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February 11, 1997 9:51 Annual Reviews AR27-06 AR27-06

Annu. Rev. Pharmacol. Toxicol. 1997. 37:119–41Copyright c© 1997 by Annual Reviews Inc. All rights reserved

TRANSGENIC ANIMALSAS NEW APPROACHES INPHARMACOLOGICAL STUDIES

Li-Na WeiDepartment of Pharmacology, University of Minnesota, 3-249 Millard Hall,435 Delaware St. S.E., Minneapolis, Minnesota 55455

KEY WORDS: pronuclear injection, ES technology, gene-targeting, animal models

ABSTRACT

Transgenic animals are becoming useful tools for pharmacological studies. Theuse of transgenic technology raises two types of questions, “How are transgenicanimals made?” and “What types of pharmacological questions can be answeredusing transgenic technologies?” Answers to these questions are discussed in thisreview. The production of animals with specific genetic alteration can be achievedby two strategies. The first involves the simple addition of DNA sequences to thechromosomes. The second strategy is to select particular genetic loci for site-specific changes. There are two well-established procedures for simple introduc-tion of DNA into an animal genome, pronuclear DNA injection and transductionusing a retrovirus. In contrast, methods for targeting specific DNA sequences todefinite sites in the chromosomes are evolving rapidly. Some of these procedurescan be used in combination to make a different variety of gene alterations in ani-mals. Pharmacological studies where transgenic technology has been extensivelyused are discussed, including studies in the cardiovascular system, the nervoussystem, the endocrine system, cancer, and toxicology.

INTRODUCTION

The use of genetically altered animals has become routine in many fields ofbiomedical science over the past decade. For investigators that utilize mammalsas experimental systems, this technical development has promised an unprece-dented opportunity for a wide variety of genetic experiments in animals to bedone in a much more sophisticated manner and within a much shorter time.

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Pharmacological research, particularly in whole animal systems, has also ben-efited largely from the progress of transgenic methodology. This review firstgives an overview of recent progress in different gene transfer methods usedfor mammalian species, followed by a discussion of the application of thesemethods in several pharmacological subjects, and finally provides a prospectiveview about these approaches in future pharmacological research.

METHODS OF TRANSGENIC TECHNOLOGY

The HistoryJaenisch & Mintz first demonstrated that foreign DNA, such as SV40 viralDNA, could be introduced into fertilized mouse eggs and detected in varioustissues of animals that developed from these embryos (1). Subsequently, usingthe Moloney murine leukemia retrovirus to infect mouse embryos, they showedthat viral sequences could be stably integrated into the genome of mouse eggsand passed to various tissues of the animals derived from these embryos (2, 3).Because of the limited capacity of the retroviral genome, retroviral infectionproved to be less applicable to generate transgenic animals despite its poten-tial use in human somatic gene therapy. In contrast, direct microinjection ofpurified DNA into the pronuclei of fertilized, one-cell eggs appeared to bean alternative method for delivering DNA molecules larger than the retroviralpackaging capacity (approximately 7 kilobases). Within four years, a number ofgroups demonstrated successful integration and expression of foreign DNA intransgenic mouse tissues using the method of pronuclear microinjection (4–9),thereby establishing the method of pronuclear microinjection as a standardprocedure to produce transgenic animals.

Despite the efficiency and consistency of pronuclear microinjection to createtransgenic animals, studies using this approach were hindered by three majorproblems: the inability to control sites of integration and copy numbers oftransgenes and embryonic lethality due to toxic effects of the expression ofcertain genes. Random integration and multiple copies of transgenes lead tounregulated expression of transgenes and cause side effects, while expressionof other genes can be cytotoxic. To circumvent these problems, a technique thatcan deliver a single copy of a mutated gene to a specific target site was developed.

Gossler first demonstrated in 1968 that embryonic cells could be isolatedand injected into blastocyst-stage embryos of different genetic backgrounds,and that these injected embryos could be transferred to the uterus of fostermothers to develop into chimeric mice composed of two cell populations withgenetic characteristics corresponding to the recipient embryos and the injectedcells, respectively (10). As embryonic cells suitable for injection could not beisolated in large quantity, a series of critical embryological techniques were used



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to test whether embryonic cells could be propagated in vitro while retainingtheir potential to colonize various mouse tissues. Finally, a procedure was suc-cessfully developed for the isolation of embryonic stem (ES) cells from certainstages of embryos such as morulae or blastocysts (11, 12), which could maintaintheir pleuripotential characteristic in culture, followed by the transfer of theseES cells back to embryos at appropriate developmental stages for the produc-tion of chimeric mice (13, 14). One important goal in the development of thistechnology was to obtain chimeric mice containing gametes derived from theseES cells so that the genetic composition of the ES cells could be passed on tothe offspring. This result is referred to as germ-line chimerism. ES technologyhas provided two advantages over the technique of pronuclear DNA injection.First, specific mutations can be made in ES cells by a gene-targeting procedure(see the following) to avoid the problems of nonspecific sites of transgene inte-gration and multiple copies of the transgenes, which eventually can be deliveredinto the mouse genome via germ-line chimerism. Secondly, most mutationscan be carried by the chimeric animals or maintained in the heterozygote statein the offspring without jeopardizing the survival of animals.

The problem of the lack of specificity in transgene integration was cir-cumvented by the development of a homologous recombination-based gene-targeting procedure (15, 16). A mutated DNA sequence can be delivered to itshomologous locus by using a recombinant DNA vector (the targeting vector)that allows homologous recombination to occur between this targeting vectorcarrying a specifically modified gene segment and its endogenous counterpartat the homologous regions, thereby altering one of the normal alleles. In theory,it would be possible to make changes in all the isolated genes and introducethese mutations into ES cells using this strategy. The genetically altered ES cellscould then be incorporated into chimeric mice and then into the genomes ofheterozygote animals derived from these chimeric mice via germ-line transmis-sion. Ultimately, homozygote animals with both alleles altered the same waycould be obtained by crossing these heterozygote animals. Two successful gene-targeting experiments in mice were reported in 1989 (17, 18). Subsequently, byintroducing multiple drug-resistance markers into the targeting vectors, the se-lection efficiency of ES cells harboring homologously recombined alleles wasimproved, allowing the gene-targeting procedure to be routinely and widelyused for different purposes.

By definition, the mice with specific genetic alteration derived from mu-tated ES cells are “transgenic” mice, even though the transgenic material ismore of a replacement than an addition to the genome, in contrast to transgenicmice generated by pronuclear microinjection. Unfortunately, ES cell technol-ogy is available only for the mouse, despite tremendous amounts of effort in-vested for other species. Currently, hundreds of gene-targeted mouse lines have



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Table 1 A brief review of transgenic techniques

Development of pronuclear injection Development of gene-targeting/ES injection

1968 Gardner (10)Embryonic cell injection into blastocysts

to produce chimaeric mice1974 Jaenisch & Minz (1)

SV40 DNA injection intobalstocyst-stage embryos

1975 Jaenisch et al (2)MMLV retroviral infection

of pre-implantation-stage embryos1976 Jaenisch (3)

Germ-line transmission of transgene1980 Gordon et al (4)

Pronuclear injection1981 Brinster et al (5); Constantini & Lacy (6) Evans & Kaufman (12); Martin (11)

Successful transgene integration/expression Establishment of pluripotent stem-cell lines1984 Bradlet et al (13)

Germ-line chimaeras from teratocarcinoma cells1986 Gossler et al (14)

Germ-line chimeras from ES cells1987 Thomas & Capecchi (15)

Homologous recombination in ES cells1989 Schwartzberg et al (17); Thompson et al (18)

Gene-targeting in mice

been generated and the number of mutant mouse lines is estimated to increasetremendously as more genes are isolated and mutated (for reviews, see 19–21).Table 1 provides a brief historical review of the progress of this technology.Currently, various gene-targeting strategies are being improved to allow moresophisticated genetic experiments to be done in animals. Although transgenicmice generated by pronuclear DNA injection are not perfect animal models,they can be suitable for certain biological questions such as consequences ofover expression or ectopic expression of specific biological molecules in ani-mals. In addition, many newly developed gene-targeting strategies require thecombined use of transgenic mice generated by pronuclear DNA injection andgene-targeted mice. Therefore, current progress in this technology includesexperimental designs for both pronuclear DNA injection and gene-targetingprocedures.

Recent Progress in Pronuclear DNA InjectionThe principle of pronuclear DNA injection to generate transgenic animals isto construct a piece of recombinant DNA with genes of interest engineered in




a specific way, followed by the injection of linearized DNA into the nuclei offertilized embryos at the one-cell stage. One major improvement made overthe years is to construct genes that can be regulated by specific and induciblepromoters. Regardless of the ultimate goals of the experiment, the immediatepurposes of generating transgenic mice generally fall into three categories: (a)the production of specific molecules, (b) the blockage of specific biologicalpathways, and (c) the studies of gene regulatory mechanisms. In the following,general considerations and recent improvements are provided for these threetypes of experiments. For technical detail, readers are referred to the lab manualby Hogan et al (22).

EXPRESSION OF BIOLOGICAL MOLECULES TO STUDY THE EFFECTS OF THEIR EX-

PRESSION IN ANIMALS, OR TO PRODUCE PHARMACEUTICAL AGENTS To achieveoptimal specificity of transgene expression, tissue-specific promoters are gen-erally used. To obtain maximal quantity of the gene product and to regulate itsexpression, inducible promoters are preferred.

A specific promoter fused to an appropriate translational control elementis critical for successful specific transgene expression. For this reason, recentprogress has been made mainly to dissect regulatory DNA elements to be usedfor tissue- and developmental stage–specific expression of the transgene. Inaddition to the 5′-flanking regulatory sequences, inclusion of introns in theexpression vector was also found to improve the efficiency of tissue-specificexpression (for reviews see 23 and references therein).

To control the expression of the transgene, improvements were made mainlyin the development of inducible promoter systems, such as the heavy metal–inducible promoter of the metallothionein (MT) gene (24), a tetracycline-inducible system (for review see 25), an estrogen-inducible system (26), andan ecdysome-inducible system (27). The human MT promoter was first tried,which appeared to lack the desired specificity and inducibility by heavy metals(24). The tetracycline-, estrogen-, and ecdysome-inducible systems are binarysystems consisting of two sets of transgenes (Figure 1). The expression of aspecific cDNA (designated as transgene-2) is defined by that of transgene-1(encoding a transactivator for the promoter of transgene-2) and by the presenceor absence of the inducer. The potential use of the tetracycline-inducible sys-tem in transgenic mice has been demonstrated in several studies (28, 29). Boththe estrogen- and ecdysome-systems were only recently developed, and theirapplication awaits further demonstrations.

TRANSGENES DESIGNED TO BLOCK BIOLOGICAL PATHWAYS RELATED TO A SPE-

CIFIC GENE PRODUCT Before the gene-targeting technique became a widelyavailable procedure, the question of lack of specific protein function was


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Figure 1 A strategy for conducting inducible, tissue-specific expression of a gene of interestin transgenic animals. Transgene-1 (Tg-1) utilizes a tissue-specific promoter-A (Pr) to directthe expression of the transactivator (TA) for the tetracycline operon (Tet-op). Transgene-2 (Tg-2)encodes the cDNA of interest under the control of Tet-op. In the absence of Tet, TA producedin Pr-specific tissues is active, thereby activating expression of Tg-2 in Pr-specific tissue. In thepresence of Tet, TA remains silent, and no Tg-2 is expressed (25).

addressed using several strategies, i.e. antisense RNA blockage, specific cellablation, and dominant negative mutation.

It was believed that antisense RNA formed a double-stranded complex withthe endogenous mRNA precursor, which could be rapidly degraded by nu-cleases specific to double-stranded regions, screwed up RNA processing, andcaused arrest of translation (30–32). Using appropriate promoters, antisenseRNA could be directed to specific cell types, thereby blocking the expressionof protein from specific endogenous mRNAs in these cells. This approach wassuccessfully applied in several studies (33, 34). Specific cell ablation made useof toxin genes linked to promoters specific to certain cell types. Two widelyused toxin genes were the viral thymidine kinase (tK) gene and the diphtheriatoxin A gene (DT-A) (35). For example, in transgenic mice expressing tK underthe control of a thyroglobulin promoter, the thyrocytes were ablated in animalsinfused with ganciclovir (36). Because the wild-type DT was too toxic when ex-pressed from a strong promoter, an attenuated form for the DT-A gene, tox-176,was engineered that proved to be more effective in achieving genetic ablation intransgenic mice (37). Dominant negative mutations were also successfully used




to address the effects of blocking the function of certain genes. For example,dominant negative retinoic acid receptors (RARs) were used in transgenic miceto address the effects of blocking specific functional domains of these receptorsin specific organ systems (38, 39). In general, the use of dominant negativemutations requires information such as detailed characterization of functionaldomains of the proteins and promoters specific to the tissues (or cells) of interest.

In summary, various approaches are available to achieve the goal of blockingspecific biological pathways in animals. In different experimental systems, thedesign of transgenes depends upon the available information for the gene ofinterest and the purposes of the experiments.

TRANSGENES DESIGNED TO STUDY GENE REGULATION Studies of only somekinds of gene regulation can be conducted in established cell lines. However,in general, to vigorously examine gene regulation in different tissues and atdifferent developmental times requires studies in animals.

One typical approach for this purpose utilizes reporter genes consisting of theregulatory sequence of a gene under investigation and a poly (A)-tailed cDNAsequence coding for an enzyme that can be assayed easily, such as chloram-phenicol acetyl transferase (CAT),β-galactosidase (lacZ), or luciferase (Luc).All of these reporters share two common features: The enzyme activity is gen-erally lacking in mammals, and the activity can be determined by simple assays.The most elegant example of using a reporter transgene to identify genetic el-ements responsible for tissue- or developmental time–specific regulation is theuse oflacZ reporter to identify temporal and spatial regulatory DNA elements.The assay forlacZactivity can be carried out by in situ or quantitative analysis(40); therefore, it is possible to follow changing patterns of promoter activi-ties in developing animals in a three-dimensional manner under a stereoscope(41, 42). Detailed expression patterns at a cellular level can be examined onselected tissue sections once specific regions are located. The assays for theother two reporters, CAT and Luc, are more sensitive; therefore, they are pre-ferred when the sensitivity of detection is a concern. However, for technicalreasons, these two systems are not as convenient as thelacZ system in the insitu analysis of transgene expression. Therefore, the choice of reporter systemsdepends primarily on the purposes of the experiments. Using this approach,DNA sequences responsible for gene expression in animals can be dissected,and the regulatory mechanism for expression, such as that of specific drugs,can be addressed in the context of whole animals.

Recent Progress in Gene-TargetingTwo principal procedures are required to perform gene-targeting experimentsin animals, to manipulate ES cells, and to design gene-targeting vectors.



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ES CELL TECHNOLOGY ES cells are generally derived from the inner cell massof blastocysts and maintained in cultures in the presence of a differentiation in-hibiting factor such as leukemia inhibition factor (LIF) (43) in order to maintaintheir totipotency. Upon introduction into host embryos by either aggregationwith eight-cell stage morulae or injection into the blastocele (for review see44), the culture-derived stem cells can integrate into the embryonic stem-cellpool and populate in various tissues of the chimeric conceptus and adult. Whenthe cells are introduced into the recipient embryos, the most critical criterionfor a good ES cell line is its totipotency, especially the potential of the cells tocolonize the germ-cell lineages (13). To date, many ES cell lines have beenestablished, but only a few of them have been demonstrated for their potential ingerm-line transmission. Most ES cells require feeder cells, such as embryonicfibroblasts, but ES cell lines that are able to maintain their totipotency withouta feeder layer have also begun to be established (45). The choice of a particularES cell line in gene-targeting experiments depends upon the individual lab’sexperience and preference. More work is needed to address the biological basisof the unique characteristic of ES cells.

Once ES cell clones containing the desired mutation are selected, they can beintroduced into embryos by two major routes, either aggregation with eight-cellstage morulae or injection into the blastocele. Although both routes have beenused successfully to generate chimeric mice, most studies rely on blastocystinjection because the success rates of aggregation method are highly variableamong individual laboratories. Since the first report of producing ES-derivedmice using a modified aggregation method (46), this technique has been underintensive experimentation to improve its consistency and efficiency in manylaboratories (A Nagy, personal communication). Therefore, the potential of theaggregation method as a routine procedure for the production of chimeric miceawaits further demonstration of its reproducibility by different labs. Currently,the blastocyst injection procedure is used routinely in most laboratories. Fortechnical detail, readers are referred to the lab manual by Hogan et al (22).

DESIGN OF GENE-TARGETING VECTORS Another critical factor for success ingene-targeting experiments is the design of targeting vectors that allow efficientselection for homologous recombinants. The principle of the prototype vector isto alter the endogenous sequence with a mutated sequence carried in a targetingvector, designed to “knock-out” the function of a specific gene. This representsthe first step to disclose the function of a gene in animals. Two prototypetargeting vectors, insertion vector and replacement vector, were designed (15)to knock-out a gene (Figure 2). One modification in designing a replacementvector is the addition of a second selection marker, such as the viral tK gene,to one end of the replacement vector in order to perform a double-selection




Figure 2 Two prototype gene-targeting strategies. (A) Replacement vector is designed suchthat homologous pairing between the linearized vector and the endogenous sequence allows twocrossing-over events to occur within the two homologous regions flanking a mutated sequence(such as by an interruption with a Neo maker). Thus, the vector sequence replaces the endogenoussequence and renders the recombined allele Neo+. (B) The insertion vector is designed such thatthe continuous genomic segment (segment 6 here, as an example) is interrupted upon linearizationof the vector. With one crossing-over event, the entire sequence is inserted into the endogenoussequence, resulting in the disruption of the continuity of the endogenous sequence. [After Thomas& Capecchi (15).]

procedure, i.e. positive (Neo+) and negative (tK−) selections (Figure 3A). ThetK gene, added outside the homologous regions, is deleted in the homologouslyrecombined alleles but retained in those alleles containing the entire vectoras a result of random integration. Thus, homologous recombinants can beenriched by using ganciclovir, which kills the cells exhibiting the Neo+/tK+

phenotype, owing to random integration of the entire vector (16). For thisreason, most gene-targeting experiments employed the double selection strategyfor the design of replacement vectors.

Because the knock-out strategy theoretically results in the complete loss ofgene function, attempts have been made to alter genes in a subtle fashion bya procedure, named “Hit and Run” (47) or “In-Out” (48), which is carried out



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Figure 3 (A) Tissue-specific gene-targeting. The endogenous sequence to be deleted is firstmodified by a gene-targeting event, resulting in the sequence flanked by a pair ofloxP sequencesthat do not disrupt the transcribed region. (B) A transgenic mouse line is produced, carrying theCre enzyme under the control of a specific promoter. (C) By breeding the two transgenic mice(from stepsA and B), the offspring animals express Cre in specific tissues, rendering segment∗deleted in these tissue as a result of site-specific recombination via the twoloxsites (stepD). [AfterGu et al (62).]




in two steps. The targeting vector containing a mutated sequence is inserted togenerate a duplication of a portion of the gene. Then, the normal sequence isexcised as a result of intrachromosomal recombination. Because very subtlemutations can be engineered in the targeting vector by standard molecular tech-niques, it is possible to introduce a subtle alteration into the genome using thisprocedure. For instance, a mouse model for familial hypertrophic cardiomy-opathy was made by introducing an Arg403/Gln mutation into theα cardiacmyosin heavy chain gene using this approach (49). Several modified proce-dures have also been developed to improve the efficiency of altering genomein a subtle manner, such as the “Tag-and-Exchange” procedure (50, 51) andan in situ gene repair by combining a site-specific recombinase system (seebelow) and homologous recombination events (for reviews and details see 52,53 and references therein). However, all these procedures were developed onlyrecently by using several test genes. More studies are required to demonstrategeneral application of these procedures.

To extend the scope of changes that can be made by simple gene-targetingprocedures, the basic scheme of positive and negative selection was furthermodified for various purposes (for review see 52), such as incorporation ofa reporter into the replaced allele, which allows the expression pattern of the“knock-out” gene to be followed (54, 55), rescue of mutation by a “knock-in” re-placement gene (56), and a wide range of modifications based upon site-specific,recombinase-mediated gene-targeting (57, 58). Two site-specific recombinasesystems have been developed, one utilizing a yeast recombinase and its recom-bination targets (59, 60) and the other utilizing the P1 phage recombinase Creand its target, theloxP sequence (61, 62). The Cre-loxP system appears to bemore effective and is also applicable for a large DNA fragment. An examplehas been reported indicating that chromosomal deletions, inversions, and du-plications extending to 3–4 cM could be constructed in embryonic stem cellsusing the Cre-loxPsystem (63). Like the modified methods for creating subtlemutations, most of these procedures are still in the experimental stage and theirfuture application awaits further demonstration in mice.

Future DirectionsThe basic tools to mutate a mouse genome, such as pronuclear DNA injec-tion, blastocyst injection, and simple gene-targeting to produce null mutations,are well established. However, other modified gene-targeting procedures andthe aggregation method to introduce ES cells into embryos need fine tuning.With these available tools, future development of genetic manipulation in micewill focus on several biological and technical problems. The first problem isthe lack of sufficient information regarding regulatory machinery for specificgene expression in animals. This is not only an important biological question,



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but also an essential piece of information required for many transgenic experi-ments. Secondly, the ES technique can be improved and simplified. The lackof good ES cell lines for other animal species has prevented gene-targetingexperiments to be conducted in animals other than the mouse. In addition, thecurrent injection procedure requires sophisticated equipment and is time con-suming. A simplified procedure such as aggregation will greatly facilitate thistype of work. Finally, the design of experimental procedures that allow thesetools to be used in combination for specific purposes can also be improved. Forinstance, the strategy utilizing thelacZ reporter in a gene-targeting experimentto follow the expression pattern(s) of a knock-out gene is very successful (54,55). With a simple modification, one can examine not only the consequences ofdeleting a specific gene but also the expression pattern of this particular gene.Thus, the defects of replacing a specific gene withlacZ can be traced at thecellular level by following thelacZstaining areas.

Another potential strategy to be further developed involves two transgenicsystems, one utilizing thecregene under the control of a specific promoter andthe other utilizing a gene-targeting method to deliver theloxP sites to specificregions of the genome (Figure 3). When these two systems are combined,recombination of a specific genomic segment via theloxP sites depends uponthe presence of the Cre enzyme, which is regulated by a specific promoter.Thus, various types of gene alteration, defined by the arrangement of theloxsites in the genome, can be controlled by the use of a specific promoter drivingthecregene. These types of experiment are ongoing in many labs.

In summary, with recent advancements in the tools for manipulating the EScell genome, a wide variety of genetic experiments can be conducted in animalsto address different questions. One critical challenge remains in the identifi-cation of tissue- and cell-specific regulatory elements. This basic informationis most important for the production of gene-targeted animals, where desiredphenotypes can be specifically regulated in order to achieve the highest levelof precision and specificity in these animal experiments.

APPLICATIONS IN PHARMACOLOGICAL RESEARCH

Essentially all pharmacological subjects have benefited from studies of trans-genic animals. It is also possible to produce pharmaceuticals from transgenicanimals (mostly large animals), although this method has proven to be success-ful only for certain products (64). Because the prerequisite for utilizing trans-genic animals is the identification and cloning of the genes that are potentialtargets of the drugs (such as the receptors) or that are involved in the signalingpathways of drug actions (such as enzymes for metabolism or components me-diating downstream events), the progress and the effectiveness of studies using




transgenic animal models to answer different pharmacological questions varieswidely. In general, most progress was made in the areas where target geneshave been characterized. Examples of transgenic application in pharmacolog-ical studies are given for several subject areas in the following sections.

Cardiovascular SystemCardiovascular drugs act on several different targets, such as the renin/angioten-sin system (RAS),β-adrenergic receptor system (βAR), ion channels, andlipoprotein metabolism. Among these target systems, the RAS, the lipoproteinmetabolism, and theβAR system are better characterized in terms of the genesinvolved; therefore, the use of transgenic animal models in these areas is betterdocumented.

The RAS system regulates hemodynamics and water and electrolyte balance,thereby controlling blood pressure and volume. Several genes involved in theproduction of the dominant peptide of this system, angiotensin II, are well char-acterized and have been used to produce transgenic animals for studies of theregulation of this system and the effects of drugs acting on this system. Thefirst transgenic animal models were transgenic mice expressing the rat reninand angiotensinogen genes (65). Because hypertension is virtually unknown inmice but is well studied in rats (66), transgenic rat models are more desirablefor functional studies (for review see 67). The first hypertensive transgenic ratmodel carried the mouse Ren-2 genomic DNA (68), which exhibited a cleargene dose-effect of renin on the development of high blood pressure and an ob-vious sexual dimorphism with respect to the level of blood pressure elevation.In addition, this hypertensive rat model responded to pharmacological manip-ulation of the RAS and allowed its hormonal interaction to be examined. Forexample, angiotensin receptor antagonists such as losartan, telmisartan, or theconverting enzyme inhibitor lisinopril were able to effectively lower blood pres-sure in these animals. In addition, studies of adrenal gland activity and steroidmetabolism in this model provided more information about the relationshipbetween renin and other hormonal systems, such as adrenal steroids (69).

In order to shed light on the role of human renin in hypertension developmentand to study drugs acting on this human protein, transgenic rats carrying thehuman renin or angiotensinogen gene were also generated (70). In these animalmodels, species-specificity of renin substrate was demonstrated, allowing thestudies of renin inhibitors and their enzyme kinetics to be conducted in vivo.As gene-targeting is not yet available for the rat, gene knock-out in this systemhas been conducted only in the mouse. Several mutant mouse lines have beendeveloped in this area. For example, the angiotensin-converting enzyme (ACE)-deficient mice exhibited a sexual dimorphism in reproductive activity and bloodpressure control (71). The angiotensin II type-2 (AT2) receptor-targeted mice



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developed an increased sensitivity to pressor action of angiotensin II (72), re-vealing an interesting relationship between the RAS and the nervous system.

The lipoproteins are responsible for transporting plasma cholesterol andtriglycerides, thereby regulating cholesterol homeostasis. Genetic and epidemi-ological studies have established a relationship between abnormal lipopro-tein/receptor content and the development of coronary heart diseases. Therefore,transgenic mouse models created for pathophysiological and pharmacologicalstudies in this system fall into two major categories: (a) models in whichreceptor function has been altered, and (b) models in which the productionof the lipoprotein ligands is affected (for reviews see 73, 74, and referencestherein). Manipulation of the receptor function included overexpression ofLDLR and receptor-associated proteins, and knock-out of LDLR and LDLR-related proteins. Manipulation of the lipoprotein ligand included overexpres-sion of ApoE, ApoB, ApoC, lipoprotein lipase, and cholesteryl ester transferprotein, and knock-out of ApoE and ApoB. Transgenic animal models withaltered LDLR provided new insights into metabolic functions of LDLR, suchas the clearance of lipoprotein. The LDLR-deficient mice accumulated signif-icant amounts of LDL and were highly susceptible to atherosclerotic lesionswhen fed a cholesterol-rich diet, reflecting the phenotype of corresponding hu-man disease. Thus, the studies of the lipoprotein transgenic mice with eitheroverexpression or deficiency of these genes highlighted the importance of mul-tiple gene interactions in the regulation of HDL, and provided strong evidencefor a direct anti-atherogenic role of HDL. These animals are potential mod-els for pharmacological studies of drugs that lower plasma concentrations oflipoproteins by either diminishing their production or stimulating removal.

TheβARs are the primary myocardial targets of the neurotransmitters nore-pinephrine and epinephrine. Activation ofβARs in the heart causes stimulationof the adenyl cyclase cascade, resulting in positive chronotropy and inotropy.Therefore, transgenic animals were created to address the role of this recep-tor system in cardiac function and to study therapeutic agents for augmentingmyocardial function via this receptor system. Several transgenic mouse mod-els were made by using the strategy of tissue-specific overexpression of thecomponents of the adrenergic system, including a normal humanβ2AR (75),a constitutively activeα1AR mutant (76), a short isoform of Gsα (77), aβARkinase (βARK), and aβARK inhibitor (βARKI) (78). All these studies uti-lized theα-myosin heavy chain (α-MHC) promoter, which was shown to bespecifically active in the atria and ventricles of transgenic mice (79). Theβ2AR transgene expression resulted in enhanced myocardial function such asincreased basal myocardial adenylyl cyclase activity, enhanced atrial contrac-tility, and increased left ventricular function, providing an experimental basisfor gene therapy in the case of heart failure. The transgenicβARK andβARKI




studies demonstrated a role of this kinase system in heart disease. The Gsα

transgenic mice exhibited increased high-affinity receptor sites and enhancedadenylyl cylase activation, allowing one to examine the consequences of recep-tor activation in animals. Mutant mice deficient in theβ3-adrenergic receptorwere also made (80), proving that the actions ofβ3 agonist CL316243 weremediated exclusively by the atypicalβ-AR, β3-ARs, which were detected pre-dominantly in white and brown adipose tissue. This animal model provided themeans for a better understanding of the pharmacology ofβ3-ARs and potentialanti-obesity drugs.

Nervous SystemThe targets for drugs affecting cognition and behavior can be neurotransmitters,their receptors, or components involved in their effector systems. Therefore,transgenic animals have been created to dissect molecular events at each targetsite by manipulation of specific genes known to be involved (for reviews see81–83 and references therein).

The first use of mouse genetics to investigate higher order cognitive functionswas based upon the hypothesis that long-term potentiation (LTP) or LTP-relatedchanges underlined the formation of memory. These changes were evoked byactivation of receptors. Several major players in these effector systems were mu-tated in the mouse by gene-targeting, including the Ca2+/calmodulin-dependentprotein kinase II (CaMKII), the protein tyrosine kinasefyn, the neuronal nitricoxide synthase (nNOS), the protein kinase C-γ (PKCγ ), and the cAMP responseelement–binding protein (CREB). These animals have provided powerful toolsfor further pharmacological and behavioral tests. For example, the CaMKII-deficient mice exhibited an abnormal fear response, aggressive behavior, andreduced serotonin release (84). The nNOS-deficient mice also exhibited aggres-sive behavior and were resistant to neural stroke damage (85, 86). Althoughthese mutant mice demonstrated that the function of these molecules was re-quired for the development of normal hippocampal LTP, spatial learning, andnormal behavior in adult mice, the possibility that structural changes of the brainas a result of deficiency in these proteins caused these electrophysiological andbehavioral deficits could not be excluded.

The second group of potential target genes in the nervous system was thegenes related to neurotransmitters and their receptors. It was less effective toalter neurotransmitter synthesis or release by manipulation of a single gene dueto multiple biosynthetic steps involved. Therefore, a more practical approachwas to manipulate the genes for these receptors. To address the function of thesereceptors, either antisense-RNA blockage or gene-targeting knock-out was thechoice. For instance, the antisense strategy was used to block the expression ofneuropeptide Y receptor, the dopamine D2 receptor, and theδ-opioid receptor.



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Although some pharmacological effects were observed in these animals, thisstrategy was generally less effective because of nonspecific and partial inhibi-tion of receptor expression. Many mutant mice were generated by using thegene-targeting approach, which lacked specific components involved in theseneurotransmitter and receptor systems, such as 5-HT1B (87), 5-HT2C (88), rap-syn (89), monoamine oxidase A (90), brain-derived neurotrophic factor (91),tyrosine hydroxylase (92), nicotinic acetylcholine receptor (93),N-methyl-D-aspartate receptor (94), and metabotropic glutamate receptor 1 (95). Despite theoverwhelming data generated from these mutant animals, concern was raisedabout the interpretation of these results (96). Indeed, many of these mutantmice exhibited phenotypes that could not be interpreted based upon our cur-rent knowledge about these molecules. One major problem could stem fromthe indirect effect of the mutation on other organ systems or that exerted dur-ing an earlier developmental stage. To circumvent this problem, an emergingtechnique using tissue-specific, conditional, or inducible gene-targeting is de-sirable. Again, the major challenge remains in the determination of regulatorymachinery for tissue- or cell-specificity.

Endocrine SystemSince the cloning of genes for peptide hormones and hormone receptors, ma-nipulation of these genes in animals has generated a great deal of enthusiasmin this field. The information generated from transgenic mouse models in thissystem is most valuable with respect to specific gene regulatory mechanismsaffected by the hormones (for review see 97). In contrast, functional studiesusing either ectopic overexpression, antisense blockage, or gene-targeting hasbeen somewhat disappointing, owing to the complexity of the endocrine sys-tem and the presence of multiple isoforms of these receptors (98–100). Amongthese hormone systems, retinoic acid (RA) and its receptors have drawn mostof the attention because of their therapeutic potential.

The nuclear RA receptors (RARs) and retinoid X receptors (RXRs) existas multiple isoforms and exhibit functional redundancy in many organ sys-tems. A number of RAR and RXR knock-out mouse lines were produced, butmost of these animals displayed no apparent phenotypes. Upon crossing twomutant lines, phenotypes of vitamin A depletion began to appear, confirmingfunctional redundancy of these receptor gene families (100). As a result, thestrategy utilizing dominant negative mutations appeared to be more effectiveto make transgenic mouse models for the studies of these receptor functions.For example, under the control of a skin-specific promoter, the mutated RARα

protein was targeted to the epidermis in transgenic mice, demonstrating the re-quirement of RA in normal skin development and providing an animal model toexamine the efficacy and mechanism of retinoid treatment in skin diseases (39).




The second group of receptors involved in RA functions is the cellularretinoid-binding protein family. These proteins are believed to be responsi-ble for retinoid uptake and metabolism. For instance, patients under retinoidtherapy frequently developed resistance, and one of the reasons was believed tobe elevated levels of cellular retinoic acid–binding proteins (CRABPs). Trans-genic mice overexpressing this protein exhibited abnormal cell differentiationin the organs that overexpressed this protein—including lung, liver, ovary, andspleen—suggesting a function of this protein in normal cell differentiation pro-cesses (101). However, CRABP knock-out mice displayed neither apparentphenotypes nor altered retinoid sensitivity, indicating that CRABP was not avital component for animal survival (102, 103). It was possible that other closelyrelated genes, such as the fatty acid–binding protein gene family, could com-pensate for the defect in CRABP genes. As in the case of the nuclear receptors,functional redundancy of these cytosolic proteins was revealed by transgenicapproaches.

CancerCancer formation involves multistep processes that result in dysregulated cel-lular growth. The fight with cancer has thus been executed by the biomedicalresearch community in a multifaceted fashion. Two major issues in cancerresearch are the mechanism of cancer formation and therapeutic efficacy ofvarious agents, both being vigorously addressed by extensive transgenic stud-ies. Our understanding of some of the mechanisms controlling cell division,differentiation, and death has advanced dramatically through recent studies us-ing animal models that permit the dissection of complex oncogenic processes ingreat detail. Particularly, in recent years, several gene-targeted mice with spe-cific oncogenes mutated have been produced, allowing the precise mechanismof each proto-oncogene action to be addressed. Readers are referred to manyexcellent reviews for the mechanism of tumor formation and progression usingtransgenic animal models (for reviews, see 104–106 and references therein).In pharmacology, most of the recent excitement has come from the develop-ment of novel therapeutic strategies for treating cancers, and the reevaluationof conventional therapeutic agents using transgenic animal models.

The best example is the development of tumor immunotherapy using tumor-specific human antigen–bearing transgenic mouse models, such as transgenicmice bearing tumor markers carcinoembryonic antigen and germ-cell alkalinephosphatase, and mice carrying ectopically expressed oncogenic proteins (105).Because these human gene products were expressed from embryonic stages,thus seen as self antigens, these animals provide ideal models in which immuno-logical therapies can be assessed without the complication of host immune re-sponses as frequently seen in conventional animal models bearing tumor grafts.



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ToxicologyIn vitro systems for the study of toxicology have the inherent problem thatextrapolation is required for in vivo conditions in whole animals. Therefore,animal models prove to be more useful for chemical safety assessment. Themajor safety assessment schemes fall into two categories, those used to screenfor toxicity and those used to elucidate mechanisms of toxicity. The applicationof transgenic animals in toxicology has recently been reviewed (107, 108, andreferences therein). For instance, several OncoMouse models for carcinogenor mutagen assessment were made that bear oncogenes (e.g.myc, ras, neu) ortumor suppresser genes (p53). However, tumor formation involves multipleplayers, and the effect of oncoproteins is dose dependent. Therefore, cautionmust be exercised in choosing the gene target and its expression level so thatthe model can be physiologically relevant. Another model made use of aneasily assayed reporterlacZ in shuttle vectors that can be recovered from themammalian genome in order to measure mutation rates, providing a tool forassessing tissue-specific mutations generated in vivo. These models all carriedthe insertion of a bacterial gene, either the reporterlacZgene or thelacI repressorgene acting onlacZ, in the genome, which serves as a target for mutations.

Several models were created in order to elucidate the molecular mechanismsunderlying toxicity. The aryl hydrocarbon (Ah) receptor gene knock-out trans-genic mouse model was created (109) to elucidate dioxin toxicity. To investigatetoxic mechanisms in developing embryos, mouse lines deficient in a number ofdevelopmental genes were established, such as families of the homeobox genes(Hox) and RAR genes (for review see 100). In these animals, the control ofdevelopmental domains could be examined at a molecular level in the contextof whole animals, allowing the fundamental questions about the mechanismsof manifestation of mutation to be addressed.

As in the case of the cardiovascular system, transgenic rat models are pre-ferred over the mouse models for toxicity assessment because most parametershave been set up for the rats. Nevertheless, the mouse models would be suffi-cient for the studies of toxic mechanisms.

THE PERSPECTIVE

Transgenic animals are produced using molecular genetic techniques to addfunctional genes, to alter gene products, to delete genes, to insert reporter genesinto regulatory sequences, to replace/repair genes, and to make tissue/lineage-specific alteration of gene expression. These genetically altered animals provideunique tools for studying a wide range of biomedical problems in vivo, allowingthe manifestation of specific genetic changes in a variety of biological systemsto be examined. The studies of pharmacology rely largely on animal systems,




and several subjects have been hindered for decades because of the lack of suit-able animal models. The established transgenic techniques undoubtedly havefacilitated the progress of animal experimentation for some pharmacologicalquestions where potential target molecules have been characterized. Becausethis technology is evolving rapidly, it is foreseeable that future development ofthis technology will allow researchers to address some difficult pharmacolog-ical questions where no specific targets are identified. However, one shouldnot ignore the complexity of mammalian organ systems in the interpretation ofdata collected from these animals, because animals do not passively receive theexperimenter’s manipulation, and endogenous gene activities may be alteredin response to the introduction of exogenous DNA sequences. Therefore, inaddition to technical advancement, more basic information about the control ofspecific gene expression in animals is required to achieve the highest level ofspecificity and precision in transgenic animal experiments.

ACKNOWLEDGMENTS

This work was supported by DK46866 to LNW. Special thanks to Drs. HHLoh, PB Hackett Jr, and BG Zimmerman for critically reading the manuscript.Thanks to Dr. A Nagy for providing unpublished information.

Visit the Annual Reviews home pageathttp://www.annurev.org.

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