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Heterogeneous Reference Populations in Animal Model Research in Aging Gerald E. McClearn INTRODUCTION For many biological disciplines, such as ecology, wild populations are the primary object of study. In other research endeavors, comparative studies examine animals of different species, either wild-trapped or laboratory-reared, and either in their natural habitat or in laboratory settings. Such comparative observations have played a very substantial role in gerontological science. The monumental study of Finch (1990) has provided a summary of this work. Advantages of the comparative approach have been succinctly argued by Austad (1993), who observes, with regret, that contemporary gerontology has "lost its comparative focus." Central among the themes he pursues is the limited perspective provided by rodent models that have provided so much of laboratory-derived data in gerontology. An underlying concern is whether the model provided by any given species will provide results that will generalize to humankind. This paper is concerned not with these broad and major issues, but with the more narrow one of generality to the species as a whole of conclusions obtained from typical laboratory observations on genotypically restricted members of that species. GENES AS VARIABLES In general, in animal model research, the basic motivation for comparing observations from laboratories to observations on "wild" populations is to determine whether controlled variables of the laboratory setting have produced an outcome that is generalizable to the real world. It is an interesting philosophical problem. The laboratory permits control that simplifies complex systems so that relationships between and among variables may be perceived. In effect, the laboratory study takes a small piece of the total causal field, isolates it as far as possible from the rest of that field, and evaluates the extent to which a change in variable X is associated with a change in variable Y. The highly controlled conditions may, however, constrict the range of circumstances under which the observed relationship between X and Y will obtain, and the issue is whether that relation- ship can be shown when controlled variables are unfettered. Such unfettering can be accomplished in a systematic way in a laboratory setting, of course, but the total immersion tactic of testing in a real world setting is sometimes championed. The process described above applies to both environmental and genetic variables. Dealing with environmental variables is at the heart of all biological disciplinary specialties, and acquisition of knowledge about identification, manipulation, control, and evaluation of these variables constitutes much of the education of the biologist. Dealing appropriately with nutritional status of the diet, caging density, light cycles, time of day of testing, dose administration, procedures for handling, frequency of cage changing, control of high frequency noise—all of these and countless more similar variables—is a mark of good practice in research in a disciplinary area. It is interesting to contemplate that the more variables that must be controlled for a relationship of X to Y to be shown reliably, the less proportional impact X must have in the more complex, uncontrolled, real world situation. Observations in a reference situation with relaxed environmental controls can determine the generality-restricting consequences exerted by controls imposed during the conduct of research. There are also genetic constraints in laboratory research. For some biological disciplines, these are the core subject matter. In others, however, genetic constraints are less well understood and appreciated than are the environmental ones. The present discussion will focus, therefore, on the logic of genetic reference groups, which relax some of the genetic controls typical of laboratory research. The notion of genetic context is of central importance. The basic point is that the influence of allelic differences at a particular locus may depend on the alleles present at other loci of the organism. A particularly apposite example of this type of interaction is that of Coleman and Hummel (1975), who showed that animals homozygous for an allele "causing" diabetes could display dramatically different symptom patterns depending on background genotype. MANIPULATION AND CONTROL OF GENES Gerald McClearn, Ph.D., is Director of the Center for Developmental and Health Genetics and Evan Pugh Professor of Health and Human Development at the Pennsylvania State University, University Park, Pennsylvania. Volume 38, Number 3 1997 It is useful to consider genes as controlled or as independent variables. Until very recently, manipulation of genotype 119 could be accomplished only by indirect means, through assignment of mates. Strong theories of Mendelian genetics, cytogenetics, population genetics, and quantitative genetics were relied on to deduce the genotypic consequences of a particular mating program. The most utilized of these mating programs for the generation of animal models have been inbreeding and phenotypic selective breeding, in which the genotypes are indirectly rearranged. Direct manipulations of genetic engineering, which can be expected to become increasingly important in gerontological research as elsewhere, will become parallel methods that will build on animal groups generated by these indirect methods but will not replace them. Inbred Strains and Derived Generations Inbreeding is the great genetic homogenizing technique. The offspring of matings of relatives are more likely to be homozygous at any particular genetic locus than offspring of unrelated parents. If inbreeding is continued in successive generations, the homozygosity increases, and in any line of descent so maintained, homozygosity becomes nearly total. As a close approximation, it can be assumed that all animals within an inbred strain are homozygous in like state at all loci. Homozygosity is closely approached after 20 consecutive generations in which each generation is derived from a single sibling pair. If the mating scheme is continued, this condition of genetic uniformity will be maintained, aside from the possibility of mutations. Inbred strains are thus quite stable and constitute the principal means of genetic control in current use. In many cases, this control is exerted simply by employing a single strain. Within a given laboratory over time, and in different laboratories, investigators using that particular strain are confident that they are dealing with the same basic biological system. Borrowing terms from the language of environmental control, the genotype of an inbred strain might be said to be fixed, or "controlled," at a single "level." The level might be better termed a configuration, because in most cases, there is no obvious metric that will permit description of the system of anonymous polygenes in terms of unidimensional levels. It is also important to note that the genotype of a particular inbred strain is achieved not by a directional process, but by a stochastic one. Thus, an inbred strain is not bred "for" some phenotype; it is simply inbred, and nothing can be inferred about the genotype except that it has approached the condition of being homozygous at all loci. Comparisons among several inbred strains will often identify one displaying a particularly desirable level of some phenotype of interest. That strain can then constitute a model system for the study of the phenotype in detail. For example, C57BL/6 mice are outstanding among the more commonly used mouse strains for their voluntary alcohol consumption when offered a choice between alcohol solution and water (McClearn and Rodgers 1959); they have become, there- 120 fore, the strain of choice for any research requiring animals to consume some alcohol. The gradually accumulating knowledge about individual strains makes them more and more desirable as new research results can be referred to an ever-expanding data base. A frequently employed research tactic is to employ several strains with different levels of the phenotype. From one perspective, use of multiple strains is an approximation to control by fixation at different levels. From another perspective, multiple strain comparisons can be thought of as manipulation of genotype as an independent variable, because choice of strain is at the discretion of the investigator. This is a quite limited manipulation, however. Strictly speaking, for genotype to be an independent variable in the classical sense, it should be possible for the investigator to assign a gene or a set of specified genes to animals at random, in the same way that drug doses or degrees of stress or caloric restriction can be assigned. This is obviously impossible, and the investigator must settle for a choice of whole, anonymous, and mostly unknown genotypes. This use of inbred strains of mice and of rats has long been a popular approach to the study of aging. Logistical difficulties encountered by individual investigators in maintaining colonies of aged animals of different strains under appropriate environmental conditions eventuated in a formal program of the National Institute on Aging to provide animals to meet the need of investigators in the field. In turn, availability of these animals has accentuated demand, so that the fixed-genotype logic has become a major component of animal model gerontology. The major strength of the inbred strain approach, as already noted, derives from their uniformity and relative stability over time. This uniformity is also the principal weakness. For example, the only source for variability within an inbred strain is the environment, broadly defined. Any attempt to describe the relationship between 2 phenotypes within a strain will be constrained by the fact that variances, and therefore covariances of measured phenotypes, are exclusively of environmental origin; genetic influences cannot be detected because there are none present. Of more fundamental import, inbred strains are very limited as representatives of their species. Each strain represents only 1 of an indefinitely large number of genotypes that can be generated from the parent population. Furthermore, it is not as though a single normal genotype was sampled randomly and then indefinitely replicated in an inbred strain. Members of a strain are homozygous at all loci and can best be considered as a single gamete that is duplicated and replicated. Such a genetic configuration could never occur in a bisexually reproducing natural population. Even worse, the gamete thus duplicated cannot be considered a representative sample from all possible gametes from the population. In the population at large, gametes that have an allele that is lethal when homozygous may persist in heterozygotes. Because of the homozygosity achieved by inbreeding, however, these alleles will be ILAR Journal eliminated. Any single inbred strain thus provides only a very limited sampling of the gene pool of its species. Along with the inbred strains, the hybrid Fl animals obtained by crossing different strains have found frequent employment in research on aging. A degree of manipulation of genotype in the classical sense is accomplished by crossing inbred strains, because the resulting Fl hybrid has known, or very strongly inferred, genotype characteristics relative to the 2 parent strains: For each locus for which the 2 strains have different alleles, the Fl animals will be heterozygotes. Again, random assignment is not possible; the investigator cannot pick an animal and assign it to be a homozygote or heterozygote, but it can be said that all Fl animals will have an allelic dose halfway between those of the parents. It is worth noting that this firm positioning of the Fl is relative to 2 mostly or completely unknown anchor points, because the nature and number of such alleles is unknown. A geographic analogy would be knowing that some place is exactly halfway between 2 other landmarks but not knowing the location of either of them. In spite of this uncertainty, the relationship of Fl to parent strains is quite useful in that it permits the most elementary of the biometric operations of quantitative genetics. In short, it becomes possible to relate the allelic dose to the phenotype. Fl animals, unlike their inbred parents, will exhibit much heterozygosity. Indeed, they will be heterozygous at all loci for which the parents had different alleles. However, all Fl animals are genetically alike, and none will be homozygous at any of these segregating loci. In this respect, the Fls are no better representatives of the species than inbred strains. Subsequent mating operations can further this type of analysis substantially. An F2 obtained by mating Fl animals, unlike their Fl parents or their inbred strain grandparents, comprises individuals that differ genetically from each other. As a consequence of the reshuffling of the parent strain alleles, each genotype of each locus occurs in a variable genetic context of the genotypes at all other loci. Insofar as the effect of genotypic differences at any one locus is dependent to some extent on the genotypes at other loci, on the heterogeneous background, this effect appears as a sort of average, more representative of the operation of the gene locus than would be evidenced on any single inbred background. In the case of F2 or other intercross groups such as backcrosses, the manipulation of genotype by assignment of mates of particular types has predictable consequences for the group of offspring but is not informative about particular individuals in the group. Analytic attention is thus focused on the variability within the group, and the objective becomes that of ascribing the measured phenotypic variance to genetic and environmental influences, to their covariances, and to their interactions. The F2 and backcrosses provide a reference context within which the inbred and Fl results can be interpreted. An elementary example can be derived from observations on 2 inbred strains and their Fl and F2 generations. The variance of the phenotype of the inbred parental Volume 38, Number 3 1997 strains and of the Fl is exclusively environmental in origin; that of the F2 has both environmental and genetic components. Subtracting the former from the latter provides an estimate of genetic variance, and these can be formed into a ratio of genetic to total phenotypic variance to generate a simple index of "heritability." The field of quantitative genetics is a highly developed conceptual and analytical model for dealing with this type of situation. Falconer and MacKay (1996) provide one of several excellent introductions to the area, and Phelan (1992) has related these issues to aging. Phenotypic Selective Breeding Selective breeding is directional. Beginning with a genetically heterogeneous foundation stock (see below), the investigator mates animals of like extreme phenotypic value. If heritability is nonzero, there will be a gradual accumulation of alleles resulting in high phenotypic scores in one line and low scores in the other line. Phenotypic selection is a comprehensive procedure, in that an ideally executed study will locate and parcel out all the structural and regulatory genes segregating in the gene pool of the foundation stock that have any influence on the phenotype in question. It remains the case, however, that genes sorted out in this way are anonymous. Although there are limited circumstances under which heterozygosity might be maintained in the selected loci, the general expectation is that the "high" line will become homozygous for the "increasing" alleles and the "low" line will become homozygous for the "decreasing" alleles. This homozygosity presents a substantially different picture from that of inbred strains, however. First, as noted, selection provides for a relative uniformity of direction of effect of the alleles within a line. All, or most, of the relevant loci in a selected line will be homozygous for the increasing (or decreasing) alleles, whereas in inbred strains, the configuration is fortuitous, with both increasing and decreasing alleles in differing proportions in the different strains. Furthermore, unlike the case in inbred strains, the nonrelevant genotype is heterogeneous. This feature offers clear opportunities for assessing hypotheses about correlated characteristics. For example, C57BL/6 inbred mice are widely reported to have longer lives than DBA/2 mice. It is tempting to test theoretical expectations (about oxidative stress, for illustration) by comparing appropriate measures (such as particular enzyme activity levels) of the 2 strains. Results from such a comparison are very weak, however. The stochastic process of inbreeding will have fixed all loci, including those pertinent to the enzymes in question. Thus, even if no relationship exists between a given enzyme activity and longevity, inbreeding will provide 4 possible outcomes with respect to the enzyme genotype (assume single locus inheritance for the enzyme activity): both strains with high enzyme activity, both strains with low enzyme activity, C57BL/6s with high and DBA/2s with low activity, and C57BL/6s with low and DBA/2s with 121 high activity. One quarter of the outcomes would falsely appear to confirm the hypothesis. The situation can be improved by employing multiple strains instead of only 2; however, the power of the analysis is limited by the number of strain means, not by the number of individuals, and the approach may not be very cost-effective compared with the alternative tactics offered by selected lines or by heterogeneous stocks. Selectively bred lines, by virtue of a more-or-less randomly segregating "residual" genotype (that genotype not relevant to the selected attribute), offer a particularly powerful means of evaluating hypotheses about correlated characteristics. Within the usual limits of sampling considerations, any trait showing a significant difference between the 2 selected lines can be regarded as being functionally involved in the causal nexus of the phenotype for which selection was performed. Because in the real world of finite breeding population size there will always be the possibility of sampling bias, replicate selected lines are frequently employed. A consistent outcome in the replicates provides compelling evidence for the relationship. One limitation of selection, however, is that the genetic array relevant to the selected phenotype, and partitioned in the upward and downward selected lines, will be limited to the allelic differences that were available in the foundation population. Another selection program for the same phenotype, but beginning with a different gene pool, might result in the same phenotypic difference, but for somewhat, substantially, or entirely different genetic reasons. Nevertheless, selective breeding procedures have proved valuable in aging research on Drosophila (see Arking [1991] and Rose [1991] for review of these studies) and are in active progress in application to mammals (Harrison and others 1995). Heterogeneous Stocks If the value of heterogeneous stocks is to be found in the extent to which they represent their entire species, it would seem that the optimal strategy would be to sample from the "real" world of mice, rats, or whatever species is involved. Certainly, the value of a genetic context will be a positive function of how varied it is. In other words, it will be advantageous to have as much allelic variation as possible. For example, it would be ideal to have in one's reference group every existing allele in all strains of mice; however, this ideal is not attainable for several reasons. In the first instance, no practical trapping program could sample all the world's mice. Any local trapping will be sampling a unique gene pool and will be no more representative of every strain of mice than sampling a village will provide all the genetic variation of humankind. Furthermore, trapping biases are well-known; trap-shy mice will be omitted, along with any of their genes that result in trap-shyness. Upon importation to the laboratory, these wild-trapped animals will suddenly encounter a stupendously different environment from their accustomed one, and natural selection will begin forthwith to weed out 122 those alleles from the wild that lead to reduced survival and fertility in the laboratory. In general, then, establishing heterogeneous stocks by wild-trapping would have the very great advantage of introducing new alleles that have been absent from the existing stocks with their idiosyncratic origins. However, these wildderived stocks would become less and less representative of the wild population as they became laboratory-adapted. This author argues that the issue of such adaptation is not really important. Indeed, for today's laboratory animals, after generations of such selection, the laboratory is the natural habitat, and the heterogeneous reference populations should be adapted to laboratories, not to fields and forests. Another route to establishing heterogeneous reference stocks does not provide for the infusion of new genetic material to the laboratory stocks but does shuffle what is already present. This route involves the generation of genetically segregating populations by intercrossing of inbred strains. Such an approach has already been hinted, with the observation that an F2 consists of genetically unique animals. It is pertinent to ask just how genetically variable an F2 really is. The answer must be stated in reference to the parent strains from which the Fl generation was derived, because genetic variability in the F2 will depend on the number of loci from which those parents differed in allelic state and for which specific alleles they differed. Thus, some F2s will be more genetically variable than others. It should be noted, however, that no F2 can offer more than 2 alleles per locus, because there are only 2 progenitor strains and each of them is homozygous for a single allele at each locus. Thus, although it provides scope for expression of genes against a somewhat heterogeneous genetic milieu, this milieu will be idiosyncratic to the particular progenitor base. If an objective is to sample more completely from the totality of genetic information available within a species, then an obvious move is to expand this progenitor base. For example, a 4-way cross can be generated by mating an Fl between 2 progenitors with another Fl from different progenitors. There is now scope for more segregating loci, and the sampling limit is now 4 possible alleles. Such expansion can proceed even further with 8-way or more comprehensive intercrosses, with advantages accruing from each increment in number of alleles per locus. The logic of using heterogeneous stocks to provide foundation stocks has been discussed in some detail elsewhere (McClearn 1979; McClearn and others 1970). Briefly, such stocks are particularly valuable when used (1) as foundation stocks for selective breeding, (2) to provide reference norms for the evaluation of means and variances of inbred strains, (3) to examine correlations between variables, or (4) to evaluate the generality of a genetic effect identified in groups with constrained genetic variability. Current use of standardized heterogeneous stocks in gerontological research is infrequent, but awareness of their advantages is growing. McClearn (1992a,b), for example, has described the use of genetically heterogeneous (HS) ILAR Journal mice, derived from 8 inbred strains, both in evaluating the heterogeneity and reliability of a panel of behavioral biomarkers of aging and in exploring the structural stability and change reflected in the patterns of relationships among the variables over the lifespan. More recently, a 4-way cross population of mice has been used to test hypotheses about interrelationships of various immune system parameters and their relationships to lifespan and cancer incidence (Miller and others 1994), to assess variation in lifespan and in lesions (Chrisp and others 1996), and to validate candidate biomarkers of aging reflecting immune system status and muscle function (Miller and others 1997). These few examples illustrate the many desirable attributes of genetically heterogeneous animal models in gerontological research. As these attributes are increasingly appreciated, it is likely that standardized heterogeneous stocks will eventually be recognized as essential complements to the inbred strains and derived generations of current popularity. REFERENCES Arking R. 1991. Biology of Aging: Observations and Principles. Englewood Cliffs NJ: Prentice-Hall. Austad SN. 1993. The comparative perspective and choice of animal models in aging research. Aging Clin Exp Res 5:259-267. Chrisp CE, Turke P, Luciano A, Swalwell S, Petersen J, Miller RA. 1996. Lifespan and lesions in genetically heterogeneous (Four-way Cross) mice: A new model for aging research. Vet Pathol 33:735-743. Coleman DL, Hummel KP. 1975. Influence of genetic background on the expression of mutations at the diabetes locus in the mouse. II. Studies on background modifiers. Israeli J Med Sci 11:708-713. Falconer DS, Mackay TRC. 1996. Introduction to Quantitative Genetics. 4th ed. Essex, England: Longman. Volume 38, Number 3 1997 Finch CE. 1990. Longevity, Senescence, and the Genome. Chicago: The University of Chicago Press. Harrison DE, Roderick TH, Paigen K. 1995. Allele capture by selection for flanking markers: A new method for analyzing multigenic traits. Growth Devel Aging 59:73-76. McClearn GE. 1979. Influence of genetic variables on means, variances, and covariances in behavioral responses to toxicological and pharmacological substances. J Toxicol Environ HI 5:145-156. McClearn GE. 1992a. The reliability and stability of biomarkers of aging. Ann NY Acad Sci 673:1-8. McClearn GE. 1992b. Heterogeneity of biomarkers of aging. In: Licastro F, Caldarera CM, editors. Biomarkers of Aging: Expression and Regulation. Bologna: Editirice Bologna, p 27-36. McClearn GE, Rodgers DA. 1959. Differences in alcohol preference among inbred strains of mice. Q J Stud Alcohol 20:691-695. McClearn GE, Wilson JR, Meredith W. 1970. The use of isogenic and heterogenic mouse stocks in behavioral research. In: Lindzey G, Thiessen DD, editors. Contributions to Behavior-genetic Analysis: The Mouse as a Prototype. New York: Appleton-Century-Crofts. p 3-22. Miller RA, Bookstein F, Van der Meulen J, Engle S, Kim J, Mullins L, Faulkner J. 1997. Candidate biomarkers of aging: Age-sensitive indices of immune and muscle function covary in genetically heterogeneous mice. J Gerontol Biol Sci 52:B39-B47. Miller RA, Turke P, Chrisp C, Ruger J, Luciano A, Peterson J, Chalmers K, Gorgas G, Van Cise S. 1994. Age-sensitive T cell phenotypes covary in genetically heterogeneous mice and predict early death from lymphoma. J Gerontol Biol Sci 49:B225-B262. Rose MR. 1991. Evolutionary Biology of Aging. New York: Oxford University Press. ACKNOWLEDGMENTS The author's research on gerontological genetics has been supported by the MacArthur Foundation Research Network on Successful Aging and by grants from the National Institute on Aging (AG04948 and AGO9333). 123