Inbred mouse strains and genetic stability: a review

Inbred mice were essential animal models for scientific research during the 20th century and will contribute decisive results in the current and next centuries. Far from becoming an obsolete research tool, the generation of new inbred strains is continuing and such strains are being used in many research fields. However, their genetic properties have been overlooked for decades, although recent research has revealed new insights into their genetic fragility and relative instability. Contrary to what we usually assume, inbred mice are far from being completely isogenic and both single-gene major mutations and polygenic mutational variability are continuously uploading into inbred populations as new sources of genetic polymorphisms. Note that several inbred strains from new major mutations are released every year, whereas small mutations can accumulate up to accounting for a significant percentage of the phenotypic variance (e.g. 4.5% in a recent study on C57BL/6J mice). Moreover, this genetic heterogeneity can be maintained for several generations by heterozygote selection and, if fixed instead of dropping off, genetic drift must be anticipated. The contribution of accidental genetic contamination in inbred strains must also be considered, although its incidence in current breeding stocks should be minimal, or even negligible. This review revisits several relevant topics for current inbred strains, discussing the latest cutting-edge results within the context of the genetic homogeneity and stability of laboratory mice. Inbred mice can no longer be considered as completely isogenic, but provide a remarkably homogeneous animal model with an inevitable moderate-to-low degree of genetic variability. Despite a certain degree of genetic heterogeneity becoming inescapable, inbred mice still provide very useful animal models with evident advantages when compared with outbred, that is, highly variable, populations.     

Hippocampal commissure defects in crosses of four inbred mouse strains with absent corpus callosum

It is known that four common inbred mouse strains show defects of the forebrain commissures. The BALB/cJ strain has a low frequency of abnormally small corpus callosum, whereas the 129 strains have many animals with deficient corpus callosum. The I/LnJ and BTBR T+ tf/J strains never have a corpus callosum, whereas half of I/LnJ and almost all BTBR show severely reduced size of the hippocampal commissure. Certain F1 hybrid crosses among these strains are known to be less severely abnormal than the inbred parents, suggesting that the parent strains have different genetic causes of commissure defects. In this study, all hybrid crosses among the four strains were investigated. The BTBR × I/Ln hybrid expressed almost no defects of the hippocampal commissure, unlike its inbred parent strains. Numerous three‐way crosses among the four strains yielded many mice with no corpus callosum and severely reduced hippocampal commissure, which shows that the phenotypic defect can result from several different combinations of genetic alleles. The F2 and F3 hybrid crosses of BTBR and I/LnJ had almost 100% absence of the corpus callosum but about 50% frequency of deficient hippocampal commissure. The four‐way hybrid cross among all four abnormal strains involved highly fertile parents and yielded a very wide phenotypic range of defects from almost no hippocampal commissure to totally normal forebrain commissures. The F2 and F3 crosses as well as the four‐way cross provide excellent material for studies of genetic linkage and behavioral consequences of commissure defects.     

Of "mice" and mammals: utilizing classical inbred mice to study the genetic architecture of function and performance in mammals

The house mouse is one of the most successful mammals and the premier research animal in mammalian biology. The classical inbred strains of house mice have been artificially modified to facilitate identification of the genetic factors underlying phenotypic variation among these strains. Despite their widespread use in basic and biomedical research, functional and evolutionary morphologists have not taken full advantage of inbred mice as a model for studying the genetic architecture of form, function, and performance in mammals. We illustrate the potential of inbred mice as a model for mammalian functional morphology by examining the genetic architecture of maximum jaw-opening performance, or maximum gape, across 21 classical inbred strains. We find that variation in maximum gape among these strains is heritable, providing the first evidence of a genetic contribution to maximum jaw-opening performance in mammals. Maximum gape exhibits a significant genetic correlation with body size across strains, raising the possibility that evolutionary increases in size frequently resulted in correlated increases in maximum gape (within the constraints of existing craniofacial form) during mammalian evolution. Several craniofacial features that influence maximum gape share significant phenotypic and genetic correlations with jaw-opening ability across these inbred strains. The significant genetic correlations indicate the potential for coordinated evolution of craniofacial form and jaw-opening performance, as hypothesized in several comparative analyses of mammals linking skull form to variation in jaw-opening ability. Functional studies of mammalian locomotion and feeding have only rarely examined the genetic basis of functional and performance traits. The classical inbred strains of house mice offer a powerful tool for exploring this genetic architecture and furthering our understanding of how form, function, and performance have evolved in mammals.     

Wild mice provide insights into natural killer cell maturation and memory

The immune system has evolved, and continues to evolve, in response to the selection pressure that infections exert on animals in their natural environments, yet much of our understanding about how the immune system functions comes from studies of model species maintained in the almost complete absence of such environmental selection. The scientific discipline of immunology has among its aims the improvement of human and animal health by the application of immunological knowledge. As research on humans and domesticated animals is highly constrained-ethically, logistically and financially-experimental animal models have become an invaluable tool for dissecting the functioning of the immune system. The house mouse (Mus musculus) is by far the most widely used animal model in immunological research but laboratory-reared mice provide a very narrow view of the immune system-that of a well-fed and comfortably housed animal with minimal exposure to microbial pathogens. Indeed, so much of our immunological knowledge comes from studies of a very few highly inbred mouse strains that-to all intents and purposes-our immunological knowledge is based on enormously detailed studies of very small numbers of individual mice. The limitations of studies in inbred strains of laboratory mice are well-recognized (Pedersen & Babayan 2011), but serious attempts to address these limitations have been few and far between. However, the emerging field of 'ecological immunology' where free-living populations are studied in their natural habitat is beginning to redress this imbalance (Viney et al. 2005; Martin et al. 2006; Owen et al. 2010; Abolins et al. 2011). As demonstrated in the work by Boysen et al. (2011) in this issue of Molecular Ecology, studies in wild animal populations-especially free-living M. musculus-represent a valuable bridge between studies in humans and livestock and studies of captive animals.     

Alloimmune reactions among recently wild syrian hamsters and classical inbred strains include alloantibody production

Partially inbred lines of Syrian hamsters, the descendants of animals caught wild in Syria within the past decade, have been studied to determine their immunogenetic relationships to the long established domestic inbred strains. These new sources of hamster genes display strong alloimmune reactions when confronted with tissues from the domestic inbred strains and vice versa: acute skin graft rejection, strong mixed lymphocyte reactions, and intense graft-versus-host reactions. While these forms of reactivity have also characterized the disparities among the domestic inbred strains, no evidence of alloantibodies specific for putative transplantation antigens has ever been found in hamsters. We report, for the first time, that immunization of recently wild hamsters with tissues from domestic inbred strain donors elicits high titer cytotoxic antibodies directed at alloantigens. Similarly, animals of the domestic inbred strains produce comparably strong alloantibody responses when immunized with tissues from the recently wild hamsters. Availability of these alloantisera will make the possibility of defining the major histocompatibility complex equivalent in this species much more likely.     

Functional polymorphisms in inbred rat strains and their allele frequencies in commercially available outbred stocks.

Polymorphisms that have been proven to influence gene functions are called functional polymorphisms. It is significant to know the distribution of functional polymorphisms in the rat, widely used in animal models for human diseases. In this study, we assessed 16 functional polymorphisms consisting of 3 coat color and 13 disease-associated genes in 136 rat strains, as a part of the genetic profiling program of the National Bio Resource Project for the Rat (NBRP-Rat). Polymorphisms of Cdkn1a, Fcgr3, Grp10, Lss, and Fdft1, which were proven to function in prostate tumorigenesis, glomerulonephritis, hyperphagia, and cholesterol biosynthesis, were shared among various inbred strains. These findings indicated that most rat strains harbored the disease-associated alleles and suggested that many unidentified functional polymorphisms might exist in inbred rat strains. The functional polymorphisms shared in inbred strains were also observed within outbred stocks available commercially. Therefore, this implies that experimental plans based on either rat inbred strains or outbred stocks need to be carefully designed with a full understanding of the genetic characteristics of the animals. To select the most suitable strains for experiments, the NBRP-Rat will periodically improve and update the genetic profiles of rat strains.     

Multiple trait measurements in 43 inbred mouse strains capture the phenotypic diversity characteristic of human populations.

The breadth of genetic and phenotypic variation among inbred strains is often underappreciated because assessments include only a limited number of strains. Evaluation of a larger collection of inbred strains provides not only a greater understanding of this variation but collectively mimics much of the variation observed in human populations. We used a high-throughput phenotyping protocol to measure females and males of 43 inbred strains for body composition (weight, fat, lean tissue mass, and bone mineral density), plasma triglycerides, high-density lipoprotein and total cholesterol, glucose, insulin, and leptin levels while mice consumed a high-fat, high-cholesterol diet. Mice were fed a chow diet until they were 6-8 wk old and then fed the high-fat diet for an additional 18 wk. As expected, broad phenotypic diversity was observed among these strains. Significant variation between the sexes was also observed for most traits measured. Additionally, the response to the high-fat diet differed considerably among many strains. By the testing of such a large set of inbred strains for many traits, multiple phenotypes can be considered simultaneously and thereby aid in the selection of certain inbred strains as models for complex human diseases. These data are publicly available in the web-accessible Mouse Phenome Database (http://www.jax.org/phenome), an effort established to promote systematic characterization of biochemical and behavioral phenotypes of commonly used and genetically diverse inbred mouse strains. Data generated by this effort builds on the value of inbred mouse strains as a powerful tool for biomedical research.     

FELASA guidelines for the production and nomenclature of transgenic rodents

The standardized nomenclature of rodent strains, genes and mutations has long been the focus of careful attention. Its aim is to provide proper designation of laboratory animals used in research projects and to convey as much information on each strain as possible. Since the development of different techniques to mutate the genome of laboratory rodents on a large scale, the correct application of current nomenclature systems is of increased significance. It facilitates not only the accurate communication of scientific results but is indispensable in controlling the dramatically increased number of transgenic animal models in experimental units, archives and databases. It is regrettable that many publications, especially on transgenic rodents, use vague and inappropriate strain designation. This situation should definitely be improved, particularly considering the increasingly emphasized importance of genetic background on the phenotype of mutations. The aim of these guidelines is to raise awareness about specific features of production and of the current nomenclature system used for transgenic rodents.     

The baboon as a non-human primate model of human schistosome infection

Over the past three decades, intensive studies of murine schistosomiasis have provided important clues to the understanding of the human disease, but growing evidence suggests that these results derived from highly inbred strains of mice might not have direct applicability to the human infection. Recent data based on the baboon indicate that infection in this non-human primate might mirror the human situation. In this review, Mramba Nyindo and Idle Farah demonstrate that baboons provide an excellent non-human primate model that produces pathology and disease closely resembling that observed in humans, and address how studies in baboons can provide insights into mechanisms regulating schistosomiasis mansoni pathology and immunity. They also address, in a general way, issues related to the use of non-human primates in biomedical research.     

Novel non-rodent models of kidney disease

Kidney disease in the 21(st) century affects increasing numbers of individuals. We continue to be challenged by our lack of understanding of the pathophysiology of acute and chronic renal disease including genetic diseases involving the kidney. Rodent knockout animals or inbred strains have greatly contributed to our understanding of many monogenetic and complex diseases. Non-rodent animal models of disease have become more attractive since genomic data has become available for a variety of organisms that offer distinct advantages over mice and rats for ease in conducting high-throughput chemical or mutagenesis screens. It is thus timely to examine the physiology and pathophysiology of the kidney or kidney equivalents in these organisms to evaluate their relevance as models for human disease. In addition to organisms whose small size and accessibility facilitate large scale screening approaches, larger animals at the other end of the spectrum offer unique physiological advantages in both size equivalency to humans as well as, in some cases, physiological and pathophysiological responses that closely mimic those of humans. Here we review a selected number of non-rodent experimental models of kidney diseases, focusing on recent advances in the use of the worm Caenorhabditis elegans, the fruitfly Drosophila melanogaster, the zebrafish Danio rerio, the little skate Leucoraja erinacea, the MGH miniature swine, merino cross sheep, and the cow Bos taurus to study kidney disease.     

Effects of laboratory culture on compatibility between snails and schistosomes

The genetic control of compatibility between laboratory strains of schistosomes and their snail hosts has been studied intensively since the 1970s. These studies show (1) a bewildering array of genotype-by-genotype interactions - compatibility between one pair of strains rarely predicts compatibility with other strains, and (2) evidence for a variety of (sometimes conflicting) genetic mechanisms. Why do we observe such variable and conflicting results? One possibility is that it is partly an artifact of the use of laboratory strains that have been in culture for many years and are often inbred. Here we show that results of compatibility trials between snails and schistosomes - all derived from the same natural population - depend very much on whether one uses laboratory-cultured or field-collected individuals. Explanations include environmental effects of the lab on either host or parasite, and genetic changes in either host or parasite during laboratory culture. One intriguing possibility is that genetic bottlenecks during laboratory culture cause the random fixation of alleles at highly polymorphic loci that control the matched/mismatched status of hosts and parasites. We show that a simple model of phenotype matching could produce dose response curves that look very similar to empirical observations. Such a model would explain much of the genotype-by-genotype interaction in compatibility observed among strains.     

Genetic variation,inbreeding and chemical exposure—combined effects in wildlife and critical considerations for ecotoxicology

Exposure to environmental chemicals can have negative consequences for wildlife and even cause localized population extinctions. Resistance to chemical stress, however, can evolve and the mechanisms include desensitized target sites, reduced chemical uptake and increased metabolic detoxification and sequestration. Chemical resistance in wildlife populations can also arise independently of exposure and may be spread by gene flow between populations. Inbreeding—matings between closely related individuals—can have negative fitness consequences for natural populations, and there is evidence of inbreeding depression in many wildlife populations. In some cases, reduced fitness in inbred populations has been shown to be exacerbated under chemical stress. In chemical testing, both inbred and outbred laboratory animals are used and for human safety assessments, iso-genic strains (virtual clones) of mice and rats are often employed that reduce response variation, the number of animals used and associated costs. In contrast, for environmental risk assessment, strains of animals are often used that have been selectively bred to maintain heterozygosity, with the assumption that they are better able to predict adverse effects in wild, genetically variable, animals. This may not necessarily be the case however, as one outbred strain may not be representative of another or of a wild population. In this paper, we critically discuss relationships between genetic variation, inbreeding and chemical effects with the intention of seeking to support more effective chemical testing for the protection of wildlife.     

Use of simple sequence length polymorphisms for genetic characterization of rat inbred strains

Genetic monitoring is an essential component of colony management and for the rat has been accomplished primarily by using immunological and biochemical markers. Here, we report that simple sequence length polymorphisms (SSLPs) are a faster and more economical way of monitoring inbred strains of rats. We characterized 61 inbred strains of rats, using primer pairs for 37 SSLPs. Each of these loci appeared to be highly polymorphic, with the number of alleles per locus ranging between 3 and 14 and, as a result, all the 61 inbred strains tested in this study could be provided with a unique strain profile. These strain profiles are also used for estimating the degree of similarity between strains. This information may provide the rationale in selecting strains for genetic crosses or for other specific purposes.     

Effect of murine strain on metabolic pathways of glucose production after brief or prolonged fasting

Background strain is known to influence the way a genetic manipulation affects mouse phenotypes. Despite data that demonstrate variations in the primary phenotype of basic inbred strains of mice, there is limited data available about specific metabolic fluxes in vivo that may be responsible for the differences in strain phenotypes. In this study, a simple stable isotope tracer/NMR spectroscopic protocol has been used to compare metabolic fluxes in ICR, FVB/N (FVB), C57BL/6J (B6), and 129S1/SvImJ (129) mouse strains. After a short-term fast in these mice, there were no detectable differences in the pathway fluxes that contribute to glucose synthesis. However, after a 24-h fast, B6 mice retain some residual glycogenolysis compared with other strains. FVB mice also had a 30% higher in vivo phosphoenolpyruvate carboxykinase flux and total glucose production from the level of the TCA cycle compared with B6 and 129 strains, while total body glucose production in the 129 strain was approximately 30% lower than in either FVB or B6 mice. These data indicate that there are inherent differences in several pathways involving glucose metabolism of inbred strains of mice that may contribute to a phenotype after genetic manipulation in these animals. The techniques used here are amenable to use as a secondary or tertiary tool for studying mouse models with disruptions of intermediary metabolism.     

A mouse-based strategy for cyclophosphamide pharmacogenomic discovery.

Genome-wide mapping approaches are needed to more fully understand the genetic basis of chemotherapy response. Because of technical and ethical limitations, cancer pharmacogenomics has not yet benefited from traditional robust familial genetic strategies. We have therefore explored the use of the inbred mouse as a genetic model system in which to study response to the cytotoxic agent cyclophosphamide. Multiple phenotypes have been assessed in response to cyclophosphamide in up to 19 inbred mouse strains, including in vitro hematopoietic progenitor cell toxicity and the mobilization of hematopoietic progenitor cells into peripheral blood. Hematopoietic progenitor cell toxicity in vitro varied 2-fold among strains, whereas in vivo progenitor cell mobilization varied almost 75-fold among strains. Males mobilized more hematopoietic progenitor cells than did females, and the low-mobilization phenotype was dominant to the high-mobilization phenotype in F1 hybrid animals. In an initial attempt to analyze candidate genes, genetic variation was assessed in three cytochrome P-450 genes involved in the metabolism of cyclophosphamide. Resequencing of eight strains identified 26 polymorphisms in these genes that may influence response to cyclophosphamide. Distinct regions of high- and low-polymorphism rates were identified, and two common haplotypes were shared among the strains for each gene that exhibited variation. This phenotypic and genotypic variation among inbred strains provides a framework for cyclophosphamide pharmacogenomic discovery.     

SNP genotyping for the genetic monitoring of laboratory mice by using a microarray-based method with dualcolour fluorescence hybridisation

Ensuring the genetic homogeneity of the mice used in laboratory experiments contributes to the Reduction aspect of the Three Rs, by maximising the quality of the data obtained from any animals that are used for these purposes, and ultimately reducing the numbers of animals used. Single nucleotide polymorphism (SNP) genotyping is especially suitable for use in the analysis of the genetic purity of model organisms such as the mouse, because bi-allelic markers remain fully informative when used to characterise crosses between inbred strains. Here, we attempted to apply a microarray-based method for a SNP marker to monitor the genetic quality of inbred mouse strains, so as to validate the reliability, stability and applicability of this SNP genotyping panel. The amplified PCR products containing four different SNP loci from four inbred mouse strains were spotted and immobilised onto amino-modified glass slides to generate a microarray. This was then interrogated through hybridisation with dual-colour probes, to determine the SNP genotypes of each sample. The results indicated that this microarray-based method could effectively determine the genotypes of the four selected SNPs with a high degree of accuracy. We have developed a new SNP genotyping technique for effective use in the genetic monitoring of inbred mouse strains.     

昆明小鼠4个可能近交系的基因分型验证

在我国,昆明小鼠作为一种实验动物广泛应用于药理和遗传学相关的研究领域。但由于昆明小鼠属于远交群,而且不同地区的种群间已经出现了严重分化,缺乏具有显著特征的近交系,这使得它在生物学上的应用受到了很大的限制。研究人员已经以昆明小鼠为背景培育出了几个可能的近交系,但由于缺乏可靠的遗传检测,至今未得到广泛的认可和应用。文章收集昆明小鼠的4个已经60代以上兄妹交配繁殖的可能近交系,并以两个标准近交系BALB/c和C57BL/6为参照,利用30个微卫星标记对每个品系的5只小鼠进行了微卫星基因分型,进而分析其遗传纯度。结果发现,品系A1和品系N4在本研究所用的30个位点均呈纯合状态;而T2和N2均在D15Mit16位点呈杂合状态。本研究第一次为我国昆明小鼠近交系的遗传学纯度提供了可靠的分子水平证据。今后应当加强昆明小鼠近交系的标准化,以扩大其在遗传学方面的应用。     

The variance in inbreeding depression and the recovery of fitness in bottlenecked populations

Theoretical analyses of inbreeding suggest that following an increased degree of inbreeding there may be a temporary recovery of fitness, because of selection either within or among inbred lineages. This is possible because selection can act more efficiently to remove deleterious alleles given the greater homozygosity of such populations. If common, recovery of fitness following inbreeding may be important for understanding some evolutionary processes and for management strategies of remnant populations, yet empirical evidence for such recovery in animals is scant. Here we describe the effects of single-pair population bottlenecks on a measure of fitness in Drosophila melanogaster. We compared a large number of families from each of 52 inbred lines with many families from the outbred population from which the inbred lineages were derived. Measures were made at the third and the 20th generations after the bottleneck. In both generations there was, on average, substantial inbreeding depression together with a highly significant variance among the inbred lines in the amount of fitness reduction. The average fitness of inbred lines was correlated across generations. Our data provide evidence for the possibility of recovery of fitness at two levels, because (i) the average fitness reduction in the F20 generation was significantly less than in the F3 generation, which implies that selection within lines has occurred, and (ii) the large variance in inbreeding depression among inbred lines implies that selection among them is possible. The high variance in inbreeding depression among replicate lines implies that modes of evolution which require a low level of inbreeding depression can function at least in a fraction of inbred populations within a species and that results from studies with low levels of replication should be treated with caution.     

Genetic analysis of endogenous xenotropic murine leukemia viruses: association with two common mouse mutations and the viral restriction locus Fv-1.

We have defined 40 endogenous xenotropic virus (Xmv) loci from several common inbred strains of mice by examining provirus-cell DNA junction fragments in recombinant inbred mice. Some inbred strains carried unique proviruses, but most Xmv loci were present in several strains, indicating that many Xmv integration events preexisted modern inbreeding. It was also clear that most Xmv junction fragment variation between inbred strains resulted from independent integration events and not modification or restriction site polymorphism following integration. Chromosomal assignments were determined for 32 Xmv loci by comparing their recombinant inbred strain distribution patterns to those of known genetic markers. The Xmv loci were generally dispersed throughout the genome, but several chromosomal regions contained more than one provirus. Furthermore, several close genetic associations with cellular genes were discovered. Four Xmv loci were closely linked to Fv-1b, a dominant viral resistance gene present in C57BL/6J, BALB/cJ, A/J, and several other strains. Xmv-28 was closely linked to rd (retinal degeneration), and Xmv-10 was closely linked to a (non-agouti), both of which are old mutations as inferred from their broad distribution in mice. We suggest that Xmv integration contributed to genetic diversity in the past and that much of this diversity exists today in common laboratory strains.     

Nucleotide variation in wild and inbred mice

The house mouse is a well-established model organism, particularly for studying the genetics of complex traits. However, most studies of mice use classical inbred strains, whose genomes derive from multiple species. Relatively little is known about the distribution of genetic variation among these species or how variation among strains relates to variation in the wild. We sequenced intronic regions of five X-linked loci in large samples of wild Mus domesticus and M. musculus, and we found low levels of nucleotide diversity in both species. We compared these data to published data from short portions of six X-linked and 18 autosomal loci in wild mice. We estimate that M. domesticus and M. musculus diverged <500,000 years ago. Consistent with this recent divergence, some gene genealogies were reciprocally monophyletic between these species, while others were paraphyletic or polyphyletic. In general, the X chromosome was more differentiated than the autosomes. We resequenced classical inbred strains for all 29 loci and found that inbred strains contain only a small amount of the genetic variation seen in wild mice. Notably, the X chromosome contains proportionately less variation among inbred strains than do the autosomes. Moreover, variation among inbred strains derives from differences between species as well as from differences within species, and these proportions differ in different genomic regions. Wild mice thus provide a reservoir of additional genetic variation that may be useful for mapping studies. Together these results suggest that wild mice will be a valuable complement to laboratory strains for studying the genetics of complex traits.