. It should be noted that in a population of cloned individuals created in this manner, the experimental unit is represented by the average performance of a line of clones. Steps in the creation of lines of clones to study side effects of nuclear transfer are given in Table 6. Multiple lines, say k lines, of male and female clones
(n1C1 to nkCk, with n1 to nk the respective numbers of identical clones generated in each line) could be derived from multiple lines of nuclear donor cells L1 to Lk, which were supplied by multiple fetuses F1 to Fk. Natural mating or AI could be used to produce many half-sib or unrelated fetuses F1 to Fk. In this case, the appropriate in vivo counterparts of lines of clones would be halfsibs of fetuses F1 to Fk, or a random sample of in vivo controls from the same population as the parents that provided the fetuses F1 to Fk. Multiple fetuses F1 to Fk could also be obtained from a limited number of females or mating pairs, using MOET. In lines of clones derived from fetuses produced by the same mating pair, individuals across lines are full-sibs. Here, testing multiple lines of clones against control animals produced by the same females or mating pairs that generated the fetuses F1 to Fk provides an unbiased estimate of the effect of nuclear transfer. The generation of adequate numbers of in vivo control animals per female or per mating pair (i.e., full-sibs of lines of clones) will also require the use of MOET. Although MOET involves some embryo manipulation, it lacks in vitro fertilization and in vitro embryo culture and has been shown to produce calf and calving characteristics that are highly similar to those obtained with AI (Van Wagtendonk-de Leeuw et al., 2000). The comparison between nontransgenic clones and proper controls should be repeated with every change of type of nuclear donor cell that is used, and with every other significant change in the experimental nuclear transfer protocol. Gene Targeting. Steps in the breeding and testing of transgenic farm animals produced by gene targeting are given in Table 7. With gene targeting it is assumed that multiple (ni) hemizygous male or female transgenic founders generated in independent nuclear transfer trials either carry the same transgene at the same site of the genome (TiO) or are deficient in one of two alleles of the same endogenous gene Pi, denoted Pi+/−. In addi-
Table 6. Steps in the creation of lines of clones to study side effects of nuclear transfer Step 1 2 3
Reproductive technology
Result
Natural mating, AI, or MOET Cell culture Nuclear transfer and ET
乆, 么 Fetuses F1, F2, . . ., Fk 乆, 么 Nuclear donor cell lines L1, L2, . . ., Lk 乆, 么 Lines of clones n1C1, n2C2, . . ., nkCk
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tion to all procedures previously described (Tables 4 and 5), gene targeting offers the possibility to create lines of homozygous transgenic animals at the earliest possible occasion while completely avoiding inbreeding, by crossing independent male and female lines of identically targeted hemizygous transgenic animals (Table 7, step 2). Homozygous transgenic animals produced in this manner are also expected to be free of defects due to recessive insertional mutations (Robl et al., 1999). Matings between hemizygous transgenic animals are assumed to produce 25% TiTi, 50% TiO, and 25% OO in case of targeted transgene insertion and 25% Pi−/−, 50% Pi+/−, and 25% Pi+/+ in case of selective gene knockout. Comparisons at G1 between homozygous transgenic, hemizygous transgenic, and nontransgenic animals provide an unbiased estimation of hemizygous and homozygous transgene effects. This latter scenario would be highly suitable, for example, for generating homozygous transgenic pigs lacking antigenic epitopes on xenografts. Prior to the production of homozygous transgenic animals, suitable lines of hemizygous males or females could be selected on the basis of a preliminary breeding experiment in each line according to the strategy in Table 4 or comparisons between hemizygous transgenic clones and similar, but nontransgenic, male and female control clones (denoted njOO 乆 and njOO 么 for female and male clones without the targeted insert and njPi+/ + 乆 and njPi+/+ 么 for female and male clones without the targeted gene knockout, with nj the number of clones, Table 7, step 2). The design of studies into the effects of nuclear transfer on animal health and welfare is identical to the design employed in the context of genetic modification by electroporation and nuclear transfer (see previous section and Table 6) and involves comparisons between multiple lines of male or female nontransgenic clones
(njOO σ, njOO 么, njPi+/+ 乆 and njPi+/+ 么; Table 7, step 2) and the appropriate in vivo controls. Treatment with Recombinant Protein. Next to breeding and testing of transgenic animals, another experimental treatment may also be important for the (early) detection of possibly deleterious effects of the intended gene-product. At a stage preceding the actual generation of transgenic farm animals, nontransgenic animals could be treated with the homologous analogue of the recombinant protein (Smith et al., 1987). Such an approach bears similarity to (immuno)toxicological studies (e.g., Martinod, 1995), and may allow for the identification of dose-response relationships between protein level and a biological response, which could be used for estimating acceptable upper limits of transgene expression levels. Significantly, problems observed in transgenic pigs expressing growth hormone transgenes could be mimicked, in several cases, in normal animals by long-term treatment with porcine growth hormone (Pinkert and Murray, 1999).
Welfare Protocol In order to be truly effective, research into health and welfare of transgenic farm animals should not only be based on sound and efficient breeding and testing procedures, but should also make use of a comprehensive welfare protocol, specifying which parameters to monitor, at which stages (pre- or postnatal) of the life of a farm animal, and in how many animals. Parameters. Although there is a lack of agreement about defining and interpreting measures of health and welfare (e.g., Rushen and de Passille´, 1992; Barnard and Hurst, 1996), it is possible to identify relevant parameters and biological functions that are clearly associated with health and welfare. A useful suggestion for a welfare protocol has been made by Broom (1993), who
Table 7. Steps in the breeding and testing of transgenic farm animals produced by gene targeting Step
Generation
Creation of clones and breedinga
1
G0
乆 么 乆 么
2
G0
Create control clones: njOO 乆, njOO 么 njPi+/+ 乆, njPi+/+ 么
founders founders founders founders
niTiO niTiO niPi+/− niPi+/−
Mating niTiO 乆 × niTiO 么 Mating niPi+/− 乆 × niPi+/− 么 3
G1
25% TiTi, 50% TiO, 25% OO 25% Pi−/−, 50% Pi+/−, 25% Pi+/+
Testing Identify characteristics in each line of identical male or female founders (clones) niTiO and niPi+/− Preliminary breeding experiment within lines of founders, or comparisons between niTiO and njOO or between niPi+/− and njPi+/++
Comparisons between TiTi, TiO and OO Comparisons between Pi−/−, Pi+/−, and Pi+/+
a Multiple (ni) identical hemizygous transgenic founder animals produced by gene targeting either carry the same transgene on the same site of the genome, denoted TiO, or are deficient in one of the two alleles of the same endogenous gene Pi, denoted Pi+/−. Homozygous transgenic animals are indicatd by TiTi and Pi−/−, in case of targeted transgene insertion and selective gene knockout, respectively. Nontransgenic animals generated by crossing hemizygous animals carrying a gene knockout are indicated by Pi+/+.
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proposed to include clinical symptoms of health and disease, measures of growth and reproduction, measures of immunocompetence, behavioral measures, and measures of hypothalamo-pituitary-adrenal (HPA) axis function. Similar measures have been suggested in the context of the assessment of health and welfare of transgenic mice (Crawley, 1999; Mertens and Ru¨licke, 1999). Some observations can be performed under baseline conditions in undisturbed animals, for example the recording of spontaneous behaviors in the home environment, the assessment of clinical health, or the measurement of resting levels of immune function. For other measures it is necesssary to subject animals to some external challenge. Examples of this latter category of observations are behavioral tests to measure various motivational states or sensimotor functions (see Crawley, 1999; Van der Meer et al., 1999, for examples in transgenic mice), experimental infections or antigenic challenges to measure dynamic aspects of immunocompetence, or physiological challenge tests to monitor the capacity or reactivity of biological systems involved in responsiveness and adaptation to environmental stress, including the HPA axis (e.g., Veissier et al., 1999). Relevant candidate parameters could also be provided by immunotoxicological (e.g., Van Loveren et al., 1998) or pharmacological disciplines (e.g., Martinod, 1995). We propose to formulate, for each of the important livestock species involved in transgenic research, a basic welfare protocol encompassing a cross-section of the most essential parameters and biological functions previously mentioned, the scope of which could then be adjusted, either extended or reduced, according to the stage of the transgenic program, the treatment under observation, or specific characteristics of the genetic modification (e.g., site of transgene expression, properties of transgene-derived protein, prospective biological function of endogenous gene inactivated after knockout, etc.). Protocols used for the evaluation of in vitro reproductive technologies should involve observations appropriate for detecting LOS symptoms, such as specific measures of neonatal vitality and viability, or ultrasound measurements to investigate disproportionate organ development (e.g., Garry et al., 1996; Van Wagtendonk-de Leeuw et al., 2000). Reduction of the scope or the degree of invasiveness of a welfare protocol would be appropriate at the initial stage of a transgenic program when relatively small numbers of (unique) transgenic animals are available, or toward the end of a transgenic program when the attention might be devoted to large numbers of animals within the framework of epidemiological experiments. With large numbers of subjects, investigated under field conditions (e.g., van Wagtendonk-de Leeuw et al., 2000), the argument of feasibility will prevail in determining which parameters will be recorded. A comprehensive and detailed survey of aspects of health and welfare should be made particularly in offspring of those transgenic founders, or in those lines of transgenic clones, that are
intended to serve as foundation stock for developing transgenic herds. We also suggest that molecular biological indices might be incorporated into a health and welfare protocol, especially those that would enable the assessment and reduction of risks of impaired health and welfare. A promising concept considers gene expression profiles of imprinted genes. There is evidence that in bovine preimplantation embryos manipulated in vitro, an aberrant epigenetic modification of imprinted genes, such as the IGF-I gene, is associated with abnormal mRNA expression patterns (Niemann and Wrenzycki, 2000). Correspondingly, there are differences between neonatal clones and normal control calves in levels of plasma IGF-I and IGF-binding protein (Chavatte-Palmer et al., 2000). Such changes may form the basis of a series of diagnostic tests allowing for evaluating the safety of embryo technologies, even at a stage preceding the transfer of embryos and the initiation of pregnancies (Young and Fairburn, 2000). Likewise, tests or paradigms might be developed to obtain information on characteristics of transgene expression before the production of transgenic livestock, for example by characterizing genetically modified cells prior to nuclear transfer (Pati et al., 1998; Wilmut et al., 1999), or by using in situ transfection based on jet injection of naked DNA into lactating mammary gland (Wall et al., 1997). Stages of Life. The stages of life of a transgenic farm animal at which to monitor aspects of health and welfare should, in our view, at least include gestation and birth, the developmental phase from birth to puberty, and a representative period of adult life, including the stage of (re)productive performance. Investigations up to senescence could have scientific value but need not necessarily be relevant for all farm animals because their productive lives usually represent only part of the entire possible life-span. Most importantly, observations on health and welfare of transgenic farm animals should be carried out during the period(s) when expression of the transgene and the exertion of the desired transgene effect are expected to occur. In line with these principles, Damak et al. (1996) monitored growth and reproductive performance from birth until after completion of yearling shearing in transgenic sheep carrying an IGF-I transgene designed to promote wool growth. Van Reenen and Blokhuis (1997) performed a longitudinal study on health and welfare of dairy cattle harboring a human lactoferrin transgene designed to express in the lactating mammary gland, and they specifically examined characteristics and composition of milk, measures of udder health, and the presence of human lactoferrin in milk and other bodyily fluids (blood, tears, saliva, and urine) in adult transgenic cows and their nontransgenic half-sibs. Remarkably, in an otherwise welldesigned comparative study by Hughes et al. (1996) on the behavior of transgenic sheep carrying a human alpha-1 antitrypsin transgene also designed to express in the lactating mammary gland, observations were
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only performed in immature animals, between the ages of 6 and 8 mo. Number of Animals Required. On mathematical and statistical grounds, when for a certain continuously distributed measure the magnitude of a relevant difference between treatment groups (e.g., between transgenic and nontransgenic animals) is small in relation to the standard deviation, the number of animals needed to detect this difference as statistically significant (P < 0.05) with a certain probability (i.e., power) is large, and vice versa. Smith et al. (1987) provided a table with numbers of progeny (half transgenic and half nontransgenic) required to significantly (P < 0.05) detect transgene effects (expressed as differences from the mean of nontransgenic controls) from 0.2 to 2.0 standard deviation units, with power of 0.80. If a transgene effect is over one standard deviation unit, a relatively small number (under 30) of progeny is required. Detection of smaller transgene effects, for example between 0.4 and 0.2 standard deviation units, requires between 198 and 790 progeny, respectively. Gama et al. (1992) presented numbers required to significantly detect differences between treatment groups in fractions, such as survival rate or fertility rate. To detect a difference in fractions between transgenic and nontransgenic animals of, for example, 10, 25, or 50%, requires 374, 76, and, 20 progeny, respectively (power is 0.80, P < 0.05, one-tailed test). Thus, the sensitivity of a welfare protocol in terms of the ability to detect relatively small effects if they do exist is greatly influenced by the number of animals investigated. It will be easy to reliably identify transgenes or in vitro embryo manipulations with extremely unfavorable effects, but large numbers of animals will be needed to detect smaller, but biologically relevant, harmful effects of transgenesis.
Future Prospects Animal biotechnology is a rapidly advancing scientific field. We will consider some technological developments that may also critically influence health and welfare of the animals involved. Although certain alternative concepts of genetic modification of animals may also be promising for the future, for example intracytoplasmic sperm injection (Perry et al., 1999), it is generally anticipated that nuclear transfer will become the dominant technology to generate transgenic farm animals (e.g., Colman, 1999/ 2000; Wilmut et al., 1999; Piedrahita, 2000). Nuclear transfer-mediated transgenesis is expected to 1) eliminate problems associated with random transgene insertion, 2) allow for the use of a wide range of cells as nuclear donors, including embryonic or ES-like cells, fetal cells, and adult somatic cells, and 3) make it possible to perform the full range of targeted genetic manipulations, including selective gene knockout. Cloning from adult rather than embryonic or fetal cells may become a method to duplicate performance-tested transgenic
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animals (McClintock, 1998). Potential applications of gene knockout in farm animals include the knockout of endogenous milk proteins to provide additional capacity for the production of transgene protein (Wall, 1999), the knockout of the prion protein (PrP) gene to make cattle and sheep resistant to BSE or scrapie (e.g., Mu¨ller and Brem, 1998; Wilmut et al., 1999; Piedrahita, 2000), the development of animal models for human physiology and disease (e.g., Petters, 1994), and the knockout of genes implicated in the rejection in heterologous recipients of porcine xenografts (Polejaeva and Campbell, 2000). Recently, the likelihood of an important part of these applications of farm animal transgenesis to become reality significantly increased when the births of the first cloned piglets generated by nuclear transfer using adult or fetal cells as nuclear donors were reported (Onishi et al., 2000; Polejaeva et al., 2000). Transgenesis in farm animals is also likely to become affected by strategies to improve regulation and control of transgene expression that are currently being examined in transgenic mice. Sophisticated strategies for tissue- or cell-specific transgene expression or gene knockout include the so-called cre/lox system, which uses cre-mediated excision of DNA flanked by repeated loxP sites, and ligand-dependent transgene expression (see Sauer, 1998; Wells and Wall, 1999). Other strategies aim at enhancing control of transgene expression by trying to overcome integration site effects, either by using insulating sequences, such as matrix attachment regions or locus control regions, that provide protection of the target transgene from surrounding sequences (Wells and Wall, 1999), or by including transgenes in very large genomic DNA fragments, such as mammalian artificial chromosomes, which may completely eliminate position effects (Vos, 1997). An initial attempt to implement this latter technology in pigs has been reported (Langford et al., 1996). The prospective use in farm animal transgenesis of both nuclear transfer-mediated gene transfer, with gene targeting via homologous recombination, and advanced systems of regulation of transgene expression may reduce some risks of impaired animal health and welfare. However, there are also reasons for concern. First, an increased reliance on nuclear transfer does not remove problems associated with the LOS syndrome. A recent study in sheep even suggested that, in comparison with in vitro embryo production (i.e., in vitro fertilization followed by in vitro embryo culture), nuclear transfer aggravated LOS symptoms such as fetal developmental retardation and deficiencies in placentation (De Sousa et al., 2000). Second, achieving complex targeted genetic modifications in cultured cells, such as with cre-mediated events, requires prolonged periods of cell culture with multiple rounds of selection. Extension of the culture period of embryonic stem or nuclear donor cells, in combination with extensive transfection procedures, may increase the incidence of chromosomal abnormalities or environmentally induced alterations in imprinted genes (Stice, 1998b; Young and Fairburn,
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2000). Dean et al. (1998) demonstrated aberrant phenotypes (i.e., poor mandible development and interstitial bleeding) in mouse fetuses derived from in vitro-cultured embryonic stem cells that were consistently associated with altered methylation patterns of imprinted genes and changes in allelic gene expression. The incidence of this type of error may also be increased when, instead of embryonic or fetal cells, adult somatic cells are used as nuclear donors (Young and Fairburn, 2000). Third, continuous enhancement of expression of transgenes in the lactating mammary gland, aided by the use of more effective genomic DNA sequences in the transgene construct (e.g., Brink et al., 2000) and the possible knockout of endogenous milk protein genes (Wall, 1999), may ultimately result in wholly unacceptable levels of foreign protein in the general circulation as far as potential effects on health and welfare are concerned. Finally, similar to transgene insertion and expression, inactivation or knockout of endogenous genes may have unexpected consequences because of disruption of an apparently essential biological function. For example, transgenic mice homozygous for disrupted PrP genes initially seemed to develop and behave normally and did not exhibit immunological defects (Bu¨eler et al., 1992). Subsequent studies, however, investigating neuronal and behavioral functions more closely, and in different substrains, have demonstrated various deleterious effects from ablation of the PrP gene (Collinge et al., 1994; Tobler et al., 1996). Thus, the production of PrP-deficient cattle and sheep has been discarded as a safe strategy to fight BSE or scrapie by some scientists specifically concerned with prion diseases (see Prusiner, 1997).
Implications Any claim as to the safety of current and future applications of farm animal transgenesis should be substantiated by comprehensive research into health and welfare of the animals involved. This type of research 1) should be multidisciplinary, involving different scientific disciplines such as reproduction, ethology, pathology, immunology and molecular biology; 2) should be logically and structurally integrated into ongoing transgenic programs, and 3) should make use of scientifically valid experimental designs and protocols, including adequate controls with sufficient numbers of animals, allowing for accurate and unbiased estimations of the essential treatment effects. Results obtained accordingly facilitate the use of the safest experimental protocols as well as the selection and propagation of the healthiest and least abnormal animals, and, thereby, enable technological progress that can be ethically justified.
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