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The Use of Transgenic Animals in the European Union

The Report and Recommendations of
ECVAM Workshop 281,2

class="maintext"T. Ben Mepham,3 Robert D. Combes,4 Michael Balls,5 Ottavia Barbieri,6 Harry J. Blokhuis,7 Patrizia Costa,8 Robert E. Crilly,3 Tjard de Cock Buning,9 Véronique C. Delpire,5 Michael J. O'Hare,10 Louis-Marie Houdebine,11 Coen F. van Kreijl,12 Miriam van der Meer,13 Christoph A. Reinhardt,14 Eckhard Wolfe15 and Anne-Marie van Zeller43

class="maintext"Centre for Applied Bioethics, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK; 4FRAME, Russell & Burch House, 96-98 North Sherwood Street, Nottingham, NG1 SEE, UK; 5ECVAM, JRC Environment Institute, 21020 Ispra (VA), Italy; 6Dipartimento di Oncologia Clinica e Sperimentale, Universitá di Genova, IST/CBA, Largo R. Benzi 10, 16132 Genoa, Italy; 7Institute for Animal Science and Health (ID-DL), Department of Behaviour, Stress Physiology and Management, Edelhertweg 15, 8200 AB Lelystad, The Netherlands; 8Instituto di Biologia Molecolare, Via Pontina KM 30.600, 00040 Pomezia, Rome, Italy; 9Department for the Study of Animal Experiments, University of Leiden, 2301 CB Leiden, The Netherlands; 10Breast Cancer Laboratory, LICR/UCL, 67-73 Riding House Street, London W1P 7LD, UK; 11Laboratoire de Biologie Cellulaire et Moleculaire, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy-en-Josas, France; 12RIVM, 3720 BA Bilthoven, The Netherlands; 13Department of Laboratory Animal Science, Utrecht University, 3508 TD Utrecht, The Netherlands; 14Swiss Alternatives to Animal Testing (SAAT), 8614 Bertschikon-Zurich, Switzerland; 15Lehrstuhl für Molekulare Tierzucht, Feodor-Lynen-Strasse 25, 81377 Munich, Germany1ECVAM - European Centre for the Validation of Alternative Methods. 2This document represents the agreed report of the participants as individual scientists.Address for correspondence: T.B. Mepham, Centre for Applied Bioethics, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics., LE12 5RD, UK.Address for reprints: ECVAM, TP 580, JRC Environment Institute, 21020 Ispra (VA), Italy.


Preface

This is the report of the twenty-eighth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organization of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses and make recommendations about the best ways forward (1). In addition, other topics relevant to the Three Rs concept of alternatives to animal experiments have been considered in several ECVAM workshops.The workshop on The Use of Transgenic Animals in the European Union was held in Southwell, Nottinghamshire, UK, on 7-11 April 1997, under the co-chairmanship of Ben Mepham (University of Nottingham, UK) and Miriam van der Meer (University of Utrecht, The Netherlands). It was held in collaboration with the Fund for the Replacement of Animals in Medical Experiments (FRAME; Nottingham, UK) and the Centre for Applied Bioethics (CAB) at the University of Nottingham, and was organised by Ben Mepham (CAB), Rob Crilly (CAB), Bob Combes (FRAME) and Anne-Marie van Zeller (FRAME). There were 16 participants from six Member States of the European Union (EU). The principal aim of the workshop was to formulate a set of guidelines to assist regulatory authorities in the EU in deciding whether to permit and/or how to regulate research involving transgenic animals. This report summarises the workshop discussions on the current status of transgenic animal research, and proposes a number of recommendations for the appropriate and harmonised control of the use of transgenic animals within the EU.IntroductionThe technique of transgenesis involves the introduction of functional genetic material (DNA) into the germline of organisms. The first "transgenic" animals, produced in 1980, were mice (2, 3). A transgenic animal is an animal which has been genetically modified by the stable incorporation, by using artificial gene transfer, of exogenous DNA into its genome, in order to introduce or delete specific characteristics of the phenotype. A transgene construct can comprise a complete gene sequence derived from a donor organism, an in vitro synthesized sequence, or a combination of both. One of the most recent definitions of transgenic animals is that suggested by Beardmore (4): "organisms containing integrated sequences of cloned DNA (transgenes), transferred using techniques of genetic engineering (to include those of gene transfer and gene substitution)". In this report, discussion is confined to consideration of transgenesis in vertebrate laboratory and farm animals.Production of transgenic animals able to transmit genetic modifications to their offspring requires germline transformation. There are several techniques for the production of transgenic animals (Tables I and II); the three most commonly used methods pronuclear microinjection, embryonic stem (ES) cell manipulation, and the cre-lox technique, are shown schematically in Figures 1, 2, and 3, respectively.


class="maintext"Table I: Methods for Producing Transgenic Animals by Introduction of Foreign DNA into the Mammalian Genome

TechniqueRemarks
Pronuclear microinjection (introduction of expressed gene)technical simplicity; low success rate; applicable to a wide range of species; most widely used; unpredictable effects due to random transgene integration.
Embryonic stem (ES) cell manipulation (introduction of expressed gene, or gene inactivation by homologous recombination)substitution of a functional gene with an inactive gene; germ-line competent ES cells have been isolated in mice; ES-like cells identified in other species, including primates, but totipotency remains to be established
Cre-lox techniquepreferred method with more control over resulting phenotype; time-consuming
Viral vectorscomplex; largely restricted to avian species
Cytoplasmic injectionless efficient than direct pronuclear microinjection
Primordial germ cellschimaeric animals result
Nuclear transferlarge potential for genetically modifying livestock
Spermatogonial manipulationtransplantation

 

Table II: Consequences of Techniques Used in Transgenesis

TechniqueRemarks
Gene additionachieved by all methods
Gene knockouttargeted inactivation of host gene by embryonic stem cell manipulation
Random insertion of mutationsachieved by all methods
Inhibition of gene expressionfor example, prevention of translation by hybridisation of anti-sense RNA with mRNA

Figure 1: Pronuclear Microinjection

figure 1


Figure 2: Embryonic Stem (ES) Cell Method

figure 2


Figure 3: Cre-lox Technology for Targeted Homologous Recombination of Transgenes

figure 3


There are several potential and actual applications of transgenic animals (3):

  1. in basic research;

  2. as a source of organs for xenotransplantation;

  3. as disease models;

  4. in the production of therapeutic proteins (that is, as bioreactors);

  5. in agriculture (for example, the manipulation of livestock production traits);

  6. for vaccine testing; and

  7. in toxicity testing.

Transgenic animals present unique opportunities for medical, agricultural, and fundamental research, and as a means of producing valuable pharmaceutical and nutrient products. Their use has increased dramatically in recent years, and is set to rise at an even more rapid rate. For example, in the U.K., there was a 525% increase in the use of transgenic animals between 1990 and 1996 (5). This increase should be considered against a background of a modest decrease in the overall use of laboratory animals during the same period of time. While transgenic animals might allow reduction and refinement in animal use via more precise gene targeting in breeding programmes, these objectives are threatened by transgenic procedures which could promote greater animal use, a greater variety of applications, and an increased likelihood of animal suffering (6-10).

There are also intrinsic ethical concerns relating to the transfer of genes between species, especially when human genes are involved (with the potential for their progressive transfer into laboratory animals [11]), and when animal organs are used in human medicine. In this regard, it is of interest that members of the general public within Europe are becoming increasingly skeptical about the use of animal biotechnology (12). This situation has prompted some to call for further ethical debate (13). On 21 May 1996, the EU Group of Advisers on the Ethical Implications of Biotechnology (GAEIB) published an opinion on the genetic modification of animals (14). One of the principal conclusions was that while genetic modification of animals might contribute to human well-being and welfare, it "is acceptable only when the aims are ethically justified and when it is carried out under ethical conditions". There was a consensus at the workshop in support of all the opinions expressed in the GAEIB report.

Current legislation on animal experimentation, such as Directive 86/609/EEC, was introduced before the full implications of transgenesis were recognised. Consequently, one of the principal objectives of the workshop was to review the current situation with regard to the development and application of transgenic animal technology, with the aim of providing a list of recommendations to key regulatory authorities and, in particular, DGXI/E/2, which is responsible for the administration of Directive 86/609/EEC. The potential benefits and animal welfare implications of animal transgenesis were discussed, together with possibilities for implementing reduction, refinement, and replacement (the Three Rs) strategies wherever feasible (15). General and specific concerns about the effects of transgenesis on animal welfare are discussed later in the report.

 

Transgenic Animal Disease Models

More than 3000 human genetic diseases are known, and there is much interest in studying their fundamental causes so that effective treatments and somatic cell gene therapies can be devised. Specific inbred mouse strains, which inherit spontaneously derived phenotypes, have provided useful models for investigating the pathogenesis of human diseases. Nevertheless, several problems are associated with studying naturally occurring human genetic diseases by using animals:

  1. animal strains showing particular disease symptoms are often difficult to obtain and expensive to maintain;

  2. their specific genetic defects can be as difficult to identify and characterize as those of their human counterparts; and

  3. affected animals often differ from unaffected controls in genetic factors additional to the gene in question.

Advantages

Over the last decade, many transgenic and knockout mutant mouse strains have been created as disease models (16). Models exist for neuropsychiatric, cardiovascular, pulmonary, oncological, inflammatory, and immunological diseases, as well as for studying mechanisms and disorders of human metabolism, reproduction, and early development. These models are documented in transgenic databases, such as TBASE (17) or Induced Mutant Resource (IMR). So far, mainly single-gene conditions have been studied, but potential applications of transgenic mice include the analysis of polygenic diseases.

Scientific limitations

One of the major problems in modeling a human disease condition is that the phenotypic effects of a mutation may vary depending on the genetic background in which it is expressed, due to the presence of specific alleles at modifying loci in different inbred animal strains. It is, therefore, important to select the correct genetic background, as well as the correct mutation(s), to produce an optimal model of any particular human disease.

It is apparent from an analysis of some transgenic disease models that the actual benefits of using the model are rarely completely equivalent to the potential benefits and that the decrease in aspects of animal welfare might be disproportionate to any benefits gained. The currently available transgenic models for cystic fibrosis (CF) illustrate this point. None of the strains is ideal, with either the genotype and/or the phenotype of the mouse failing to accurately model the human condition (18). For example, in the case of the Edinburgh CF mouse model, the transgenic animals have a normal life-span, a normal body weight gain, and a phenotype which is generally nonlethal. Nevertheless, to model the human disease as closely as possible, it is likely that the mice suffer to some degree. In addition, one of the major potential benefits of the work, gene therapy, remains elusive. It might, however, be feasible to study the molecular effects of the genetic lesion at the cellular level, to understand the mechanistic basis of the disease, and to develop other forms of therapy.

There are several limitations in relation to the usefulness of the current approaches to developing transgenic disease models, particularly since many diseases are multifactorial. Problems persist when extrapolating data obtained by using such transgenic animals to the disease condition in humans.

 

Transgenic Animal Models in Toxicology

The majority of transgenic animal strains in toxicology have been used for genotoxicity and carcinogenicity testing. In the case of genotoxicity, current methods for detecting gene mutations are restricted mainly to in vitro. In vivo approaches have principally involved the analysis of chromosomal damage in a single tissue type for mutagenic/genotoxic effects, which has made their application limited, particularly when other target tissues are involved. The rationale for using transgenic animals was to develop an assay that would detect a mutagen/genotoxin in vivo in a range of different tissues, including germ cells.

The transgenic approach to genotoxicity testing

Commercially available transgenic mouse models include Mutamouse® and Big Blue®, which contain the Escherichia coli lac Z and lac I transgenes, respectively (19). The transgenes are cloned in bacteriophage lambda vectors that are integrated in the genome. Following the treatment of the transgenic mice with a test chemical, the integrated bacteriophage vectors are rescued from the total genomic DNA by in vitro packaging. Mutant phage, with disrupted lac genes, are recognised by their ability to grow on susceptible E. coli host strains and by the colour of the resulting plaques.

Agents which are strong mutagens are detected with a high degree of accuracy, but the ability of these assays to correctly detect non-carcinogens requires further investigation (20). Also, failure to detect compounds that cause predominantly large deletions has been reported (21), probably due to the fact that only DNA fragments of a specific length are efficiently packaged, so that large deletions or insertions will probably not be detected. To overcome this problem, transgenic mice have been generated that contain a plasmid-based lac Z system, in which large deletions are detectable (22).

Carcinogenicity testing

The chronic rodent bioassay uses a large number of animals (400-500 per compound), and is prone to generate spurious data at high dose levels (23). The principle underlying the use of transgenic animals for carcinogenicity testing is that the presence of an appropriate transgene will not directly provoke tumours, but will establish a high predisposition to carcinogenesis. Since the emergence of a malignant clone requires several additional genetic changes in affected cells, the time needed for this to occur is shortened. This predisposition to carcinogen-induced tumorigenesis, without a concomitant increase in spontaneous turnout rate, might not only allow shorter times for exposure to the test compound, but also a considerable reduction in the numbers of animals required relative to the conventional bioassay.

Three different types of transgenic strains have been employed in the generation of transgenic mice for carcinogenicity testing:

  1. the Eµ-pim-1 transgenic mouse, containing the activated pim-1 oncogene (which has a low spontaneous tumour rate and appears to be very sensitive to carcinogen-induced tumorigenesis by genotoxic carcinogens that target the Iymphoid system [24]);

  2. those containing an activated oncogene (v-H-ras, c-H-ras) or an inactivated tumour suppressor (p53) gene (25, 26); and

  3. mice with an inactivated DNA repair (XPA) gene (27).

However, transgenic animals bearing single cancer-associated mutations might provide misleading information. In rodent cells, single mutations may lead to a transformed phenotype but not be sufficient to cause transformation of human cells. The transgenic models may consequently be oversensitive to additional carcinogenic events, such as exposure to carcinogens, and thus overestimate human risk.

Insufficient studies have been conducted to assess the suitability of using transgenic mice models in carcinogenicity testing as alternatives to the chronic two year rodent bioassay. However, assessment may be facilitated when the results of a recently initiated international collaborative validation study become available. Consensus has, nevertheless, been reached at the International Conference on Harmonisation on a recommendation that the chronic mouse bioassay be replaced by an assay based on transgenic mice for the regulatory carcinogenicity testing of pharmaceuticals (28).

One important potential problem with transgenic models for carcinogenesis is that the effects of mutations in certain genes, including tumour suppressor genes and oncogenes, might be influenced by species variation. As a corollary, mice bearing specific oncogenic mutations might not necessarily be sensitive to the same secondary events as occur in human carcinogenesis.

 

Transgenic Farm Animals

Currently, microinjection is essentially the only method which can be used for producing transgenic farm animals. The applications of these animals fall into three broad categories:

  1. as bioreactors;

  2. for xenografts; and

  3. in animal production (29-31).

Bioreactors

There are two approaches to generating bioreactors, here defined as transgenic animals producing pharmaceutical proteins. The most effective approach is to express a protein in the mammary gland by using a promoter from a milk protein gene to direct expression. An example is the production of the ovine ?-lactoglobulin promoter for use in expressing a variety of human proteins with pharmaceutical applications, such as a-1-antitrypsin (AAT). Thus, expression is directed to the mammary gland of the lactating mammal, commonly a sheep, cow, goat or pig, and the human protein is secreted directly into the milk (32). In the second approach, the desired therapeutic protein is produced in non-mammary body fluids, such as blood. To date, this approach has only been used in pigs for producing haemoglobin (33).

Xenotransplantation

A key element in producing transgenic farm animals to provide organs (xenografts) for transplantation into human patients is preventing rejection of the transplant through activation of complement factors belonging to the human immune system. This objective has been pursued, for example, by producing transgenic pigs expressing genes coding for human complement inhibitory factors, such as decay-accelerating factor (34).

Production traits used for transgenes

The following livestock production traits are currently subject to manipulation by transgenesis:

  1. growth and body composition;

  2. improvements in the quality and yield of wool; and

  3. increased disease resistance (35-39).

 

Consequences of Transgenesis for Animal Welfare

Three factors that may negatively influence the health and welfare of transgenic animals have been identified by van Reenen & Blockhuis (40, 41):

  1. reproductive and other biotechnological interventions;

  2. mutations; and

  3. expression of the transgene.

These factors are discussed below, followed by an assessment of their impacts in relation to specific applications.

Reproductive and other biotechnological interventions

Studies involving certain species (for example, sheep and cattle) have shown that in vitro procedures employed both before and after microinjection (in vitro culture, embryo transfer) might lead to increased gestation length, body weight, incidence of dystocia, and perinatal loss and anomalies, relative to in vivo (artificial insemination) procedures (41-43). Moreover, there is evidence that microinjection, irrespective of successful integration of the foreign DNA, increases embryonic and fetal losses in manipulated mouse embryos (44, 45). The culling of mice for embryo recovery, and other surgical interventions used in generating transgenic animals, also compromise welfare.

Mutation effects

Following microinjection, foreign DNA often becomes integrated within or near an endogenous gene, thereby creating a new ("insertional") mutation and causing a loss of host gene function. Integrated microinjected material is sometimes associated with chromosomal translocations, and with other rearrangements leading to developmental defects. While insertional mutations can be of either a dominant or a recessive nature, it is assumed that detrimental insertions usually result in early prenatal deaths, and hence are unlikely to be detected (46). Implicit in the unpredictability of both the integration site of the foreign DNA and of the number of DNA constructs which are successfully integrated, is the notion that each transgenic founder animal is unique in terms of both genetic makeup and the nature of any defects resulting from insertional mutations. Gene targeting in ES cell manipulations might reduce the uncertainty to a degree, by directing integration to a particular site.

Expression of the transgene

The extent to which transgenic animals express harmful consequences from exposure to foreign proteins and/or expression of a transgene is dependent on the following, interrelated, factors:

  1. the biological properties of the resulting protein;

  2. the tissue(s) in which transgenes are expressed;

  3. the route of secretion of the gene product; and

  4. the level of transgene expression.

Thus, in terms of the severity of potential risks, any particular transgenic animal model is situated at some point on a more or less continuous spectrum between, at one extreme, a condition in which inert proteins are synthesised at a low level in a limited number of specific tissues insulated from the bloodstream and, at the other extreme, a condition in which a biologically highly active protein is synthesised in large amounts in many tissues with abundant access to the bloodstream. An example of the latter extreme was the notorious Beltsville pig, which suffered from a range of pathological conditions (47).

Additional problems can be caused by the incorrect or unpredicted expression of the transgene. This can be due to pleiotropic effects of the gene itself, or may result from epistatic effects -- interactions with endogenous genes and their gene products (48). The location of a transgene in a chromosome (that is, a position effect) can also affect gene expression (49), especially if it is integrated close to endogenous DNA control regions, such as enhancers. As a result, enhanced transgene expression, no expression, or expression in an inappropriate cell type or tissue, may occur.

Several strategies are being developed to improve the control of transgene expression. Thus, it might be possible to eliminate position effects by incorporating insulators, which are portions of DNA which act as boundaries, preventing interference with the transgene by endogenous sequences (49). Furthermore, the use of complete promoters or the presence of control regions within introns and other untranslated regions, could enhance the control of transgene expression. Inducible promoters have been developed which could restrict transgene expression to those cells containing an inducer molecule (50).

The cre-lox system also allows tissue-specific expression of transgenes. This method uses the bacteriophage P1 recombinase enzyme and its lox P target sequences (Fig. 3; 51). Lox P sequences are recognizable by the bacteriophage P1 Cre recombinase enzyme which, although capable of being active in eukaryotic cells, is not normally present. The transgene sequence is constructed from the DNA coding sequence of the gene to be targeted together with flanking lox P sequences. The modified DNA is introduced into ES cells, in which homologous recombination occurs to replace the target host gene with the modified gene copy. This genetically modified ES cell line is used to develop a transgenic mouse strain. A second strain of chimaeric mice, which exhibit tissue-specific expression of the Cre recombinase, is produced by introducing the coding sequence for Cre into specific cells in late stage embryos or in early adult animals. When the two strains of mice are crossed, homologous recombination occurs between the two lox P sites, under the influence of the enzyme, but this occurs only in those tissues expressing the recombinase within the resulting hybrid mice. This results in elimination of the intervening gene sequence, leading to specific gene inactivation. In those cells unable to express the enzyme, no such recombination occurs, and the gene is expressed.

Systematic effects

In addition to welfare issues relating to the modification itself, subsequent housing husbandry and production systems (that is, systematic effects) can affect the well-being of a transgenic animal (7). For example, pigs generated as sources of organs for human transplant surgery, or cows kept for producing human pharmaceuticals in their milk, will need to be maintained under strict hygienic conditions. This could increase the risk that transgenic animals will be deprived of certain environmental conditions necessary for accommodating normal behavioural needs (for example, proper bedding, rooting material, and exercise space), and hence welfare might be reduced (52). However, provided that the behavioural needs of transgenic animals are taken into consideration sufficiently, optimal care and hygiene could improve their level of health and welfare.

Welfare implications of disease models

Animals generated in such a way that they develop a human disease raise a distinct set of welfare issues. Reduction in animal welfare is intrinsic to the objective and is, therefore, inevitable while, for other applications animal suffering, where it occurs, might be seen as incidental. The development of transgenic animal disease models has led to some unexpected effects, such as glomerulo-sclerosis in the growth hormone (GH) model (53). It is sometimes difficult to distinguish such effects from the symptoms of the disease being modelled. Models are also being developed to study the control of blood pressure (54), and animals of such strains could well suffer from cryptic effects.

The replacement of conventional animal models of disease by improved transgenic models reflecting more accurately the human disease, could conceivably benefit animal welfare via a reduction in the number of animals needed to achieve statistically significant results. Furthermore, it should be possible to generate animals that have a number of salient features of the disease but do not develop the full disease condition. However, this objective has so far received very little attention.

Welfare implications of mutagenicity testing

There do not appear to be any well-documented studies of animal welfare in the Mutamouse and Big Blue transgenic strains currently in widespread use for mutagenicity testing. The numbers of animals used routinely in genotoxicity testing could be reduced by the use of a transgenic rodent assay that would enable the detection of mutations in the whole animal in every tissue, following exposure by different routes to a potential genotoxin. A reduction in animal numbers will be achieved more readily, however, if protocols involve detecting mutations in a wide range of tissues using the minimum number of animals consistent with achieving statistical significance. Also, the ability to investigate the induction of mutations in germ cells might obviate the need to use currently available methods for detecting heritable mutations, which require large numbers of animals.

Welfare implications of carcinogenicity testing

The use of transgenic rodents for carcinogenicity testing could also lead to an overall reduction in the large numbers of animals that are used in the conventional rodent bioassay. In addition, the duration of such tests would be shortened, resulting in an overall decrease in suffering.

However, there have been reports of adverse health effects which go beyond those due directly to the testing procedure itself. Thus, high mortality rates have been reported for c-neu and c-myc strains (25), and v-H-ras mice are prone to papilloma development following minor skin abrasions (55).

Welfare implications of using transgenic animals as bioreactors

Data on transgenic bioreactor farm animals suggest that potential welfare problems could be related to the actions of biologically active proteins after transgene expression (35, 47). For example, ectopic expression of putative mammary-specific expression has been observed in rabbits carrying human erythropoietin (hEPO) genes (56), sheep harbouring human AAT genes, and mice carrying the human GH (hGH) fusion gene. In the case of potent transgene-derived proteins like hEPO and hGH, ectopic expression was associated with severe detrimental effects on animal health. Moreover, ectopic expression of even a putatively non-detrimental protein, such as mouse whey acidic protein in sheep, could have contributed to unusually high morbidity rates (57). In addition to ectopic expression, proteins may leak from milk into the bloodstream or, in some cases, exert detrimental effects locally in the mammary gland (58, 59).

Welfare implications of using transgenic animals for xenotransplantation

There appear to be no published data relating to the welfare implications of generating transgenic pigs for the provision of xenografts. The principal concerns relate to the housing conditions of these animals, particularly where pigs are maintained under strict disease-free (gnotobiotic) conditions to avoid any transfer of disease to patients. In the UK, these issues are about to be addressed by introducing a code of practice on the husbandry of such animals as part of the government's response to the Nuffield Report (52).

Productivity promotion

Addition of transgenes encoding elements affecting growth such as GH, to sheep and pigs have generally had detrimental effects. For example, the Beltsville pigs suffered from lethargy, lameness, uncoordination, exopthalmus, gastric ulcers, severe synovitis, degenerative joint disease, pericarditis and endocarditis, cardiomegaly, paraketosis, nephritis, and pneumonia (47). Such severe problems are thought to be the result of the continuous elevation of circulating GH levels, but attempts to control expression (by the use of inducible promoters) have not yet been successful. Limiting transgene expression to a particular region of the animal is another strategy for reducing welfare impacts. For example, insulin-like growth factor 1 expressed in the skin of transgenic sheep stimulated wool production without any apparent welfare problems beyond those inherent in the process of transgenesis (52).

Animal welfare might also be improved by the addition of genes encoding disease resistance. However, to date, only one example of transgene-derived disease resistance has been demonstrated (avian leukosis virus in chickens [60]), and this was associated with other health problems (61).

Monitoring welfare

The animal welfare concerns identified above indicate the need for a substantial effort to monitor the health and welfare of transgenic animals in a systematic manner. Although there is extensive literature on indices of animal welfare (62), the lack of consensus on their relevance to transgenic animals might hamper progress. This suggests the need to identify a minimum number of indices covering a broad spectrum of biological responses (for example, ethological, physiological, and pathological). Moreover, to assess risks of impaired welfare there may be merit in monitoring molecular biological variation (for example, by measuring transgene products and RNA in various tissues).

Since each founder animal generated after microinjection is unique, the effects of transgenesis need to be evaluated on a case-by-case basis. This requires half-sib comparisons (transgenic versus non-transgenic), in both sexes, of the progeny of founder animals (41, 63). As founders are hemizygous for the transgene, breeding founders with wild-type animals will result in the transgene being transmitted to 50% of the offspring. To detect any deleterious effects on welfare due to insertional mutations, another necessary step will involve breeding homozygous transgenic animals from half-sib x half-sib, or half-sib x founder, matings. Although there may be a tendency for early disposal of non-transgenic animals, it is imperative when studying welfare implications to maintain control animals in sufficient numbers to ensure reliable statistical analysis.

The first and foremost strategy to avoid harmful consequences of transgenesis must be systematic screening of all aspects of animal health and welfare, this will form the basis of critical decisions (for example, a decision not to breed homozygotes, or to refrain from enhancing transgenic expression beyond a certain level). The availability of ES cells would both increase the number of potential applications and reduce the risks to animal health and welfare.

Before generating transgenic animals, insights into potential problems may be gained by using alternative techniques to introduce the gene product into the animal. This may involve treatment with the exogenous protein, or its homologous analogue (where immunological consequences can be avoided), or the addition of transgenes to somatic tissues of the adult animal.

 

Public Acceptability and the Role of Ethical Analysis

There seems to be a significant lack of public support for animal transgenesis. For example, in a European Commission poll on attitudes to biotechnology (64), it was reported that nearly 50% of Europeans considered that, if they were able to, they might not allow production and use of certain forms of transgenic animals. Moreover, a majority of respondents opposed farm animal transgenesis. This situation was endorsed in a more recent European poll, in which a minority approved the use of transgenic animals (12). Some believe, however, that scientists, as guardians of scientific knowledge, possess the only correct view, while the public are ill-informed or misled. Others argue that considerations within the scientific community are considerably influenced by self-interest and professional loyalties. Public concerns, however, go beyond issues of pain, suffering, and the welfare conditions of animals.

Criteria for public acceptability

Several criteria determine the public acceptability of animal transgenesis. First, there is the need of the scientific community for sound methodology, combining optimal validity and sensitivity with full accountability for scientific work designed to achieve set objectives. Some argue that the application of transgenic technologies is incompatible with the Three Rs concept. Thus, it is claimed that welfare problems encountered in the early phases of the development of new transgenic strains are often overlooked (65), and that animal welfare considerations are secondary to commercial criteria.

A second criterion is the need to show animals respect. The housing, husbandry, handling, and use for experimental purposes of laboratory animals might be said to be further violations of their species-specific life (their "telos"), as can substantial alterations to the genome.

Other key ethical concepts about which the general public and scientists often disagree are "naturalness", "integrity", and "intrinsic/inherent value" (66-70). Thus, while the public might perceive animals as linking humanity to nature, some scientists may tend to consider laboratory animals as tools to manipulate and exploit.

A further criterion for public acceptability relates to the possible adverse effects of animal transgenesis on future generations and the environment. One way of addressing these concerns is to invoke the precautionary principle, which states, broadly, that one should not proceed with a new process unless consideration has been given to the worst-case scenario (71). If scientists do not anticipate potential problems, it is likely that public views will continue to contribute to and possibly distort, the outcome of any debate, thereby increasing polarity (72).

A final criterion concerns respect for lifestyle and religious orientation. Determining one's own lifestyle depends on the availability of relevant information, for example via unambiguous product labeling. In the case of genetically modified products, such as foods and pharmaceuticals derived from transgenic animals, the situation regarding labeling has yet to be resolved.

These differences in values, views, and lifestyles can be said to constitute different "world views" (73). Clearly, there is no right or wrong position concerning these world views but, in democratic societies, radical technological change, such as the widespread production and application of transgenic animals, should only be introduced with explicit public consent.

The Decision-making process

All groups in society have a role to play in democratic decision-making procedures, which should be based on rational, ethical, and scientific arguments. While several ethical schemes exist to aid assessment of the costs and benefits of research proposals involving animal experimentation, these were developed before the full implications of transgenesis were recognized.

As part of an ECVAM/FRAME/CAB project, the U.K. Institute of Medical Ethics (IME), and Canadian and Dutch ethical schemes (74-76), have been evaluated for their abilities to deal with proposals involving transgenic animals. The schemes were applied to several different transgenic model systems, such as Immortomouse (77), and the CF (78) and GH (79) mouse models. The results of the study suggest that transgenic technology raises a number of unique issues which are not addressed by the application of any of the ethical schemes. For example, the IME and Dutch schemes consider the suffering imposed on animals solely in terms of the procedures and housing conditions. This results in an inability of the schemes to address issues relating to animals specifically designed to suffer, such as disease models which spontaneously develop a pathology in the absence of any scientific procedure.

Furthermore, none of the three schemes distinguish between the generation and subsequent use of transgenic animals. For example, in establishing a transgenic animal line, the surplus animals required, and the possible random side-effects of the transgene on the host, need to be considered separately from surgical interventions.

In terms of the benefits likely to accrue from the use of transgenic disease models, questions relating to the importance and complexity of the genetic component (multifactorial and polygenic diseases), and problems linked to the production of a phenotype in relation to a genotypic condition, are not specifically addressed by the schemes. The next stage of the collaborative project will be, therefore, to further develop the existing schemes so that the above questions and issues specific to transgenic animals are adequately considered. As an alternative, it might prove necessary to develop a novel scheme to cope with the unique problems encountered.

One possibility is to promote the use of ethical frameworks to aid reflection. The term "ethical" is used here in the broad sense of concerning "what we should do", and thus encompasses risk, cost-benefit, welfare, suffering, and intuitive responses. Such frameworks should attempt to encompass the concerns of both the scientific community and the general public which, as shown earlier, may differ considerably.

An example of such a framework is the "Ethical Matrix" (80), based on the principles employed by Beauchamp & Childress (81) for medical ethics. The aim is to construct a comprehensive framework of ethical issues which is both philosophically coherent and comprehensible to the lay-public. The three principles involved (corresponding to the major ethical theories of utilitarianism, deontology, and Rawlsian "justice as fairness", are claimed to form the basis of the "common morality" or "commonsense ethics". For example, in the case of experimental animals, respect for the three principles of well-being, autonomy and justice translates into respect for "animal welfare", "behavioural freedom" and "telos", respectively. Decisions regarding final ethical acceptability depend, however, on the manner in which the ethical matrix is applied and, in turn, on the user's world view.

The use of a common framework throughout the EU would standardize the way in which decisions regarding ethical acceptability are taken, while allowing local differences in attitude to be expressed. It would also improve the accountability of regulatory processes by making policy decisions transparent and comprehensible to a wider society, in turn promoting public debate and education.

 

Concluding Remarks

The technique of animal transgenesis appears to offer the prospect of considerable advances in biomedical science and biotechnology. Moreover, in some cases, the use of transgenic animal models could lead to refinement and reduction in the numbers of animals used in experiments. There is, however, a substantial risk that the current intense interest in developing novel transgenic strains will, in fact, result in an overall increase in experimental animal use. It should also be remembered that many additional animals are required during the generation of new transgenic strains (for example, surrogate mothers and vasectomised males). It is, therefore, crucially important that measures are taken to ensure that the production and application of transgenic animals is subject to close monitoring and control. The technique of transgenesis also raises serious ethical concerns, since it is possible to induce irreversible and often potentially far-reaching alterations in the genetic constitution of animals, for example, producing strains which express human genes, or which, in the case of disease models, are designed to suffer.

Urgent attention needs to be given to establishing committee structures throughout the EU to harmonize the regulation of the production and use of transgenic animals. National ethical and scientific committees would be charged with the task of interpreting relevant EU Directives and monitoring the activity of local (institutional) ethics and scientific committees. The latter committees would be responsible for ensuring that individual research proposals conform to agreed ethical principles. Institutional committees would also ensure that all possible measures were taken to protect the welfare of transgenic animals, working together with any existing statutory bodies charged with regulating laboratory animal experimentation. In this way, implementation of comprehensive measures to protect and continuously monitor the welfare of transgenic animals should be guaranteed. Measures taken might include application of accurate gene targeting techniques, storage of cryopreserved transgenic embryos, and increased application of in vitro methods for characterizing new gene constructs. These objectives would be facilitated by establishing an international database (accessible on the Internet), which would include information on:

  1. animal welfare;

  2. the validation and regulatory status of transgenic animal models for toxicity, carcinogenicity, and mutagenicity testing; and

  3. the extent to which the stated objectives and potential benefits of the work were achieved.

Such details should be used to assist with the ethical and scientific assessment of further research proposals.

It seems clear that existing regulations and legislation for animal experimentation do not adequately provide for the development and varied applications of genetically modified animals. It is hoped, therefore, that the recommendations of this workshop will contribute to the formulation of an appropriate set of harmonised regulations which take account of the important public and scientific concerns raised by this form of technology. A possible scheme for regulating the production and use of transgenic animals in the EU is shown in Figure 4.


Figure 4: A Possible Scheme for Regulating the Production and Use of Transgenic Animals in the European Union (EU)

figure 4


Summary and Conclusions

General considerations

  1. The recommendations of the EU Group of Advisers on the Ethical Implications of Biotechnology on the ethics of genetic manipulation of animals are endorsed.

  2. The objectives of this workshop report are to build on the analysis of the EU Group of Advisers, by providing more specific recommendations for the regulation of procedures involving transgenic animals in the EU, with the aim of ensuring inter alia that levels of animal welfare are maintained at the highest possible, while permitting the continued legitimate use of transgenic animals.

  3. Attempts should be made to reduce unnecessary duplication of developments in animal transgenesis, without unduly compromising the competitive nature of scientific investigation.

  4. Surveys of public opinion within the EU suggest that special consideration is required for the production and use of transgenic animals within national and international legislation.

  5. Existing controls on the production and use of transgenic animals in some Member States of the EU may be inadequate, and EU legislation needs to be harmonised and rationalized since it is too variable between Member States.

  6. As current EU legislation (Directive 86/609/EEC) does not explicitly refer to transgenic animals, a distinction needs to be made between the different (prospective) applications of animal transgenesis, as this could have important implications for the acceptability of certain procedures.

  7. Certain uses of transgenic animals (for example, the use of higher nonhuman primates) should be considered, in principle, to be unacceptable.

  8. Technological opportunity should not drive the production and use of new transgenic strains in the absence of any genuine social need.

  9. Any proposed work involving transgenic animals needs to have a clearly stated purpose so that its justification can be assessed.

  10. It is important not to allow a narrow cost-benefit approach to assessment which limits consideration of other important ethical factors, such as:

    1. respect for cultural values;

    2. responsibility for the protection of the physical environment; and

    3. the preservation of species integrity and diversity.

    Certain schemes for ethical assessment and review already exist, and it is recommended that their suitability in the context of transgenic animal procedures should be further investigated.

  11. While some may consider the progressive introduction of increasing numbers of human genes into transgenic models to be of minor concern at present, the technology exists, or could soon be developed, to allow this to attain a level of "humanization" which many would consider unacceptable. This matter should be kept under very close review at national and international levels.

  12. The labeling of products derived from transgenic animals should allow informed consumer choice.

  13. Even where proposals for the use of transgenic animals are acceptable in principle, the existence of a valid alternative non-transgenic method, which meets the stated objectives, should result in the rejection of the proposal to produce and use transgenic animals. Consideration should be given to an alternative procedure, at a late stage of development, if it is likely to become reasonably and practicably available in the near future.

Proposals following prima facie approval of a proposal to use transgenic animals

  1. It should be recognized that the genetic modification of animals has the potential to cause unexpected detrimental effects in progeny, which are sometimes severe.

  2. Approval of an application for a program of work with transgenic animals should be reviewed periodically, with regard to the alleged costs to the animals involved and the benefits to society.

  3. Independent, competent, and continuing assessment of the health status of animals should be undertaken, and this information should be freely available and used to assess future applications.

  4. Existing databases in the public domain should contain up-to-date information regarding the effects of transgenic procedures on animal welfare, to include details of:

    1. microbiological status;

    2. breeding, health, and behavioural problems;

    3. specific problems encountered in generating new transgenic strains;

    4. the actual benefits to be derived from the program of work; and

    5. details of any new alternative methods.

  5. Full records of all relevant information relating to scientific and welfare status should be maintained at each research establishment for inspection for two full generations of the natural life span of the transgenic animal strain when kept under optimal husbandry conditions. In the absence of adverse effects, the model should be considered equivalent to a non-transgenic animal model, but only for purposes of implementing relevant legislation.

Production and use of transgenic animals

  1. More research is needed into:

    1. the basis for differences in gene expression in vivo and in vitro;

    2. methods for facilitating the screening of transgenic embryos; and

    3. improved methods for gene targeting and inactivation.

    Such methods should be used to increase the use, when appropriate, of cell culture methods before, or instead of, the use of whole animals.

  2. To maximise scientific benefit and animal welfare, studies involving transgenic animals should be conducted by teams of individuals with relevant experience in, for example, molecular biology, genetics, pathology, cell biology, and animal care and welfare.

  3. It is important to ensure that housing and transport requirements appropriate for specific transgenic animal strains are strictly applied, to minimise adverse effects on their welfare.

  4. Measures should be taken to avoid unnecessary deleterious side-effects in stocks of transgenic animals, for example, by maintaining breeding colonies in the heterozygous state and, subsequently, producing limited numbers of animals homozygous for the prospective transgene for use only when strictly required.

  5. Transgenic animal models could be stored as cryopreserved embryos instead of in breeding colonies, in which they might exhibit deleterious side effects. The advantages and disadvantages of local, rather than centralized, embryo banks should be investigated.

  6. To promote refinement of procedures, all personnel handling transgenic animals should be required to attend training courses which stress the problems unique to, or frequently encountered in, such work.

Specific considerations relating to animals used as basic research/disease models

  1. Clarification of the concept of "models" (for both disease and basic research) in the context of animal transgenesis will assist both scientists and regulators in assessing the relevance and feasibility of research proposals.

  2. In view of the fact that substantial numbers of new transgenic strains can be produced, great care should be taken to select the most promising models. Account should be taken of:

    1. the need to identify critical factors which are indispensable for establishing the relevance of a model; and

    2. any existing or possible alternative methods of studying the human disease in question, or for developing therapies (for example, analysing clinical samples and disease conditions, the use of specific cell lines, and gene expression in vitro).

  3. Models should mimic as closely as possible the human condition being studied (for example, in terms of aetiology, pathology, and molecular mechanisms involved), although it may not always be necessary for exact parallels to exist. Furthermore, attempts should be made to model the earliest stage of the disease which is required to produce effective therapies, without necessarily producing a model which exhibits all symptoms of the disease.

  4. When the levels of suffering experienced by a transgenic animal are not considered disproportionate, continued studies to improve the model in question might be justified, but only where the amount of anticipated suffering remains within acceptable limits.

Specific considerations relating to animals used in toxicity and carcinogenicity testing

  1. When developing transgenic animal strains for toxicity studies, it is important to bear in mind the specific purpose for which the model is intended (for example, whether for routine regulatory testing or for specialized mechanistic studies).

  2. The use of transgenic animals in toxicity testing could lead to reduction and refinement in existing in vivo test protocols (for example, with respect to the rodent bioassay for carcinogenicity).

  3. Although the use of transgenic animals might reduce and refine existing in vivo toxicity and carcinogenicity assays, this should not be regarded as a reason to diminish efforts to use and further develop currently available in vitro methods (see point 32, below).

  4. To answer specific questions and to characterise particular mechanisms of toxicity, as well as for routine toxicity testing, panels of cell lines should be derived from any available genetically modified animals and from clinical samples.

Specific considerations relating to transgenic farm animals

  1. Relevant measures of animal well-being should be identified during the early stages of a program to generate new transgenic farm animals. The systematic use of these measures should involve:

    1. the use of general indices such as those relating to behaviour, immunological and disease status, and measures of growth and reproductive efficiency; and

    2. more specific indices, chosen according to the anticipated consequences of the expression of a particular transgene.

    To assess the extent of any adverse effects of the transgenic procedure, sufficient numbers of appropriate controls will be required, for example, by retaining non-transgenic siblings alongside transgenic offspring of the same founder animals.

  2. It might be deemed appropriate to grant official permission for using transgenic farm animals only for a limited period, pending the results of continued welfare monitoring. The period for which the resulting "provisional licence" was to be issued would depend on the species of animal in question and the conditions under which the animals were to be kept.

  3. The maintenance of adequate records on the absence of adverse effects in transgenic offspring before they are released from automatic protection under animal procedures legislation might be especially important in the case of farm animals, in view of the greater likelihood of their being released into the environment for routine use.

Other conclusions

  1. Improved dialogue is needed between scientists and the general public concerning the scientific rationale and perceived ethical justification for applying transgenic techniques to animals.

  2. The independent assessment of proposals to use transgenic animals should be undertaken in ways appropriate to the nature of the application. Some issues involving transgenic animals are of such fundamental and/or general importance as to require regulation by EU or national committees. Other more specific, issues dealing with particular programs of work, are more appropriately considered by local or a regional ethical and scientific assessment, or by other systems (such as the Home Office Inspectorate operating in the UK).

  3. The composition of ethics committees should:

    1. allow both the general public and scientists to be confident that their views receive proper consideration; and

    2. genuinely reflect the range of viewpoints within society, while taking into account the need to reach a consensus.

  4. The accountability of ethics committees should be ensured by rendering the decision-making process as open and transparent as possible (while recognizing legitimate provisions for commercial confidentiality). Consideration should be given to the use of a widely accepted and published framework, such as that termed the "ethical matrix", to facilitate this process.

Recommendations

The above list of conclusions has been used to derive a series of key recommendations designed to assist regulatory authorities in formulating legislation for the production and use of transgenic animals throughout the EU.

  1. Agreement should be sought throughout the EU concerning the degree and level of continuing legal protection to be afforded to transgenic animals. As part of this process, and as current legislation does not specifically cover transgenic animals, regulations for the control and use of such animals throughout the EU should be harmonised and rationalized. If necessary, existing legislation should be modified or replaced.

  2. Adequate and clear statistics on the numbers, species, types, production, breeding, and uses of transgenic animals should be collected and published at least biannually by each EU Member State, and by the European Commission to promote transparency and accountability.

  3. A list should be compiled of procedures involving transgenic animals which, while technically feasible, would not be considered ethically acceptable under any circumstances.

  4. There should be a broad and systematic ethical assessment of all proposals for work involving transgenic animals which have not been excluded by applying recommendation 3, above.

  5. Principles concerning matters of fundamental and general importance about the use of transgenic animals should be agreed at EU and national levels. A local institutional ethical and scientific committee should ensure that each individual research proposal conforms to these principles.

  6. When an alternative, non-transgenic method is at a late stage of development and is likely to be reasonably and practicably available in the near future, it should be given consideration as a replacement for the proposed use of a transgenic animal method. For example, appropriate cell culture methods should be developed and used wherever possible before, or instead of, using whole animals in transgenic research and application.

  7. Legal approval for any research involving transgenic animals should be regarded as contingent on the outcome of independent and continuing assessment of the health and welfare of the animals involved, and of the benefits being derived from the results obtained. Such information should be recorded in a database, be publicly accessible wherever possible, and be used in the appraisal of future research proposals.

  8. To maximise scientific benefit and animal welfare, all studies involving transgenic animals should be conducted by teams of individuals covering the full range of relevant expertise in, for example, molecular biology, genetics, pathology, cell biology, and animal care and welfare.

  9. All personnel directly involved in using transgenic animals should be required to attend training courses which emphasize issues and problems unique to, or frequently encountered with, such animals.

  10. Approval of any research proposals involving transgenic animal models of human disease should be based primarily on a requirement for the model to parallel as closely as possible the human condition being studied. The relevance of all such transgenic models should be kept under continuous review, and should not be assumed because of any superficial similarity with the disease being modeled, or solely on the basis of the importance of this disease.

  11. During the production of new transgenic animal disease models, efforts should be directed toward modelling the earliest stage of a disease which will allow the objectives of the research to be achieved.

  12. When transgenic animals are released from protection under the terms of laboratory animal legislation, such as transgenic farm animals kept for agricultural production, the maintenance and central storage of adequate records of the breeding and location of such animals, even during their routine husbandry, should be required.

  13. Where biomedical applications of transgenic farm animals require that they are kept in quarantine, or specific pathogen-free or gnotobiotic conditions, the maximum possible provision for the behavioural needs of the animals should be guaranteed.

  14. While the application of bioreactor and xenograft technology might offer significant medical benefits to society, the production of foreign biologically active molecules within the transgenic animals concerned might reduce the standard of welfare, and monitoring for any adverse effects should be mandatory.

 

Acknowledgements

Thanks are due to Mr & Mrs B.H. Annett (AMA Services, London, UK) and to Mrs M. Smith (FRAME, Nottingham, UK) for undertaking organizational aspects of the workshop. We thank Dr R.J. Lysons (Home Office, London, UK) who was invited to attend the workshop as an observer and who contributed information and advice for the report. The assistance of Dr C.G. van Reenen (Institute for Animal Science and Health [ID-DL], Lelystad, The Netherlands) in providing information on transgenic farm animals is also gratefully acknowledged.

 

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MeetingS

 
FRAME Training School in Experimental Design and Statistical Analysis
May 31-June 2, 2017
Saskatoon, Canada

Advances in Cell and Tissue Culture 2017
May 22-24, 2017
Manchester, UK

CAAT Academy
In Vitro Skin and Eye Models Hands-on Training
June 1-2 2017
Brussels, Belgium

CAAT Academy
Tools for Read-Across
June 15-16, 2017
Helsinki, Finland

ecopa SSCT Workshop 2017
June 14-16, 2017
Helsinki, Finland
Thomas Hartung will deliver a keynote address

CAAT Academy
Updates on Hepatotoxicity AOP Landscape and on the ADME Field - Season 2
June 22-23, 2017
Rennes, France

9th Euro-Global Summit on Toxicology and Applied Pharmacology
June 22-24, 2017
Paris, France
Email for Details

6th Annual Meeting of the American Society for Cellular and Computational Toxicology (ASCCT)
September 21-22
College Park, MD

SAVE THE DATE:
10th World Congress on Alternatives and Animal Use in the Life Sciences
August 20-24, 2017
Seattle, Washington

CAAT Academy
Hands-on Training in Quantitative Human Cell and Effect-based In Vitro Bioanalysis for Assessing Endocrine-disrupting Compounds (EDCs)
September 7-8. 2017
Amsterdam
 

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