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Journal Vol 47(2)

Mouse Phenome Research: Implications of Genetic Background

Atsushi Yoshiki and Kazuo Moriwaki

Atsushi Yoshiki, Ph.D., is Head of the Experimental Animal Division, Department of Biological Systems, RIKEN BioResource Center. Kazuo Moriwaki, Ph.D., is a Special Consultant to RIKEN.

Abstract

Now that sequencing of the mouse genome has been completed, the function of each gene remains to be elucidated through phenotypic analysis. The “genetic background“ (in which each gene functions) is defined as the genotype of all other related genes that may interact with the gene of interest, and therefore potentially influences the specific phenotype. To understand the nature and importance of genetic background on phenotypic expression of specific genes, it is necessary to know the origin and evolutionary history of the laboratory mouse genome. Molecular analysis has indicated that the fancy mice of Japan and Europe contributed significantly to the origin of today's laboratory mice. The genetic background of present-day laboratory mice varies by mouse strain, but is mainly derived from the European domesticussubspecies group and to a lesser degree from Asian mice, probably Japanese fancy mice, which belong to the musculus subspecies group. Inbred laboratory mouse strains are genetically uniform due to extensive inbreeding, and they have greatly contributed to the genetic analysis of many Mendelian traits. Meanwhile, for a variety of practical reasons, many transgenic and targeted mutant mice have been created in mice of mixed genetic backgrounds to elucidate the function of the genes, although efforts have been made to create inbred transgenic mice and targeted mutant mice with coisogenic embryonic stem cell lines. Inbred mouse strains have provided uniform genetic background for accurate evaluation of specific genes phenotypes, thus eliminating the phenotypic variations caused by mixed genetic backgrounds. However, the process of inbreeding and selection of various inbred strain characteristics has resulted in inadvertent selection of other undesirable genetic characteristics and mutations that may influence the genotype and preclude effective phenotypic analysis. Because many of the common inbred mouse stains have been established from relatively small gene pools, common inbred strains have limitations in their genetic polymorphisms and phenotypic variations. Wild-derived mouse strains can complement deficiencies of common inbred mouse strains, providing novel allelic variants and phenotypes. Although wild-derived strains are not as tame as the common laboratory strains, their genetic characteristics are attractive for the future study of gene function.

Key Words: inbred strain; genetically engineered mice; mouse resource center; subspecies; wild-derived strain

Introduction

The 21st century began with an analysis of the complete nucleotide sequence of the human genome, followed soon thereafter by that of the mouse. All of the individual genes in the genome, which comprise the “genetic background,” are now understood to be DNA molecules. Although traditional methods in molecular biology have allowed investigators to work on a single gene in an experiment, current high-throughput systems enable simultaneous monitoring of thousands of genes over the whole genome on a single chip. For investigations that focus on a specific gene, the “genetic background” can be defined as the genotypes of all other related genes over the genome that may interact with the gene of interest and potentially influence the specific phenotype.

More than two decades ago, Goodenough (1978) stated in his textbook on genetics, “A particular gene product will normally be operating in the presence of countless different combinations of other gene products.” This notion implies that a series of related genes could be working as “hidden alleles,” even in a single Mendelian phenotype. In 1979, Vogel and Motulsky mentioned in their book on human genetics that genetic polymorphisms that are biochemically and pathophysiologically related to a disease may represent the genetic background that makes certain individuals more likely to be affected. As illustrated in Figure 1, a specific phenotype is determined by a particular combination of alleles in the related gene loci of the genetic background. This concept explains variable susceptibility to a specific disease among different human individuals, even when they carry the same allele that determines the phenotype.

Figure 1. Contribution of multiple gene loci (1-5) with multiple alleles (a-e) for the onset of a given disease. Reprinted from Moriwaki K, Miyashita N, Yamaguchi Y, Shiroishi T. 1999. Multiple genes governing biological functions in the genetic backgrounds of laboratory mice and Asian wild mice. In: Hiai H, Hino O, eds. Vol 35: Progress in Experimental Tumor Research. Animal Models of Cancer Predisposition Syndromes. Basel: Karger. p 6. With permission from S. Karger AG Medical and Scientific Publishers, Basel, Switzerland.

In the first half of the 20th century, the genetic background of laboratory mice was a matter of concern in biomedical research. The major concern in the early days was heterozygosity in many gene loci. Great effort was therefore made to develop inbred strains (see the monograph by Festing 1979). Each of the highly inbred strains thus established were selected for unique phenotypic characteristics, and thus had unique genetic backgrounds. They have been quite useful for the genetic analysis of single Mendelian traits.

For multigenic traits such as histocompatibility, cancer, hypertension, diabetes, and other adulthood diseases, the use of multiple single-gene mutants has contributed to understanding the biochemical pathways involved in pathogenesis (Moore 1999). To evaluate the effect of a single gene and eliminate the effect of the genetic background, one may use a breeding protocol of successive backcrosses to create a congenic strain. In the field of immunogenetics, the presence of many minor histocompatibility antigen loci in the genome interfered with effective genetic analysis of the major histocompatibility complex (MHC¹). The establishment of H2 congenic strains by Snell (1948) was a seminal contribution to genetic research, in which he created congenic strains with the same genetic background except for the H2 region. The structure and function of mouse MHC loci were clearly elucidated with congenic strains by eliminating the differences of the genetic background. Another strategy to dissect the multigenic or oligogenic quantitative trait loci (QTL¹) is to use the recombinant inbred (RI¹) strains (Silver 1995). Phenotypic data of a series of RI strains and high-density strain distribution patterns can now be analyzed by computer software to map statistically significant QTLs simultaneously in the genome (Ohno et al. 2003).

The elimination of genetic background effects for analysis of spontaneous or genetically engineered mutations can be carried out by successively backcrossing the mutant strain to a standard inbred strain for more than 12 generations while selecting for the mutation. Many standard inbred strains may be used, and it is important for the investigator to choose the appropriate strain for a particular phenome study. For example, when investigating coat color phenotypes in the mouse, one should never choose albino strains whose melanin synthesis is genetically defective. The C57BL/6 mouse is the most widely used in biomedical research. However, in auditory research, one should be aware of the auditory deficiency in the common laboratory strains, including C57BL/6, DBA/2, 129, and others (Zheng et al. 1999). Moreover, C57BL/6 mice are deficient in pineal melatonin, which plays important roles in the vertebrate circadian rhythm (Ebihara et al. 1986). Some inbred strains such as C3H, which carries a recessive retinal degeneration mutation (Sidman and Green 1965), are not appropriate to use for the study of visual function. Thus, in mouse phenome research, the choice of background mouse strain is critical to the result of the experiment, and the genetic background of the strain should be carefully considered in the interpretation of the experimental results.

Since the early 1990s, large-scale N-ethyl-N-nitrosourea mutagenesis programs have been carried out in several countries to create thousands of new mutant mouse lines (Cordes 2005). Moreover, in the coming years, it is predicted that new embryonic stem (ES¹) cell lines that carry targeted mutations or gene trapped mutations will continue to be created until multiple alleles for all mouse genes (∼ 30,000 genes) will be available (Abott 2004). Thus, the environment of biomedical research using mouse models continues to advance and is establishing a full set of research resources. In this article, we review the evolutionary origin of laboratory mice to increase readers' understanding of the importance of genetic background in mouse phenome research and to inform mouse users of the unique and invaluable resources of wild-derived mouse strains for the scientific community.

Genetic Background from an Evolutionary Perspective

Since the early 1970s, knowledge of the molecular evolution of the gene has been based extensively on the concept of a molecular clock (Zucherkandl and Pauling 1962) and also the neutrality in gene mutation (Kimura 1968). Subsequently, since completion of the mouse genome sequencing, it is now possible to approach the evolutionary history of the genetic background of each laboratory strain and their ancestral wild mice by applying molecular biology.

Genetic Differentiation of Mouse Subspecies

More than 50 yr ago, Schwarz and Schwarz (1943) classified Mus musculus into 11 subspecies based on morphological characteristics. Moriwaki and colleagues (1979) used biochemical markers to estimate that the time line of the genetic divergence between the European domesticus and the Asian musculus subspecies is roughly one million years. Yonekawa and colleagues (1980) and Ferris and coworkers (1983) demonstrated a similar order of time divergence among four subspecies of M. musculus—domesticus, bactrianus, castaneus, and musculus—using restriction fragment length polymorphisms (RFLPs¹) in mitochondrial DNA (mtDNA¹). Bonhomme and colleagues (1984) proposed five biochemical groups in M. musculus—Mus-1, -2A, -2B, -2C, and -5—using 42 biochemical markers. These groups largely correspond to the four subspecies mentioned above, except for -2B, which is an intersubspecifc hybrid (Yonekawa et al. 1988).

As illustrated in Figure 2, we proposed classifying M. musculus into at least three subspecies groups—domesticus-, castaneus-, and musculus (Moriwaki 1994). This pedigree of genetic divergence in mouse subspecies groups has been confirmed by whole genome scanning using microsatellite DNA markers (Kikkawa et al. 2001; Sakai et al. 2005). Geographically, the domesticus subspecies group inhabits Western Europe and North Africa, the musculus group Eastern Europe and northern Asia, and the castaneus group Southeast Asia and southern China. In addition to the three groups, a new mitochondrial lineage has been reported in Yemen (Prager et al. 1998). Although the mice belonging to this lineage have been designated taxonomically as M. gentilulus, molecular analyses of mtDNA, Y-chromosome Zfy-2, and p53 have revealed a closer relationship with M. musculus.—It is not yet known whether this lineage has any connection to the genetic background of laboratory mouse strains.

Figure 2. Genetic diversification of major mouse subspecies groups and possible origin of the laboratory mouse strains. Reprinted from Moriwaki K. 1994. Wild mouse from a geneticist's viewpoint. In: Moriwaki K, Shiroishi T, Yonekawa H, eds. Genetics in Wild Mice. Tokyo: Karger Japan. p xvii. With permission from Japan Scientific Societies Press, Karger Japan, Inc., Tokyo, Japan.

Origins of the Laboratory Mouse from Written History

In the pedigree of laboratory mouse strains (Potter and Lieberman 1967), many strains stemmed from Abbie Lathrop's fancy mouse stocks (Granby Mouse Farm) in the United States. English fanciers are mentioned in the 1800s and Japanese fanciers are mentioned even earlier. In the Edo era, 100 to 400 yr ago, fancy mice were very popular in Japan. Many Japanese people bred fancy mice as a hobby, and famous artists such as Katsushika Hokusai and Kawanabe Gyousai used fancy mice as subjects in their drawings. Mice were also commonly used in Netsuke, a tiny carving of wood or ivory used by men as an accessory. We may assume that those mice were exported to Europe and later to the United States.

The booklet “Chingan Sodategusa,” translated as “how to breed fancy mice,” was published by Zeniya Choubei in Kyoto in 1787. Keeler made reference to this booklet in his book (1931), and Tokuda (1935) translated its outline into English. The booklet contains descriptions of the characteristics of many fancy mouse mutants as well as the breeding methods for maintaining the mutant genes.

In 1987, we obtained old Japanese fancy mice from Denmark as a courtesy of Dr. Nielsen at the University of Aarhus. The morphological and genetic characteristics of the mice were those of the musculus subspecies group (Koide et al. 1998). Since then, those mice have been bred at the National Institute of Genetics, Mishima, as the JF1 strain.

In Europe, and particularly in the United Kingdom, there has been a long tradition of mouse fanciers (Morse 1978). Even today, there is a “National Mouse Club.” At the beginning of the 20th century, two major groups of mouse stocks existed in the United States—the Japanese waltzing mice and the English stocks (Gates 1926).

Origin of the Laboratory Mouse from a Genetic Perspective

The fancy mice in both Europe and Asia were most likely derived from wild mice in each region. Genetic analyses on mtDNA (Yonekawa et al. 1982) clearly indicated that the maternal origin of most common laboratory mouse strains was the European domesticus subspecies group. In regard to the paternal origin, Bishop and colleagues (1985) suggested the contribution of the male musculus subspecies from results using a Y-specific DNA probe. There have been several reports that Japanese fancy mice were bred and used in research (Gates 1926). Using the RFLPs of the Sry gene, Nagamine and colleagues (1992) demonstrated that the musculus-Y chromosome in the laboratory strains was likely derived from the Japanese fancy mouse.

Other than the Y chromosome, there is other evidence of genetic contribution of the musculus subspecies group to the laboratory mouse. Chromosome C-band patterns have suggested that some of the chromosomes were derived from the musculus subspecies group (Moriwaki et al. 1990). The Akv gene in the AKR mouse strain appears to have originated from the musculus subspecies group on the Chinese mainland, based on molecular and geographical survey (Inaguma et al. 1991). Blank and coworkers (1986) documented the statistically significant contributions from Asian mice in the genomes of common laboratory mouse strains.

Recent all-inclusive surveys by microsatellite DNAs and single nucleotide polymorphisms (SNPs¹) in the nuclear genome have clarified the enormous contribution of the domesticussubspecies group to the common laboratory mouse strains (Kikkawa et al. 2001; Lindblad-Toh et al. 2000; Sakai et al. 2005; Witmer et al. 2003). Their findings strongly supported the domesticus-origin hypothesis by mtDNA (Yonekawa et al. 1982), in addition to the previous studies on ribosomal DNA (Suzuki et al. 1986) and chromosome C-band patterns (Moriwaki et al. 1985).

The possibility that the common laboratory inbred mouse strains originated from a relatively small number of founder mice has been investigated. Ferris and colleagues (1983) carried out an extensive survey on Scandinavian wild mice and suggested that the mtDNAs found in Scandinavian M. musculus were derived from a single M. domesticus female. Tanooka and coworkers (2001) surveyed mutations in the exon 4-5 region of the p53 pseudogene in wild mice and common laboratory mice. They found that laboratory mice probably originated from one of two domesticus subspecies groups of a northern European type, which suggested that their original number was small. Whiltshire and coworkers (2003) recently defined haplotype patterns in many laboratory strains by SNPs analysis. They found a limited number of haplotype blocks in the laboratory mouse strains, indicating that these strains were derived from a small founder population. Witmer and colleagues (2003) also analyzed 54 inbred laboratory mouse strains by 314 simple sequence length polymorphism (SSLP) markers and found that those strains consisted of five groups. These results suggest that laboratory mice are descended from a very small number of progenitor M. m. domesticus, a possibility also mentioned earlier by Potter and Lieberman (1967) and Kondo (1983b).

In summary, all of the data obtained to date have confirmed that the fancy mice of Japan and Europe contributed significantly to the origin of the laboratory mice currently used. The genetic background of the present-day laboratory mice was mainly derived from the European domesticussubspecies group and in a small part from Asian mice, probably Japanese fancy mice belonging to the musculus subspecies group. The contribution of the Asian mice is believed to be more than 20% from the large-scale genome analysis (Lindblad-Toh et al. 2000; Wade et al. 2002). Abe and colleagues (2004) have estimated it to be approximately 5% from a large-scale bacterial artificial chromosome-end sequence analysis. A recent report on the whole genome scanning analysis of eight common inbred strains and eight wild-derived inbred strains in the polymorphic 1,226 loci demonstrated that the average ratio of domesticus to non-domesticus alleles was 3:1 (Sakai et al. 2005). Most of the non-domesticus alleles are likely from the musculussubspecies group. These data indicate that the common laboratory mice are actually intersubspecific hybrids.

Effect of Genetic Background on the Phenotypes of Genetically Engineered Mice

As a result of recent remarkable developments in the technologies of both gene and embryo manipulation, it is now possible to isolate genes for study of normal biological functions and diseases from the mouse model. Cloned genes can be injected into the nuclei of zygotes to create transgenic mice with random insertions (Palmiter and Brinster 1986). The successful establishment of ES cell lines has made it possible to “knock-out” or “knock-in” a given gene by homologous recombination to create targeted gene mutations and observe their phenotypic effects in vivo (Capecchi 1989; Nagy et al. 2003; Smithies 1993).

Efficient production of transgenic mice is highly dependent on the successful recovery of a large number of good-quality zygotes. To maximize the yield of good-quality zygotes, F1 hybrid females and males are commonly used to recover F2 zygotes for microinjection (Nagy et al. 2003). The most widely used F1 hybrids are C57BL/6 x SJL and C57BL6 x DBA/2. However, phenotypic evaluation of these hybrids is complex because each individual transgenic mouse has a different genotype due to the segregating background genes of the two parental strains. There are many genetic and phenotypic differences between C57BL/6 and DBA/2 strains (Belknap et al. 1992; Klein et al. 1998; Peleg and Nesbitt 1984; Plomin et al. 1991; Zidek et al. 1998). Therefore, it is necessary to have a statistically significant number of transgenic and nontransgenic animals of mixed genetic backgrounds to distinguish the phenotype of the transgene from variations in the genetic backgrounds. The only practical method to remove the background effects is to produce congenic strains from standard inbred mouse strains.

Recently, inbred mouse strains such as FVB/N, C57BL/6, BALB/c, and C3H have been reported to be preferable as embryo donors for the creation of transgenic mice (Nagy et al. 2003; Taketo et al. 1991). The use of zygotes from inbred mouse strains enables more precise evaluation of the effect of the transgene because the difference between the transgenic and nontransgenic control mice is only the transgene.

Mice carrying targeted mutations have also been developed with mixed genetic backgrounds. The most widely used ES cells are derived from the 129 substrains (Nagy et al. 2003; Silver 1995). Usually, the targeted ES cells are injected into C57BL/6 blastocysts to form chimeric mice. The chimeric mice are crossed to C57BL/6 to recover heterozygous deficient mice of C57BL/6 x 129 background. Because C57BL/6 mice are good breeders, and the 129 substrains have poor reproduction (Festing 1979), most of targeted mutations have been developed in a mixed genetic background of C57BL/6 and 129 mice, or have been successively backcrossed to C57BL/6 as congenic strains. The mixed genetic background produces a potential source of variability in experiments, as mentioned above.

It should be noted that a germ cell-competent ES cell lines derived from a standard C57BL/6 inbred mouse strain have been established successfully (Schuster-Gossler et al. 2001). The generation of targeted mutations using C57BL/6 ES cells enables the establishment of a mutant strain of a pure genetic background without the labor-consuming backcrossing to create a congenic strain.

A number of reports to date have described the effect of genetic background on the results of transgenesis and targeted gene disruption. Threadgill and colleagues (1995) created epidermal growth factor receptor (Egfr¹) gene knock-out mice and demonstrated the effect of genetic background on embryonic lethality. The Egfr deficiency in CF-1 mice resulted in early embryonic death, but on a 129/Sv background, the homozygous embryos died at mid-gestation. Moreover, the homozygous Egfr-deficient mice survived to 3 wk of age on a CD-1 background. Overexpression of a transforming growth factor alpha transgene induced hepatocarcinogenesis in adult male mice (Takagi et al. 1992). In this hepatocarcinogenesis model, the incidence of tumor formation was found to be dependent on the genetic background. A hypoxanthine-guanine phosphoribosyltransferase (HPRT¹)-deficient mouse is a model for Lesch-Nyhan disease associated with dysfunction of basal ganglia dopamine systems. The analysis of striatal dopamine of the HPRT-deficient mice clearly demonstrated that the degree of dopamine loss was dependent on the genetic background (Jinnah et al. 1999).

The recent abundance of DNA markers in the mouse has made it possible to detect secondary genes, called modifier genes, using mating experiments. Baribault and coworkers (1994) reported that the embryonic lethality in the targeted mutation of the keratin 8 gene was greatly influenced by the mouse strain genetic background (FVB/N v.s. C57BL/6 x 129/Sv). They emphasized the importance of using several inbred strains to reveal the polygenic contribution to mutant phenotypes, and they demonstrated that modifiers of K8/K18 filament functions operated differentially in mice with FVB/N and C57BL/6 genetic backgrounds. The survival of the cystic fibrosis transmembrane conductance regulator (Cftr¹)-deficient mice was also affected by the background strains (Rozmahel et al. 1996). A genome scan among backcross and intercross progeny with different inbred strains revealed the major modifier locus for the Cftr gene.

The phenotypes of mutant mice are thus influenced by their genetic background. Other variable factors should also be taken into consideration for the correct interpretation and comparison of the phenotypes of genetically engineered mice (Lahvis and Bradfield 1998; Mahler 2000). Such factors include ES cell lines (129 substrains), linked genes around the induced mutations, insertional mutagenesis or genomic alteration associated with random transgenesis, gene targeting strategy, expression of targeted alleles (protein, RNA), microbiological status, pathogens and pollutants in the environment (bedding, food, water), modifier loci, and strain-dependent phenotypes.

Wild-derived Strains as a Unique Genetic Background

Experimental animals used as models for human diseases should constitute genetic variations similar to human populations. Well-known inbred strains, however, represent only a relatively small part of the genetic divergence of the Mus species (Potter and Lieberman 1967). At the time of this writing, more than 400 inbred strains can be found in the present public database of major mouse strains (http://www.informatics.jax.org/external/festing/mouse/STRAINS.shtml). In addition, many spontaneous mutations for metabolism and immunity have also been recovered among laboratory mice over the last century. These mutant mice have been used as models to dissect human diseases genetically. However, Kondo (1983b) noticed the lack of a “normal” mouse strain that represents the M. musculus “wild-type,” due to the fact that inbred and mutant mouse strains are genetically homozygous for accumulated mutations and other genetic loci, and are therefore too specialized to be “normal” mice. Kondo emphasized the need to establish strains derived from wild mice, especially for behavioral and brain sciences, and described attempts to establish inbred strains from Japanese wild mice, M. musculus molossinus.

Kondo (1983a) had also attempted to develop RI strains from two genetically remote subspecies, the C57BL/6 and a Japanese wild-derived MOA strain. In spite of great effort, most of the RI strains exhibited reproductive difficulty between the inbreeding generations 3 to 8. No RI strain has been successfully established. Later, similar trials using different combinations of inbred laboratory and wild-derived mouse strains were performed at the National Institute of Genetics, but they also were not successful. This phenomenon is likely related to hybrid breakdown or genetic incompatibility between remote subspecies; however, the precise mechanism is unknown. A novel approach has been the use of a genetically remote genome to study complex genetic traits through the establishment of consomic strains by Dr. Shiroishi at the National Institute of Genetics and by Dr. Yonekawa at the Tokyo Metropolitan Institute of Medical Science. They introduced each chromosome of the Japanese wild-derived strain MSM into C57BL/6 by repeated back crossing. This trial has been mostly successful, except for the X-chromosome. Genetic incompatibility was detected between some C57BL/6 autosomes and the MSM X-chromosome. Recently, the significant X-linked and autosomal gene loci responsible for the sterility have been mapped by QTL analysis (Oka et al. 2004).

A number of wild-derived inbred mouse strains from different Mus species and M. musculus subspecies captured from locations worldwide are currently available from mouse resource centers such as the Jackson Laboratory (http://www.jax.org) and the RIKEN BioResource Center (http://www.brc.riken.jp) (Figure 3). Koide and colleagues (2000) clearly demonstrated that a variety of wild mouse strains provide novel behavioral phenotypes in several different tests. Totsuka and coworkers (2003) found that physical performance of muscle fibers of the wild-derived MSM was superior to those of the laboratory mouse strains. Masuya and colleagues (1997) found that the polydactylous phenotype of the mutants Rim4, Xt, Ist, and lx could be modified by crossing with the wild-derived MSM. Moreover, a novel tumor suppressor gene was detected in skin tumors by using the MSM congenic strain of p53 KO mice (Miyazawa et al. 2002). Thus, the wild-derived inbred mouse strains can be used successfully as a unique resource to find novel allelic variations and modifiers of spontaneous, randomly induced or genetically engineered mutations.

Figure 3. Wild-derived strains of Mus species available from the RIKEN BioResource Center (RIKEN BRC), Tsukuba, Japan. Most of the strains, except Car, Mus caroli, are inbred (http://www.brc.riken.jp/lab/animal/en).

Concluding Remarks

The genome of laboratory mice is considered to be derived from two different M. musculus subspecies, mostly domesticus and partly musculus. For the establishment of common laboratory mouse strains, fancy mice from England and Japan might have contributed to some part of the genome. The various laboratory inbred mouse strains have become genetically uniform as a result of extensive inbreeding and have accumulated characteristic mutations and genetic backgrounds during the course of breeding. The inbred strains have contributed greatly to genetic analyses of many Mendelian traits, and have also provided uniform genetic background for the spontaneous and genetically engineered mutations to evaluate the effect of a specific gene. However, mutations that have accumulated under laboratory conditions appear to be biologically atypical. Because the common laboratory mouse stains have generally been established from relatively small gene pools, common inbred mouse strains have limitations in their genetic polymorphisms and phenotypic variations.

Wild-derived strains can complement these defective points in the laboratory inbred mouse strains by providing novel phenotypes, probably a “normal” phenotype of the M. musculus, as well as new allelic variants. The wildness of the wild-derived strains is sometimes regarded as a disadvantage for a good experimental animal. However, for the future study of the biological functions of genes, this wild character has great potential value, particularly for the study of normal neuronal and physiological functions.

For these reasons, it is important to recognize the value of genetic background to the interpretation of experimental results. Knowledge about the origin of the laboratory mouse and the evolutionary history of its genome will hopefully inspire the scientist to select and refine their mouse strains and thus contribute to the reduction and refinement of animal experimentation.

Acknowledgments

The authors wish to thank Drs. J-L Guenet (Institut Pasteur, Paris) and T. Shiroishi (National Institute of Genetics, Mishima) for providing wild-derived mouse strains to the RIKEN BioResource Center. The support of Drs. Yuichi Obata (Department of Biological Systems, RIKEN BioResource Center) and Toshihiko Shiroishi (Genetic Strains Research Center, National Institute of Genetics), as well as all staff members of the Experimental Animal Division, RIKEN BioResource Center, is gratefully acknowledged. The RIKEN BioResource Center, which collects and preserves the mouse resources as a core facility for the National Bio-Resource Project in Japan, is supported by the Ministry of Education, Culture, Sports, Science and Technology.

Abbreviations used in this article: Cftr, cystic fibrosis transmembrane conductance regulator; Egfr, epidermal growth factor receptor; ES, embryonic stem; HPRT, hypoxanthine-guanine phosphoribosyltransferase; MHC, major histocompatibility complex; mtDNA, mitochondrial DNA; QTL, quantitative trait loci; RFLP, restriction fragment length polymorphism; RI, recombinant inbred; SNP, single nucleotide polymorphism.

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