Use of Genetically Engineered Mice in Drug ...

SYMPOSIUM

Use of Genetically Engineered Mice in Drug Discovery and Development: Wielding Occam’s Razor to Prune the Product Portfolio Brad Bolon1 and Elizabeth Galbreath2 1 2

Amgen, Inc., Thousand Oaks, California, USA Eli Lilly & Co., Indianapolis, Indiana, USA

Genetically engineered mice (GEMs) that either overexpress (transgenic) or lack (gene-targeted, or “knock-out”) genes are used increasingly in industry to investigate molecular mechanisms of disease, to evaluate innovative therapeutic targets, and to screen agents for ef cacy and/or toxicity. High throughput GEM construction in drug discovery and development (DDD) serves two main purposes: to test whether a given gene participates in a disease condition, or to determine the function(s) of a protein that is encoded by an expressed sequence tag (EST, an mRNA fragment for a previously uncharacterized protein). In some instances, phenotypes induced by such novel GEMs also may yield clues regarding potential target organs and toxic effects of potential therapeutic molecules. The battery of tests used in phenotypic analysis of GEMs varies between companies, but the goal is to de ne one or more easily measured endpoints that can be used to monitor the disease course—especially during in vivo treatment with novel drug candidates. In many DDD projects, overt phenotypes are subtle or absent even in GEMs in which high-level expression or total ablation of an engineered gene can be con rmed. This outcome presents a major quandary for biotechnology and pharmaceutical rms: given the signi cant expense and labor required to generate GEMs, what should be done with “negative” constructs? The 14th century philosophical principle known as Occam’s razor—that the simplest explanation for a phenomenon is likely the truth—provides a reasonable basis for pruning potential therapeutic molecules and targets. In the context of DDD, Occam’s razor may be construed to mean that correctly engineered GEMs lacking obvious functional or structural phenotypes have none because the affected gene is not uniquely essential to normal homeostasis or disease progression. Thus, a “negative” GEM construct suggests that the gene under investigation encodes a ligand or target molecule without signi cant therapeutic potential. This interpretation indicates that, at least in a market-driven industrial setting, such “negative” projects should

Received 13 July 2001; accepted 10 October 2001. This symposium is from the ACT 2000 annual conference. Address correspondence to Dr. Brad Bolon, Amgen, One Amgen Center Drive, M/S 5-1-A, Thousand Oaks, CA 91320-1789 , USA. E-mail: [email protected]

be pruned aggressively so that resources may be redirected to more promising DDD ventures.

Keywords

Drug Discovery, Exploratory Toxicology, Functional Genomics, Knock-Out, Mouse, Phenotype, Transgenic

The molecular biology revolution of the past two decades has provided the grist for great leaps in our understanding of disease etiologies and mechanisms. In particular, the recent advent of genetic engineering techniques that allow intentional engineering of novel animal models for human diseases has had a profound impact on basic biomedical research. Additionally, these methods have accelerated the pace at which pharmaceutical and biotechnology rms can discover new targets and innovative drug candidates (126). However, our understanding has lagged with respect to the most ef cient application of this knowledge in drug discovery and development, especially the choice between validation or cancellation of novel targets and therapeutic molecules during ef cacy and exploratory toxicology studies. The present paper brie y reviews the utility of genetically engineered mice (GEMs) in drug discovery and development and then proposes a strategy by which new GEM models may be used to streamline product development decisions and prune unproductive programs at earlier stages of preclinical assessment. GENETIC ENGINEERING: BASIC FACTS FOR EXPERIMENTAL TOXICOLOGISTS Concepts and Denitions Genetic engineering technology is a series of procedures whereby foreign genetic material is introduced into the genome of an organism. Current methods can overexpress, partially suppress, or totally delete a gene’s function (Table 1). In the literature, GEMs that overexpress foreign DNA typically are referred

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B. BOLON AND E. GALBREATH

TABLE 1 Common genetic engineering strategies

Manipulation

Technique

Transgenesis

Gene microinjection Gene therapy Adult animal Conceptus Speci c tissues Conditional transgenesis Gene Targeting Conventional knock-out Knock-out by coaggregation Knock-in Conditional knock-out

References

13, 49, 123, 138 122, 146 12 5, 61, 128 79 16, 106, 110, 130, 154 21, 68, 124 66, 160 4, 53, 54, 63, 103, 104, 133, 134, 148, 149 1, 36, 52, 75, 96, 145

to as transgenic, whereas those in which foreign DNA has replaced an endogenous gene are termed gene targeted (or knockout). In the strict sense, however, both these procedures yield a transgenic mouse (i.e., one with added genetic material); it is this latter de nition (speci ed in the text using quotation marks, i.e., “transgenic”) that is used most frequently in the current review. Foreign DNA may be added at any stage of an animal’s life, either by injecting the “transgene” or “transgene”-containing cells into the embryo or by treating adult animals with a “transgene”bearing gene therapy agent. Genes typically are investigated rst by creating a transgenic model as this task can be accomplished more quickly and at less cost than can a knock-out model for the same gene. Recent innovations have been developed to more speci cally target “transgene” activity to given tissues or stages of development. Two new strategies in particular are gaining widespread acceptance as basic research tools and offer considerable promise for generating speci c models of importance to toxicology. Conditional transgenic mice harbor “transgenes” that contain and are controlled by an inducing or repressing element. Conditional transgenes can be used as a means of avoiding any confounding in uences that result from widespread expression of the engineered gene throughout development (16, 96, 110, 130, 133, 154). In addition, it is now feasible to replace a given mouse gene with its human homolog (103, 104, 133, 145, 148, 162, 164), thereby providing a means of investigating the impact of xenobiotics in vivo on human proteins of interest. This technique reduces a major limitation in animal modeling experiments, i.e., dif culty in de ning the extent to which the intact mouse recapitulates the human response. We anticipate that these techniques will have great utility in drug discovery and development programs in the near future. Any phenotype induced by a novel GEM construct depends on the distribution and extent to which the foreign DNA is ex-

pressed as a functional protein, which in turn varies with many factors. First, the presence of foreign DNA in a given cell population depends on the stage of development at which the “transgene” was added. Integration into a single-celled embryo prior to DNA replication will yield copies in all the GEM cells, including germ cells. Alternatively, integration after replication is nished or division has occurred will result in “transgene” insertion in some but not all cells, yielding a cell lineage – speci c, or mosaic, pattern of gene expression. Second, the nature of the foreign DNA will dictate the integration site. In contrast, targeting (“knock-out” or “knock-in”) protocols employ homologous recombination between identical anking sequences of nucleotides on a targeting construct and the endogenous chromosome within murine embryonic stem cells, thereby replacing the existing gene with a single copy of an engineered sequence. Targeted stem cells are then used to generate chimeric and, when passed in the germ cells, heterozygous and homozygous mice that carry the engineered gene. The properties of the manipulated “transgene” will determine what its functional signi cance will be in the gene-targeted animal. Disruption of the normal coding sequence prevents protein expression and creates a null mutation (a “knock-out”). Insertion of a targeting sequence that contains a “transgene” with constitutive activity may replace the role of the deleted endogenous gene and modify or prevent the knock-out phenotype [a “knock-in”; (53, 104)]. In contrast, transgenic DNA microinjected into a zygote (a fertilized singlecelled ovum) is incorporated into a GEM’s genome at random (83), usually as multiple copies (13) at a single site. Because each integration site is different, each transgenic GEM founder has a unique genetic background. Expression levels will vary greatly between different founder lines containing the same transgene due to divergence in the types of transcriptional control elements that reside in the nearby genomic sequences (9, 26, 107). Finally, numerous genetic and physiological differences may in uence the impact of an engineered gene in the organism (Table 2). For example, differences in the level of transgene expression of certain cytokines in the central nervous system results in the development of either acute (high expression) or chronic progressive (low expression) disease associated with a spectrum of clinical, physiologic and pathologic manifestations (17 ). Phenotypes may decline or become enhanced in later generations (142). TABLE 2 Genetic and physiological factors that in uence “transgenic” phenotypes

Factor

References

Age Breeding history Gender Genetic background Gene dosage Gene function (pleiotropy) Genetic instability

19, 73 142, 153 28, 151 13, 57, 62, 64, 67, 76, 138 19, 78, 80, 108, 118, 131, 144 46, 118, 121 83, 143

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EXPLORATORY TOXICOLOGY WITH ENGINEERED MICE

ANALYSIS OF GENETICALLY ENGINEERED MICE Most GEM models used in toxicological research have been moderately well characterized with respect to both genetic manipulations and phenotypic changes. In contrast, GEMs produced for use in drug discovery and development programs are novel. Thus, their genetic and phenotypic composition must be de ned before useful information may be gathered regarding the mechanism, ef cacy, and potential toxicity of the engineered molecule. Expression Analysis of mRNA and Protein in Genetically Engineered Animals Foreign DNA incorporated into a GEM’s genome can exert its function only if the “transgene” is expressed, or transcribed into message (mRNA) and then translated into functional protein. Either mRNA or protein may be used for expression analysis, but detection of the protein is preferred (if feasible) as the level of “transgene”-derived mRNA often does not correlate with the extent of protein production. In addition, expression patterns for mRNA and protein may not overlap in some tissues. An example of this phenomenon is the nervous system, in which centrally located cell bodies contain RNA and much of the machinery for protein production whereas protein activity often occurs only after transport to far-distant cellular processes. In tissue homogenates, expression of the gene of interest usually is quanti ed by hybridization with a nucleotide probe (for mRNA) or by antibody binding (for protein). Analytical techniques of choice for RNA are the Northern blot assay, reverse transcriptase – polymerase chain reaction (RT-PCR), and the ribonuclease protection assay (RPA). The RT-PCR and RPA methods are more sensitive and speci c because their reactions occur in solution rather than on a membrane. Typically, several probes are prepared to react with sequences spanning the normal and engineered genes, the promoter that drives the engineered gene, and/or the vector (usually bacterial or viral genetic material); the use of multiple probes increases the assay speci city. Common procedures for protein quanti cation are the Western analysis and enzyme biochemical procedures. The former method detects the presence of the molecule, whereas the latter measures both the presence and activity of functional protein. In addition, secreted molecules may be assessed in body uids using enzyme-linked immunosorbent assays (ELISA) or enzyme kinetic tests to detect the physical or functional presence, respectively, of the protein. A special case adopted for many knock-out experiments is to use a targeting vector in which the manipulated gene contains a nonmammalian marker protein, such as the bacterial enzyme ¯-galactosidase (95, 128) or green uorescent protein (65), to increase the discriminating capacity of the assay. Once expression has been con rmed, “transgene” mRNA and/or protein is localized qualitatively either in intact organs or embryos (“whole mount” procedures) or in tissue sections (slide-based procedures). The principal method for demonstrating mRNA is in situ hybridization (158). Methods in which probes are labeled with isotopes are considered to be more

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sensitive but can seldom be used to specify the exact cells in which expression occurs. In contrast, probes conjugated to uorochromes or enzymes often may be used to de ne the cellular and even subcellular distributions of “transgenic” mRNA. Protein distribution is assessed by immunohistochemistry to detect the presence, and/or enzyme histochemistry to demonstrate the presence and activity of functional protein. Antibodies used for immunohistochemistry may be available commercially for more common antigens (84, 156), although cross-reactivity often occurs in GEM tissues due to the mouse origin of many such reagents. In most instances, however, antibodies do not exist for proteins derived from novel “transgenes,” so they must be made and validated before protein can be localized. This latter issue is a common occurrence in drug discovery and development programs that are engaged in research that is based on screening expressed sequence tags (ESTs) for potential therapeutic molecules. (An EST is a mRNA sequence resulting from transcription of a given gene in a speci c tissue; the EST represents all the coding elements of the gene that are needed to manufacture the functional protein.) The laborious and time-consuming nature of this process coupled with the likelihood that the reagent for protein detection will be useless if the novel “transgene” is dropped as a therapeutic target or drug candidate often dictates that “transgene” expression in vivo be inferred only from the mRNA data in high-throughpu t GEM programs. Functional and Structural Phenotyping of Genetically Engineered Mice Expression of an engineered gene in GEMs may induce a functional change, a structural alteration, both of these effects, or neither. Endpoints may be examined either in the intact GEM in vivo or in likely target tissues in vitro. The consequences of genetic modi cation typically are investigated in young adult GEMs using various combinations of conventional anatomic, biochemical, clinical, and molecular methods. The battery of tests used in phenotypic analysis of GEMs varies considerably between companies. However, the goal is always to de ne one or more easily measured endpoints that can be used to monitor the disease course—especially during in vivo treatment with novel drug candidates. Phenotypes have been identi ed in GEM models using many assays. Functional test batteries (Table 3) are selected based on a company’s speci c research focus (e.g., behavioral testing for neuroscience programs) and/or to con rm a pattern of defects predicted by the resemblance of a novel gene’s sequence to a gene family for which a given phenotypic pattern is known. The major advantage of many functional assays is the capacity for repeated in-life measurements. However, the high degree of variation between individual biological responses necessitates large animal numbers; in all cases, data should be compared to the ndings for normal mice, ideally using age-matched wildtype littermates as controls. Morphology often is considered the “gold standard” for phenotypic analysis (101). Speci c endpoints that might be noted at the gross or microscopic level


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TABLE 3 In-life tests used in phenotypic analysis of genetically engineered mice

Assay

References

Behavior Clinical pathology Electrophysiology Flow cytometry Metabolism Imaging Cardiac Gene expression Neural

30, 31, 131 25, 77, 141 19, 131 50, 67, 74 25, 48 58, 87, 125 24, 33, 100, 157 163 8, 59, 72, 119

include alterations in the size (larger, smaller, absent), shape, color, or location of organs, or the presence of aberrant elements (e.g., extra organs, tumors). Any “transgene”-induced phenotype may be further investigated at the cellular and subcellular levels to de ne the mechanism for a given lesion by using in situ molecular pathology methods that correlate anatomic with biochemical data at a given point in time. The latest re nements in imaging rodent organs in vivo have further bridged the gap between functional and structural testing by providing real-time, dynamic assessment of “transgene” expression or function in vivo (8, 25, 58, 87, 100, 119, 125, 157, 163). Pharmacological challenges are another means of discerning subtle phenotypes in GEMs. “Transgenic” animals often appear normal during functional and structural screens until challenged (48, 82). This paradigm seems to be of particular importance in the eld of neurobiology. For example, deletion of a neurotransmitter receptor gene represents the ultimate antagonist, a level of blockade that is seldom reached using pharmacological intervention alone. However, drugs often do not elicit the same effects as a gene knock-out (46, 114, 129). The explana-

tion for this effect is not known, but one possible explanation is that the complete absence of the gene throughout development (knock-out ) engenders compensatory mechanisms that cannot develop during transient blockade provided by administration of the antagonist during adulthood (114). Strategies have been developed to increase throughput in functional genomics screening programs. One approach is to generate individual lines of mice in which multiple genetic alterations have been introduced. In this scheme, several “transgenes” are screened at one time. Discovery of a phenotype in one of these “multimutant” mice is then assigned to the function of a speci c “transgene” by breeding for or engineering mouse lines that contain only one of the different transgenes. In this scenario, loss of the “multimutant” phenotype in single-“transgenic” lines suggests that the previous phenotype resulted from interactive effects between two (or more) of the original transgenes. Another possibility is to analyze founder mice for phenotypes (102). If a potential “transgene”-induced effect is found in multiple animals, additional mouse lines can be engineered and expanded. This approach can greatly decrease the time required for initial characterization of a “transgene” and limits animal husbandry efforts to those lines with potential utility. GENETICALLY ENGINEERED MICE IN EXPLORATORY TOXICOLOGY The use of GEM models in toxicology is relatively recent, but the contributions of these animals have been vitally important to recent advances within the eld. In general, toxicologists select between two main strategies when employing GEMs. First, investigators can choose to utilize an existing model (Table 4). For example, GEMs have been used to assess drug speci city (116), to investigate mechanisms of toxicity (48, 127, 162), and to screen for mutagenic (3, 43, 98, 137 ) and carcinogenic (37, 92, 140) activities of xenobiotics. The genetic and phenotypic effects of these recognized GEM models have been reasonably well characterized. However, the phenotypic response of

TABLE 4 Genetically engineered mouse models in toxicology

Function

Research

Examples

Cell injury

Metabolism Signal Transduction

Testing

Toxicogenetics Mutagenesis Carcinogenesis

Cytokines: interferons (IFN), interleukins (IL-x), tumor necrosis factors (TNF) Death Factors: amyloid precursor protein (APP), huntingtin Enzymes: caspases, matrix metalloproteases (MMP), nitric oxide synthase (NOS), superoxide dismutase (SOD) Phase I: cytochromes P450 Phase II: gamma-glutamyltransferase (GGT) Receptors: AhR, EGF, estrogen, PPAR, RXR Transducers: connexins, cyclases, cyclins, kinases lacI (Big Blue), lacZ (Muta Mouse) Hras2, p53 C=¡, Tg.Ac, xpa Downloaded from ijt.sagepub.com at OhioLink on November 15, 2013

References

18, 20, 38, 117, 127

6, 48, 55, 82, 147, 150, 162 47, 71, 111 104 3, 43, 98, 137 37, 92, 140


“transgenic” mice to a given xenobiotic may depend on such experimental variations as duration and route of exposure. Even for “transgenic” mice in which lesions have been induced by expression of human genes, one must exercise caution in accepting the phenotype, or lack thereof, as a strict analog of naturally occurring lesions in humans (27, 83). In other words, a “humanized” mouse—a “transgenic” animal that contains human genetic material—is still a mouse. Alternatively, GEMs can be made from scratch by manipulating a suitable gene to create a useful phenotype for speci c toxicologic analyses. This latter approach has seen more use in industrial drug discovery and development programs, where novel GEM constructs are continually produced. The GEM effort in such programs generally serves two purposes. First, GEMs may be created to de ne the contribution of a given gene to a disease process (the hypothesis-driven approach). This tactic is chosen because of prior suppositions about gene function as de ned by previous work in detailing gene and protein structure, expression pattern, and any functional studies. Second, GEMs may be generated from novel ESTs to de ne the functions of previously uncharacterized proteins (a biology-driven approach). EST-transgenic GEMs are made by using RT-PCR to transcribe the EST mRNA back to complementary DNA (cDNA), after which the cDNA is microinjected into a mouse embryo. Phenotypes discovered in EST-transgenic GEMs are used principally to understand a transgenic protein’s functions and/or to de ne whether the molecule has any potential as a biopharmaceutical agent; in most instances, toxicity data relevant to the novel transgene is a serendipitous outcome rather than the primary focus of the experiment. Over time, however, robust GEM models may become characterized with enough thoroughnes s to provide new tools for toxicologic pro ling as well. INTERPRETATION OF GENETICALLY ENGINEERED PHENOTYPES “True” Phenotypes In many cases, new GEM models have obvious functional and/or structural phenotypes that can be attributed to the in uence of the engineered gene. Even so, most GEM phenotypes exhibit no known, or at best imperfect, homologies to human disease conditions (51). Ideally, true phenotypes will be robust, meaning that the same genetic manipulation will produce the same lesion pattern in multiple laboratories, even if applied to multiple strains of mice. An example of such robustness is found in mice in which deletion of the serotonin 1A receptor (5-HT1A) leads to an anxiety-like syndrome (56, 109, 118). However, unexpected patterns of gene expression (41) or phenotypes (e.g., paradoxical changes based on current physiological knowledge) are not uncommon (46, 63, 77, 94, 144). In addition, many true phenotypes are quite subtle (34, 77). Concordance should exist between phenotypes resulting from changes to the effector (e.g., ligand and receptor) or the target (e.g., ampli cation cascades for signal transduction) molecules.

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For example, mice transgenic for osteoprotegerin (OPG), a soluble receptor that inhibits osteoclast activity by preventing the binding of OPG ligand (OPGL; also known as RANK ligand, RANKL) to the membrane-bound receptor RANK (receptor activator of NF-·B), were shown to have high circulating OPG levels in conjunction with profound osteopetrosis (135). Subsequently, severe osteoporosis was identi ed in OPG knock-out mice (15, 99), whereas mice with deletions of OPGL and RANK genes were demonstrated to develop extensive osteopetrosis (35, 70, 81). Thus, these GEM-based mechanistic studies provide in vivo evidence that OPG preserves bone and offer a strong rationale for using OPG as a therapeutic agent in bone-eroding diseases. Follow-up studies in other animal models of bone loss using either recombinant OPG (22, 69, 97) or OPG gene therapy (12) have con rmed the ef cacy of this treatment strategy. Comparable rapid-throughpu t experiments can be used as tools for exploring toxicologic issues. For example, neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) promote survival of diverse groups of neurons in both the central (CNS) and peripheral (PNS) nervous systems and, therefore, are being investigated as potential therapies for neurodegenerative conditions. However, certain neurotrophic factors [e.g., NGF (159)] have been demonstrated to induce proliferative lesions in the CNS as a potential side effect of treatment. Recent studies of artemin, a newly discovered GDNF-like ligand (7, 93), and its receptor, GFR®3 (105), demonstrate that both molecules are limited to the PNS in mammals, thereby suggesting that artemin therapy might be employed as a PNS-speci c neuroprotective agent. However, subsequent injection of recombinant artemin into adult mice for 2 weeks induced multifocal hyperplasia of the adrenal medulla during one of three studies. Next, artemin-transgenic mice were generated by microinjection of EST-derived cDNA into embryos to determine whether or not this factor actually resulted in adrenal gland lesions. The founders as well as their progeny developed profound neural crest dysplasia characterized by widespread involvement of the autonomic nervous system, including the adrenal medulla (11). Finally, irradiated adult mice were injected with transfected stromal cells containing the artemin transgene to delineate whether or not this ligand could actually initiate neural expansion in the mature (as opposed to the developing) adrenal gland. Some of these latter transgenic mice also exhibited adrenal medullary lesions, including neuronal metaplasia. Taken together, the ndings in these GEM models indicate that the autonomic nervous system represents a potential target for side-effects associated with artemin administration. Confounding Phenotypes Phenotypes in GEMs cannot be presumed to represent true consequences of the genetic modi cation without rigorous molecular and physiological testing. This fact is predicated by the existence of numerous confounding factors that can in uence or that even mimic the induction of a true phenotype


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TABLE 5 Confounding factors on phenotypic expression

Factor

Environment

Genetic

Examples

Diet Maternal care Maternal malformations Microbial ora Genetic drift Insertional mutagenesis Multiple mutations Species-speci c transgene Strain differences Nonresponders

References

164 14, 42, 46, 85, 86 39 139 113, 142 94, 112, 120, 123, 124, 161 23, 144 44 29, 45, 89 – 91, 134 10, 143, 155

(Table 5). For example, transgene integration into a critical site (insertional mutagenesis) may produce a knockout in which the effects of the unintended mutation are superimposed on changes (if any) induce by the transgene (94, 112, 120, 123, 161). In such cases, the insertion-related phenotype is distinguished from the transgene-related one because insertional events occur in only a single animal line, whereas true transgenic phenotypes are comparable across all lines. In like manner, transgene expression may differ based on the speci c integration site (26) or the presence of regulatory elements within or adjacent to the added material (9, 107). Physiological differences associated with factors such as diet (164), environment (139), and maternal in uences (39, 85, 86) may require investigation of apparent phenotypes under a variety of husbandry conditions. In addition, phenotypes may be altered by background strain (29, 57, 134) and genetic drift (113, 142, 143). Therefore, the genetic milieu of GEMs and their wild-type controls must be identical—ideally by backcrossing to a constant genetic background. This step, although often ignored in high throughput drug discovery efforts, would be essential if a new GEM model were to be used for extended periods in a toxicology screening program. Finally, a consideration for toxicity experiments in which animals are made transgenic using a gene therapy protocol is to demonstrate that a potential phenotype is associated with the activity of the foreign DNA and not with the action of the delivery agent. Adenoviral gene therapy vectors, for example, induce chronic hepatic in ammation and hepatocyte apoptosis, regardless of whether they express a functional transgene or a non-toxic marker protein (12). Negative Constructs Successful introduction of foreign DNA, even if con rmed by genotypic (DNA) and expression (mRNA and/or protein) analyses, may produce no apparent effect on function or structure. Phenotypes may be quite subtle (34, 73, 77) or absent (46, 78, 88), even if a transgene is highly expressed or a targeted gene is completely knocked out. The most frequent explanation for such events is that other molecules compensate for the

presence of the engineered mutation (2, 40, 46, 77, 88, 127, 132). For example, single null mutations of either neuronal or endothelial nitric oxide synthase (NOS) yield no alteration in hippocampal long-term potentiation, whereas deletion of both NOS forms signi cantly impairs neural conduction (136). Genetic background also plays a role in attenuating GEM phenotypes; for example, penetrance may be incomplete (115) unless modi ed by an additional mutation (40). A contrasting view [stated in (32)] is that most “negative” GEMs actually have phenotypes. The alterations are missed because the phenotyping effort relied on insensitive techniques, probed the wrong tissues, or omitted methods (e.g., pharmacological challenges) that could detect the “transgene”-related changes. By this view, inconsistencies between transgenic practices (e.g., use of different mouse strains) and phenotypic test batteries between laboratories could account for the apparently unsuccessful outcomes of many GEM experiments. In some settings, the absence of a “transgenic” phenotype might represent the hoped-for outcome of a drug development program. For example, the generation of viable and fertile lines of mice with null mutations for a potential target protein is a welcome result because it implies that pharmacological blockade of the molecule in vivo will elicit no major adverse effects. In like manner, the apparent lack of an in vivo phenotype could conceivably be used in conjunction with substantial evidence of in vitro ef cacy to support the selection of a likely “noobservable-effect level” (NOEL) for use in preclinical pharmacology and toxicology studies. In most instances, however, the absence of a pronounced phenotype is a serious setback in the context of a drug discovery program based on characterizing the functions of novel genes. In this setting, GEMs— especially knock-out mice—often are considered to have little value because they so frequently develop an unexpected phenotype, a subtle phenotype, or no visible phenotype. This outcome presents biotechnology and pharmaceutical rms with a major quandary: given the signi cant expense and effort required to generate a single GEM, let alone a host of GEMs, what should be done with the large numbers of “negative” constructs?

ANALYSIS OF GENETICALLY ENGINEERED MICE IN A HIGH VOLUME SETTING In the rapid-throughput industrial setting, a reasonable basis for managing such “failed” experiments may be found in the philosophical proposition known as Occam’s razor, or the law of parsimony. Occam’s razor (60, 152) is a 14th century principle attributed to the English logician and theologian William of Ockham (Occam), who lived ca. 1285 to ca. 1347. His law, “Entia non sunt multiplicanda sine necessitate,” may be translated as “The number of entities required to explain a thing should not be increased beyond what is necessary.” Although promulgated originally as a theological precept, this principle may be applied in a modern scienti c context to mean that researchers should make no more



assumptions than necessary when interpreting an experiment. In other words, for a set of otherwise equivalent models of a given data set, the simplest explanation is likely the model that is closest to the truth. Of what utility is this law to GEM programs in drug discovery and development? A major obstacle in pharmaceutical produc tion is distinguishing at an early stage between potential targets and therapeutic candidates and the many molecules that will be discarded at some later premarket stage. In a high-volume setting, Occam’s razor may be construed to mean that novel GEM models lacking obvious phenotypes have none because the affected gene is not suf ciently essential to homeostasis or centrally engaged in progression of a given disease to substantially impact the animal’s physiological status. Thus, a “negative” GEM construct suggests that the gene under investigation likely encodes an effector or target molecule without ef cacy, and therefore without signi cant therapeutic potential. This interpretation indicates that, at least in a market-driven industrial setting, such “negative” projects should be pruned aggressively so that resources may be redirected to more promising drug development ventures. As noted above (32), it is feasible that expanding the number of screening tests employed during phenotypic analysis could discover one or more subtle phenotypes. However, we submit that in the context of the biopharmaceutical and pharmaceutical industries, novel GEMs should be discarded if prominent functional and/or structural changes are not detected using a targeted test battery that is selected deliberately to discover phenotypes relevant to organ systems or disease conditions of interest to that speci c laboratory. In this manner, human and capital resources can be reallocated rapidly to characterize other GEMs with potential phenotypes that might yield new animal models of disease, therapeutic targets, or drug candidates. REFERENCES 1. Akagi, K., et al. 1997. Cre-mediated somatic site-speci c recombination in mice. Nucl. Acids Res. 25:1766 – 1773. 2. Arnold, H. H., and T. Braun. 1996. Targeted inactivation of myogeni c factor genes reveals their role during mouse myogenesis : A review. Int. J. Dev. Biol. 40:345 – 353. 3. Ashby, J., and H. Tinwell. 1994. Use of transgenic mouse lacI/Z mutation assays in genetic toxicology. Mutagenesis 9:179 – 181. 4. Askew, G. R., T. Doetschman, and J. B. Lingrel. 1993. Site-directed point mutations in embryoni c stem cells: A gene-targeting tag-and-exchang e strategy. Mol. Cell. Biol. 13:4115 – 4124. 5. Baldwin, H. S., C. Mickanin, and C. Buck. 1997. Adenovirus-mediate d gene transfer during initial organogenesi s in the mammalian embryo is promoter-dependen t and tissue-speci c. Gene Ther. 4:1142 – 1149. 6. Ballatori, N., W. Wang, and M. W. Lieberman. 1998. Accelerated methylmercury elimination in gamma-glutamyl transpeptidase-de cient mice. Am. J. Pathol. 152:1049 – 1055. 7. Baloh, R. H., et al. 1998. Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFR®3-RET receptor complex. Neuron 21:1291 – 1302. 8. Benveniste, H., K. Kim, L. Zhang, and G. A. Johnson. 2000. Magnetic resonance microscopy of the C57BL mouse brain. Neuroimage 11:601 – 611.

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