Xenobiotica the fate of foreign compounds in biological systems

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Transgenic animals in the evaluation of compound efficacy and toxicity: Will they be as useful as they are novel? H. D. Liggitt & G. M. Reddington To cite this article: H. D. Liggitt & G. M. Reddington (1992) Transgenic animals in the evaluation of compound efficacy and toxicity: Will they be as useful as they are novel?, Xenobiotica, 22:9-10, 1043-1054, DOI: 10.3109/00498259209051859 To link to this article: http://dx.doi.org/10.3109/00498259209051859

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Date: 16 March 2016, At: 23:44

XENOBIOTICA,

1992, VOL. 22, NOS 9/10, 1043-1054

Transgenic animals in the evaluation of compound efficacy and toxicity: will they be as useful as they are novel? H. D. L I G G I T T S and G. M. R E D D I N G T O N ? Department of Comparative Medicine, School of Medicine, T-142 Health Sciences, SB-42 University of Washington, Seattle, WN 98195, USA tConsultants for Applied Biosciences, Inc., Wilton, C T 06897, USA

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Received 5 October 1990; accepted 1 March 1992 1. Construction of transgenic mice is predicated upon inserting foreign DNA into native host DNA and having this expressed in the germline. This may be accomplished by nuclear injection, retroviral vectors or use of embryonic stem (ES) cells.

2. Expression of novel structural genes may be reasonably directed by the judicious use of an accompanying promoterlenhancer sequence. Insertion of foreign genes may be designed to result in phenotypic expression of a novel trait or ablation of a native gene or gene product. 3. Resulting transgenic mice offer significant utility as models of human diseases and a unique opportunity for investigating immune and metabolic pathways as well as for exploring mechanisms of development, mutagenesis and teratogenesis. 4. Use of transgenic animals in drug development has considerable potential although realization of this potential will take time. Constructing transgenics is only the first step in a complex series of events culminating in understanding the consequences of imposing novel genetic material on an intact, highly integrated living system. Practical use of transgenic animals will depend upon substantial effort being spent in investigating and validating the phenotypic consequences of gene transfer.

Introduction Twelve years ago the idea that exogenous genetic material could be injected into a pronucleus of an embryo, integrated, retained and expressed in a newborn animal was so novel that it lacked a name. Since then the proof of concept of what has become known as transgenic animals has been fulfilled and the technology has erupted into an extremely active area of investigation with significant and broad potential. As with any new technology, initial investigatory efforts have been predominantly directed toward exploration of the varied techniques, breadth and tolerances of the system rather than applications. Hence, currently there is more promise than proof available that transgenic mice and other species will prove to be useful particularly in the area of drug development/evaluation. None-the-less one need not look too hard to uncover numerous potential applications in this area (see reviews by Babinet et al. 1989, Capecchi 1989, Connelly et al. 1989; Cuthbertson and Klintworth 1988, Evans et al. 1985, Gordon 1989, Hanahan 1989, Jaenisch 1988, Palmiter and Brinster 1985,1986, Scangos and Bieberich 1987, Tilghman and Levine 1986, Westphal 1989). T h e objective of this review is to briefly examine

t T o whom correspondence should be addressed. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd

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various methods for the construction of transgenic mice and, based upon examples, explore some potential applications for transgenic mice in the valuation of drug efficacy and safety.

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Methods for construction of transgenic animals One of the surest indications that a new technology has caught the fancy of the scientific public is the number of citations available concerning a given topic. Before 1987 transgenic animals did not warrant a separate subject heading. In 1988 the number of journal citations listed in Index Medicus under ‘mice, transgenic, was 52 and this escalated to 84 in 1989. Several of these articles go into significant detail concerning the construction and other aspects of transgenics (Baribault and Kemler 1989, Frohman and Martin 1989, Gordon 1990, Hogan et al. 1986, Palmiter and Brinster 1986, Scangos and Bieberich 1987) and readers are referred to these sources for a more detailed examination of this issue. Regardless of the technique used the usual goal in constructing transgenic animals is to introduce new (foreign) DNA (in the form of a defined gene sequence) into the DNA of the host animal and have this new DNA expressed in host somatic and germ cells so that it may be propagated across generations. Implicit in this goal is the need to have the new genetic material expressed phenotypically or, alternatively, have it result in the mutation or ablation of native genetic material. One of the obvious benefits of this is that the host will be tolerant to proteins encoded by the foreign DNA (Arnold et al. 1990). Introduction of foreign DNA into a host animal can be accomplished in several ways. T h e most commonly used method was devised by Gordon and Ruddle (1981) and involves the direct micro-injection of specially prepared foreign DNA into the pronucleus of a single cell embryo which is then implanted into a pseudopregnant mouse which serves as a foster mother. Resulting offspring are tested for evidence of integration of the new DNA by Southern blotting or, more recently, the polymerase chain reaction (PCR). Gene expression efficiency of this method is high with up to 30% of mice originating from microinjected embryos being transgenic. It does have some drawbacks however including (1) it is technically difficult and requires special equipment, (2) it can result in the disruptive integration of multiple copies of the new DNA fragment leading to expression stability problems, and (3) it can be associated with development of unanticipated mutational events (for example see McNeish et al. 1988) and sterility. In addition if injection occurs past the single cell embryo stage or if integration is delayed the mice generated will be mosaic (chimeric) for the foreign gene. Another method for directing placement of foreign DNA into the host nucleus utilizes retroviruses as a shuttle mechanism. In this system small (generally < 9000 base pairs) segments of foreign DNA are inserted into the retroviral genome the results of which are then used to infect mouse embryos. This results in the production of single integrants which are minimally disruptive to host DNA but rather inefficiently expressed. This coupled with size limitations and the difficulty in making the recombinant retroviruses has made this a less attractive approach for generating transgenic mice. T h e DNA fragment intended for use in constructing a transgenic animal is assembled in the laboratory in the form of a fusion or chimeric transgene. This is made by linking the regulatory portion of one gene to the structural portion of another. It is not necessary that these two elements be active in the same tissue or

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even be from the same species. In many regards the success and utility of the transgenic animal derived from the process is dictated by proper selection and availability of the promoter and enhancer elements-principal components of the regulatory region. It is the regulatory region which controls initiation of transcription and, fundamentally, tissue-specific expression. Some promoters drive expression in a wide number of tissues (examples would be the promoters for mouse mammary tumour virus and metallothionein) while others are much more tissue-specific (such as elastase or insulin promoters). Other elements (reporters, etc.) can be incorporated into the fusion transgene to, for instance, aid in detection of its presence in the genome and differentiate it from an endogenous gene. By coupling regulatory and structural gene elements purposely and selectively an investigator can, to some degree, regulate expression (in contrast to integration which occurs in all cells) of a transgene. Several examples of promoter/structural gene constructs are presented in table 1 . While judicious selection of promoter/ structural gene combinations can encourage tissue-specific expression it should be understood that the fusion genes are integrated randomly into the genome and the chance of them integrating into their ‘normal’ site is extremely small. In spite of this, if a tissue-specific gene is expressed it will generally be subjected to reasonably proper controlling elements which determine expression in the appropriate tissue and at the appropriate time in development. Unfortunately, because the regulatory portion of the fusion gene may be incomplete the quality of the expression of a foreign gene can vary from making nothing to unpredictable levels of encoded product. I t should also be understood that insertion of a transgene will integrate only rarely directly interfere with production of the analogous natural protein (although it may interfere indirectly via feedback inhibition). In spite of these limitations transgenic mice produced by pronuclear insertion techniques have opened the door to enormous scientific possibilities. A third method for generating transgenic mice recently has come to the forefront because it offers the possibility for targeting transgenes to particular sites

Table 1 .

Examples of transgenic constructs exhibiting either widespread or localized expression. ~~~

~

Fusion (Promoter/structural)gene Expression widespread SV40/mutant dihydrofolate reductase Metallothionein/growth hormone and others Metallothionein/growth hormone Metallothionein/bcr/abl p 190 Expression localized Insulin/class I 1 or interferon Elastase/diphtheria toxin Elastase/ras oncogene Albumin/hepatitis B surface antigen Lens crystallin/diptheria toxin

Phenotypic expression

Reference

Resistance to methotrexate

Isola and Gordon 1989

Non-allometric growth pathology of overexpression Hepatocellular neoplasia, excessive growth Acute leukemia

Quaife et al. 1989

Insulin-dependent diabetes Ablation of pancreas Pancreatic neoplasia Hepatic injury, neoplasia

Sarvetnick et al. 1988 Palmiter et al. 1987 Quaife et al. 1987 Chisari et al. 1989

Microphthalmia

Breitman et al. 1987

Orian et al. 1990 Heisterkamp et al. 1990

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in the genome. This technique, which is likely to become very popular, involves the use of embryonic stem (ES) cells. ES cells are pluripotent cells established from normal embryos (blastocysts). These cells can be cultured and manipulated in vitro and will resume normal development when implanted into blastocysts from foster mothers. Transfer of genetic material to ES cells is accomplished via microinjection, electroporation or retroviral infection (Capecchi 1989, Frohman and Martin 1989, Gordon 1989, Baribault and Kemler 1989). One advantage of the ES-based technique is that foreign genetic material can be transferred into ES cells and ‘positive’ cells selected before generation of animals. This makes feasible another significant advantage of the ES system, which is the ability to select for cells where homologous recombination has occurred. Homologous recombination is a spontaneous but infrequent event (in the range of 1 : 1000 odds in mammalian cells) where donor DNA is integrated directly into the site of the endogenous DNA (i.e. it replaces its chromosomal homology) rather than randomly as with other systems. This propensity toward homologous recombination has been exploited to ‘knock-out’ or inactivate targeted endogenous genes (Capecchi 1989) as well as, more recently, to replace defective genes (Thompson et al. 1989). ES cells positive for the homologous construct are selected and used to establish transgenic mouse lines. Selecting ES cells where homologous recombination has occurred is a rapidly-evolving but currently rather tedious process (Capecchi 1989, Williams 1990) but it makes practical targeting of gene expression-a powerful tool and something not feasible with other methods. Mice generated from ES-inoculated blastocysts are chimeric in somatic and germ cells for the selected, novel trait. Inbreeding of heterozygotes permits generation of homozygotes and phenotypic expression of the trait.

Uses of transgenic animals in the evaluation of drugs Ultimately transgenic animals are likely to display their real use in elucidating fundamental genetic factors regulating normal and abnormal development including factors that influence disease resistance and susceptibility. As part of this, and much more immediately accessible, is the use of transgenic animals as models of disease (and therapy/toxicity) in their own right or for investigating mechanisms of disease. A somewhat separate use for transgenic animals is utilization of the technology to encourage greater production of animal proteins (e.g. meat and milk) or as bioreactors (‘factories’) for the production of designed, species-specific peptides, proteins or cells. T o understand the benefits as well as limitations of transgenic animals it is helpful to investigate some examples.

Models of neoplasia From the point of view of immediate utility appropriate for the applied investigation of xenobiotic drugs, the ‘off-the-shelf’ selection of transgenic animals are rather limited. An exception to this might be found in the area of transgenic cancer models as illustrated by the recently described transgenic model of acute leukemia (Heisterkamp et al. 1990). T h e evaluation of xenobiotic (or any other) drugs can involve the use of rodents as either models with which to evaluate the efficacy of potential chemotherapeutic (or other) drugs or as sentinels with which to monitor for carcinogenic potential as a component of a safety assessment. Non-transgenic in vivo tumour models which are typically available include tumours of spontaneous origin, transplanted tumours and those induced by

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Transgenic animals in the evaluation of compound efficacy and toxicity 1047 chemical or physical agents. Many investigators feel that tumours occurring spontaneously in laboratory animals may be more relevant for investigating the efficacy of chemotherapeutic agents because, among other reasons, their growth behaviour, location, tissue context and histological diversity are more akin to the clinical situation. Unfortunately, spontaneously-occurring tumours, even in susceptible laboratory strains, are of low numbers, of poorly-predictable occurrence and site, and generally require aged animals which may have complicating problems. T h e ability to generate the development of oncogene-driven tumours in transgenic mice holds great promise for providing investigators with a model that approaches closely a ‘spontaneous’ tumour. Linking specific combinations of promoter and structural gene (oncogene) elements can result in the reasonably predictable, site-specific development of tumours. One of the earliest successes in transgenic technology involved using the SV40 T-antigen gene linked to its own promoter to induce choroid plexus tumours (Brinster et al. 1984). Since then numerous constructs have directed SV40 T-antigen-driven neoplasms to pancreatic acinar cells (elastase promoter), islet cells (insulin promoter), lens of the eye (A crystallin promoter), liver (metallothionein promoter), etc. (reviewed in Gordon 1989, Jenkins and Copeland 1989). T h e proto-oncogene c-myc also has been used to generate tumour-bearing mice. When fused with the mouse mammary tumour virus promoter (MMTV) and then used to construct transgenics, multiparous mice developed mammary tumours (Stewart et al. 1984) and in other lines additional tumours occurred in testis, and lymphoid tissues (Leder et al. 1986). It is a variation of the myc-transgenic mouse that has been made commercially available to investigators. Other fusion genes employing various oncogene elements have been used to generate a variety of different tumours in varied locations and have provided significant information concerning oncogenesis. In addition, these studies also have demonstrated that foreign oncogenes escape immune surveillance and rejection because their early expression induces tolerance; although if expression is delayed, immune rejection can occur (Hanahan 1989). Cordaro (1989) has reviewed the potential for using transgenic mice expressing human oncogenes in risk assessment studies. Investigators obviously should exercise caution in unconditionally accepting tumours in transgenic mice as strict analogues of naturally-occurring tumours. T h e biological behaviour of most of these transgenic tumour systems has been investigated only superficially. For instance pancreatic tumours derived from elastase-ras constructs lack features typically associated with malignancy including aneuploidy, invasiveness and metastasis (Quaife et al. 1987, Freeman et al. 1989). It is also obvious that factors in addition to oncogene expression are necessary for tumour development. Hence, additional study is necessary on the variable role that secondary factors play in promoting development of tumours in transgenics carrying foreign oncogenes (Freeman et al. 1989). An illustration of this is provided by the work of Breuer et al. (1989) where they constructed a transgenic mouse overexpressing the pim-1 oncogene. These mice are predisposed to develop lymphosarcoma but the spontaneous occurrence of this tumour is only about 10% by 240 days of age. If these mice, on the other hand, are exposed to a single low dose of a chemical carcinogen (ethyl-nitrosurea; ENU) nearly 100% of the pim-1 transgenics develop tumours versus 15% of ENU treated non-transgenics. An obvious implication is that this system, once further characterized and validated, may be useful for the screening of carcinogens in industrial or environmental settings.

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Tumours also can be generated in transgenic mice by the overexpression of some growth factors. One transgenic model which has marked similarities to the situation observed with chemical carcinogens is that seen with a metallothioneinovine growth hormone fusion gene. This construct results in a chronic proliferative process in the liver which manifests as megalocytosis, oval cell proliferation, hyperplasia, adenoma and, after 43 weeks of age, hepatocellular carcinoma (Orian et al. 1990). The authors suggest that these mice may be useful for testing for carcinogens since they are, in effect, primed to develop carcinomas. This would result in a significantly more sensitive and timely assay than the classical 2-year chronic study (Rao and Huff 1990). While time savings would be valuable, the increased sensitivity may not be universally embraced by those in the pharmaceutical/chemical industries, and certainly would require much study and validation. It has also been recently observed that transgenic mice constructed using transforming growth factor-alpha as the structural gene develop a variety of hyperproliferative events including neoplasia of the mammary gland and liver (Jhappan et al. 1990, Matsui et al. 1990, Sandgren et al. 1990). It is likely that similar findings will occur when other growth factors/cytokines are removed from a natural regulatory context and overexpressed or expressed inappropriately in transgenic models. While immediate uses for these transgenics in the evaluation of xenobiotics may be limited they can demonstrate target organ propensities and toxicities for those evaluating protein/peptide-based drugs, or perhaps, provide models for testing antagonists of such drugs or their endogenous analogues. At a more fundamental level, however, the system provides a novel in vivo system for evaluating the basic role of growth factors and others in carcinogenesis (Hanahan 1988, Jenkins and Copeland 1989, Messing et al. 1985).

Models based on inappropriate expression of protein Evaluation of pharmaceutical compounds for efficacy frequently requires the use of animal models. Among their uses are, in dissection of disease pathogenesis with an aim toward targeting development of new drugs or indications. Until recently, animal models that were available depended upon the generation and identification of naturally-occurring mutations, or the induction of a disease state by chemical or surgical methods. With the advent of transgenic animals it becomes possible to develop animal models by selectively expressing human (or other species) gene products in a non-human species. This permits the generation of animal models that may be more appropriate or relevant for drug testing because they recapitulate the pathogenesis of human diseases or allow expression of human proteins, or infectious disease agents, in non-human systems. An example of directed animal model development is provided by the work of Sarvetnick et al. (1988, 1990). This effort involved generation of a mouse model of Type I diabetes (insulin-dependent) to test the hypothesis of whether inappropriate expression of MHC class I1 antigens on beta cells could mediate development of insulitis and, ultimately, diabetes. Class I I expression was generated either by constructing a transgenic mouse that expressed these antigens on beta cells directly (insulin promoter/MHC class I1 structural gene), or by expressing class 11 indirectly via stimulation by a cytokine (insulin promoter/interferon-gamma structural gene).

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Both types of transgenic mice became diabetic. However, transgenic mice constitutively expressing class I1 did not develop inflammatory lesions, while those expressing class I I via cytokine induction did develop progressive insulitis/ pancreatitis, similar to that seen in human with Type I diabetes. Additional studies demonstrated that the diabetes in this IFN-gamma transgenic mouse was likely due to a lymphocyte-mediated destruction of beta cells rather than by IFNgamma alone or interferon-induced expression of class I1 antigens. Evidence suggests that islet lesions may be due to IFN-gamma activating quiesent autoreactive T-lymphocytes normally present in the periphery. This model of diabetes is unique because it is generated by a known genetic event (expression of IFNgamma driven by a transgene active in beta cells) which culminates in islet inflammation and insulin-dependent diabetes. I n mutational models of diabetes the initial genetic event triggering development of the disease is not known. T h e IFN-gamma diabetic model offers the possibility for testing compounds related to the treatment and management of diabetes, as well as more futuristic compounds directed at disrupting cytokine traffic involved in the generation of autoimmune or autoreactive disease. A transgenic system which will undoubtedly find immediate utility in evaluation of drugs and, potentially, vaccines, are constructs which carry human immunodeficiency retrovirus (HIV) DNA sequences. Work in this area has been reviewed recently (Lassam et al. 1989, Cordaro 1989). One construct that is particularly interesting is the model developed by Vogel et a1.(1988) where HIV LTR is fused to the tat gene coding sequence. Male mice derived from this construct developed Kaposi sarcoma-like tumours in their skin suggesting that Kaposi sarcoma (a frequent component of HIV infection) may be directly mediated by the virus rather than it occurring as a consequence of immunosuppression or other cofactors. Developing a transgenic model which recapitulates faithfully the entire clinical spectrum of HIV infection is a formidable task that has yet to be accomplished. Even so, the efforts underway offer possibilities for testing drugs for their abilities to suppress or activate viral expression or modulate sequelae of retroviral infection (Field and Brown 1989). Another model demonstrating the potential utility of transgenic animals in the evaluation of efficacy and/or toxicity of drugs is provided by Galaski et al. (1989). This group generated transgenic mice expressing the human multidrug resistance gene (MDRI) under the chicken beta-actin promoter. MDRI encodes a protein which acts as a multidrug transporter to pump drugs (chemotherapeutic agents) out of resistant cells. T h e investigators reasoned that if they could cause expression of this M D R I protein on relevant cells, they might be able to protect these cells from the toxic effects of certain chemotherapeutic compounds. T h e transgenic mice constructed using this rationale did express high levels of M D R I glycoprotein on the surface of bone marrow and spleen cells. These cells were resistant to a challenge with the cytotoxic drug daunomycin such that total white blood cell counts of treated, non-transgenic mice fell three-fold compared with treated transgenics (whose counts were actually slightly above normal). These M D R I transgenics displayed no phenotypic abnormalities associated with gene expression, either clinically or histologically. This model, or similar types of models (Isola and Gordon 1986, 1989), could be of significant utility for testing high-dose chemotherapeutic strategies, developing new agents and regimens for

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avoiding drug resistance, and as models for possible somatic cell gene therapy in human.

Models based on genetic ablation Up to this point we have considered the consequences of insertion of novel genes into host DNA. An additional method of manipulating the host genome and producing transgenic animals involves ablation of either selected cells (and tissues) or genes. A number of transgenic animals have been developed which display phenotypically the results of having selected populations of cells removed by coupling a given promoter to a structural gene which encodes a toxin. One of the first illustrations of this was provided by Palmiter et al. (1987). This group linked the elastase promoter to a gene for diphtheria toxin A and constructed transgenic mice using this fusion gene. Diphtheria toxin is synthesized by Corynebacteriurn diptheriae as two subunits, A and B. T h e A subunit is toxic and the B subunit is responsible for cell surface binding of the A-B complex. By using only the A component the investigators were assured of having only producing subunit-A cells dying, since without subunit B bystander death would not be possible. Indeed mice generated from this construct had only a rudimentary pancreas consisting of scant islet and duct cells. This indicated to the investigators that all three cell types in the pancreas may have a common precursor or, alternatively, that elastase-producing cells may contribute to a microenvironment that drives pancreatic differentiation via production of growth of vascular factors, etc. Other cell ablation techniques making use of other toxic elements and promoters have been used to generate transgenic mice lacking lenses, pituitaries, lymphoid systems and nervous system components (Breitman et al. 1987, Bernstein and Breitman, 1989). This toxin-based system is associated with some problems including the virtual certainty that some targeted cells will escape elimination, and the possibility that embryo lethality can result due to use of an inappropriate promoter. Regardless, this technique is likely to be used not only to construct animal models of deficiency and disease but also to investigate cell and tissue development, and mechanisms of organogenesis, morphogenesis and teratogenesis. A much more powerful ablation technique using ES cells has been developed recently. This technique is different fundamentally from the toxin-based systems described previously, in that it depends on eliminating a targeted gene rather than cells or tissues (Baribault and Kemler, 1989, Capecchi 1989, Frohman and Martin 1989, Williams 1990). An illustration of this technique is provided by the work of Thomas and Capecchi (1987) who used ES cells and homologous recombination to disrupt the gene encoding the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). T h e lack of this enzyme in humans results in the severe neurological disorder, the Lesch-Nyhan syndrome. T h e chromosomal H P R T gene was ablated by using a targeting vector consisting of an exogenous H P R T gene containing a neomycin resistance gene. This neomycin gene serves to both disrupt the H P R T coding sequence and act as a selectable marker. Insertion of such a vector by homologous recombination results in inactivation of the native H P R T gene. Embryonic stem cells containing such a vector are inserted into blastocysts and used to generate chimeric mice which transmit the mutation in their germ line. Cross breeding of these mice results in homozygous expression of the trait. A similar strategy employing a non-disrupted gene as a vector has been

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used to correct an H P R T deficiency by gene targeting (Thompson et al. 1989). Clinical phenotypic expression of either one of these events (ablation or correction) is not possible in the mouse since normal neurological function does not depend upon presence of the H P R T enzyme. Nevertheless, this model demonstrates the potential for specifically altering the genome by gene targeting. Using similar strategies it should be possible to alter essentially any metabolic pathway involved in drug metabolism, to create models to assess efficacy, toxicity or mechanism of action.

Other directions T h e list of applications and potential applications of transgenic technology in pharmaceutical science is expanding rapidly. For instance, generation of larger species of transgenic animals for milk-based production of proteins and peptides for pharmaceutical use has been proposed and tested (Westphal 1989). Transgenics also have found application as sources of novel in vivo and ex vivo cell lines (and their products) generated by gene transfer (Bowman et al. 1990, Chisari et al. 1989, Cordaro 1989). Also, as briefly mentioned earlier, the feasibility of treating human diseases by gene transfer can be tested and challenged by not only restoring the deficient gene product, but also inducing the specific deficiency to begin with (Mason et al. 1986, Williams 1990). T h e list of potential applications grows daily, being limited only by the patience, perseverance and imagination of the investigator.

Conclusions Several potential uses for transgenic animals in evaluation of pharmaceutical compounds have been mentioned. Real possibilities exist for the use of these genetically altered animals in such diverse areas as: (a) Development of relevant disease models. (b) Investigation of disease and lesion pathogenesis and pathophysiology. (c) Investigation of gene expression and control including mutation analysis. (d) Establishment and study of immune tolerance. (e) Investigation of organogenesis/teratogenesis. (0 Evaluation of potential gene therapy. (g) Synthesis of custom cells and proteins. (h) Evaluation of mechanisms of metabolism, resistance, and toxicity. Generating the founder transgenic animal, is just the first step in a long road to characterize and validate the expression of a novel trait which has been imposed upon the complex milieu of a ‘foreign’ living system. T h e possibilities associated with the development and use of transgenic mice are nearly endless (including the high probability that several scientific ‘black holes’ will be thoroughly investigated). T h e biggest challenge with this technology, as with most new technologies, is being able to ask the proper question and have it be answerable without, in the process, generating a finely tuned artifact. While the potential for practical use of transgenic animals in drug development is significant several caveats remain including: (1) There is currently very limited understanding of a complex system. There is tremendous advantage in knowing the novel genetic event that initiates a cascade of changes resulting in a particular phenotype. However, past ‘knowing’ the structure of the fusion gene that is transferred, the investigator remains at the

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mercy of the host genome and controlling genetic and epigenetic elements for integration and expression of the novel gene. ( 2 ) Physiological and pharmacodynamical relevance of transgenes is variable. Transfer of novel genetic material from one species to another must be undertaken with the understanding that this is happening ‘out of context’. It should not be assumed, for instance, that native physiological control or defence mechanisms will interact with the novel gene product. Furthermore quantities and sites of synthesis of transgene products can differ significantly from the physiological or pharmacological norm. ( 3 ) Transgenic mice may carry human genetic material but they remain mice. T h e power of the transgenic approach to animal modelling is considerable and while it does remove some of the liabilities associated with naturally occurring mutant models (including the propensity for transgenics to be tolerant to foreign proteins) some of the same factors limiting use of more traditional animal models remain. These include lack of genetic diversity, and variations in non-human biology of ageing, size, behaviour, natural disease and disease agents and environmental influences among obvious others.

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