What is a mouse? Ask any small child and you will hear the answer — a small furry creature with big ears and a big smile. Today, in Japan, Europe, America, and elsewhere, this question evokes images of that quintessential mouse icon — Mickey. But even before the age of cinema, television, and theme parks, mice had entered the cultures of most people. In the English-speaking world, they have come down through the ages in the form of nursery rhymes sung to young children including "Three blind mice" and Hickory, dickory dock, the mouse ran up the clock . . . . Artistic renditions of mice in the form of trinkets, such as those shown in figure 1.1 and on the cover of this book, are sold in shops throughout the world. Why has the mouse been in the minds of people for so long? The most obvious reason is that one particular type of mouse — the so-called house mouse — has lived in close association with most, if not all, human populations since the dawn of civilization.

This dawn occurred at the end of the last ice age, some 10,000 years ago, across a region retrospectively called the Fertile Crescent that extends from modern-day Israel up through Lebanon and Syria and curves back down through Iraq toward the Persian Gulf (figure 1.2). It was in this region at this time — known as the neolithic transition — that tribes of nomadic hunters and gatherers began to cultivate plants and domesticate animals as a means for sustenance (Ammerman and Cavalli-Sforza, 1984). Farming eliminated the need for constant migration and brought about the formation of villages and the construction of permanent shelters for both people and their livestock. With the seasonal planting of crops, families needed to store dry food, in the form of grain, for both themselves and their animals. With food reserves in granaries and cupboards, the house mouse began its long interwoven history with humankind.

The ancestors of the house mouse, who were concentrated in the steppes of present-day Pakistan at that time (figure 1.2), had been living happily oblivious to people for eons, but suddenly (in terms of evolutionary time), migrants to the new Neolithic villages found mouse paradise in the form of a secure shelter with unlimited food (Auffray et al., 1990). With their ability to squeeze through the tiniest of holes — adults can pass through apertures as small as a single centimeter in width (Rowe, 1981) — our furry friends were clearly pre-adapted to take advantage of these Neolithic edifices, and with their agility and speed, they were able to stay one step ahead of the cleaver wielded by the farmer’s wife. This pre-adaptation, and the opportunistic ability to eat almost anything, has allowed the house mouse to become the second most successful mammalian species living on earth today (Berry, 1981; Sage, 1981).

When people wandered out from the Middle East in search of new lands to cultivate, mice followed as stowaways within the vehicles used to carry household belongings. Later, they would travel with ship-borne merchants going to and from distant lands. In this millennium, it is not too farfetched to imagine mice traveling on the Santa Maria with Columbus to the new world, and on horse-drawn buggies with families emigrating from the original American colonies to the Western part of the continent. As people overcame harsh environments through the construction of artificial habitats, these became the natural environment for the house mouse. Freeloading on people has allowed the house mouse to enjoy a wider range than all species but one. Today, house mice can be found wherever there are permanent populations of people (as well as many places where there are none), in both urban and rural areas, on all of the continents, at altitudes as high as 15,600 feet (4750 m), as far north as the Bering Sea and as far south as sub-Antarctic islands (Berry and Peters, 1975; Sage, 1981).

The fact that many "grown-up" humans and mice have had an adversarial relationship through most of history is evident in the derivation of the name that English speakers use to describe these creatures. Mouse can be traced back through the Latin mus and the Greek mys to the ancient Sanskrit mush, meaning "to steal". There was little that adults in the ancient world could do to prevent mice from overrunning granaries until the discovery of the natural predilection of cats for rooting-out and destroying small rodents. In fact, Keeler (1931) has suggested that the deification of the cat by the ancient Egyptians was due mostly to the role that it played in reducing house mouse populations. And an ancient Persian legend, from the millennium before Christ says that "the moon chases the clouds as a cat chases mice" (Keeler, 1931). In somewhat later times (900 AD), the Welsh fixed the price of cats based on their mouse-catching experience (Sage, 1981). This image of the cat as a veritable biological-pesticide is prevalent in many early cultures, and could explain the original rationale for its domestication.

Although mice and farmers may not have seen eye-to-eye, one can imagine the potential for a very different type of relationship between mice and people not directly affected by their dastardly deeds. This is because mice are often viewed in a very different light than other animals as best summed up in the words of a contemporary artist:

"The mouse is a great friend to artists, then, because we like him. He doesn’t seem to have any specially bad characteristics — at worst, his life is a little drab, but we all suspect our lives of being just that . . . Not enough like us to unnerve us, he is a tiny creature (therefore clearly inferior) who looks up to us and fears us (therefore reassuring), who is not directly useful to us (therefore not a menial), and can be a pleasant furry companion without making extensive demands on us (therefore a true friend). No wonder artists appreciate the mouse; put him in a work and you win your human audience instantly . . ."

The house mouse was highly visible to children growing up on farms as well as in towns, and legend has it that the tame animals wandering in and out of Walt Disney’s original cartoon studio in Kansas provided the inspiration for the creation of Mickey Mouse (Updike, 1991). House mice can express a high level of interesting activity in a small amount of space when presented with various playthings. They can breed easily in captivity, their diets are simply satisfied, they can be housed in small spaces, and one can select artificially for increased docility in each generation. With continuous human contact from birth, mice acclimate to touch and can be handled quite readily.

Early instances of mouse domestication, and even worship, by the ancient Greeks and Romans is described in detail by Keeler (1931). From the classical period onward, the domesticated mouse has appeared in various Eurasian cultures. Of particular importance to the history of the laboratory mouse was the fondness held for unusual-looking mice by the Chinese and Japanese. This fondness led Asian breeders to select and develop a variety of mutant lines with strikingly different coat colors, some of which can be seen in detailed paintings from the eighteenth and nineteenth centuries. During the nineteenth century, the house mouse became "an object of fancy" in Europe as well (Sage, 1981), and British, Chinese, and Japanese traders brought animals back and forth to develop new breeds. By the beginning of the twentieth century, European and American fanciers were familiar with lines of mice having fanciful names like white English sable, creamy buff, red cream, and ruby-eyed yellow (Sage, 1981).

A critical link between the mouse fanciers and early American mouse geneticists was Miss Abbie Lathrop, a retired school teacher who began, around 1900, to breed mice for sale as pets from her home in Granby, Massachusetts (Morse, 1978). Conveniently, Lathrop’s home and farm were located near to the Bussey Institute directed by William Castle of Harvard University (see section 1.2.2). Not only did Lathrop provide early mouse geneticists — including Castle and his colleagues at Harvard and Leo Loeb at the University of Pennsylvania — with a constant source of different fancy mice for their experiments, but she conducted her own experimental program as well with as many as 11,000 animals breeding on her farm at any one time between 1910 and her death in 1918 (Morse, 1978). Many of the common inbred lines so important to mouse geneticists today — including C57BL/6 and C57BL/10 (commonly abbreviated as B6 and B10) — are derived entirely from animals provided by Abbie Lathrop. A more detailed account of her contributions along with photographs of her breeding records and her farm can be found in a historical review by Morse (1978).

The mouse played a major role in early genetic studies begun immediately after the rediscovery of Mendel’s laws in 1900. All of the initial findings were based on work carried out entirely with plants and there was much skepticism in the scientific community as to how general Mendel’s Laws would be (Dunn, 1965 p.86). Did the laws explain all aspects of inheritance from individuals? Were there some species groups — such as ourselves and other mammals — where the laws did not apply at all? In particular, the competing theory of blending inheritance was defended by Galton during the latter part of the 19th century. The main tenet of this theory was that a blending of the traits expressed by each of the parents occurred within each offspring. Blending inheritance and Mendelism have strikingly different predictions for the future descendants of a cross that brings a new "character" into a pure-bred race. According to the blending theory, the new character would remain in all of the descendants from the original "contaminating" cross: even upon sequential backcrosses to the pure-bred parental strain, the contaminating character would only slowly be diluted out. Of course, the Mendelian prediction is that a contaminating allele (to use current language) can be eliminated completely within a single generation.

The main support for blending inheritance came through a cursory observation of common forms of variation that exist in animal as well as human populations. It can certainly appear to be the case that human skin color and height do blend together and dilute from one generation to the next. However, skin color, height, and nearly all other common forms of natural variation are determined not by alternative alleles at a single loci, but instead by interactions of multiple genes, each having multiple alleles leading to what appear to be continua of phenotypes. Mendel’s leap in understanding occurred because he chose to ignore such complicated forms of inheritance and instead focused his efforts on traits that came in only two alternative "either-or" forms. Of equal importance was his decision to begin his crosses with pairs of inbred lines that differed by only a single trait, rather than many. It was only in this manner that Mendel was able to see through the noise of commonplace multifactorial traits to derive his principles of segregation, independent assortment, and dominant-recessive relationships between alleles at single loci.

How could one investigate the applicability of Mendel’s laws to mammals with the use of natural variants alone? The answer was with great difficulty — not only does natural variation tend to be multifactorial, there’s just not very much of it that is visible in wild animals, and without visible variation, there could be no formal genetics in 1900. The obvious alternative was to use a species in which numerous variants had been derived and were readily available within pure-breeding lines. And thus begun the marriage between the fancy mice and experimental genetics.

Evidence for the applicability of Mendel’s laws to mammals — and by implication, to humans — came quickly, with a series of papers published by the French geneticist Cuénot on the inheritance of the various coat color phenotypes (Cuénot, 1902; Cuénot, 1903; Cuénot, 1905). Not only did these studies confirm the simple dominant and recessive inheritance patterns expected from "Mendelism", they also brought to light additional phenomena such as the existence of more than two alleles at a locus, recessive lethal alleles, and epistatic interactions among unlinked genes.

The most significant force in early genetic work on the mouse was William Ernest Castle, who directed the Bussey Institute at Harvard University until his retirement in 1936 (Morse, 1985). Castle brought the fancy mouse into his laboratory in 1902 and with his numerous students began a systematic analysis of inheritance and genetic variation in this species as well as in other mammals (Castle, 1903; Morse, 1978; Morse, 1981; Snell and Reed, 1993). The influence of Castle on the field of mammalian genetics as a whole was enormous — over a period of 28 years, the Bussey Institute trained 49 students, including L.C. Dunn, Clarence Little, Sewall Wright, and George Snell; thirteen were elected to the National Academy of Sciences in the U.S. (Morse, 1985), and many students of mouse genetics today can trace their scientific heritage back to Castle in one way or another.

A major contribution of the Castle group, and Clarence Little in particular, was the realization of the need for, and development of, inbred genetically-homogeneous lines of mice (discussed fully in section 3.2). The first mating to produce an inbred line was begun by Little in 1909, and resulted in the DBA strain, so-called because it carries mutant alleles at three coat color loci — dilute (d), brown (b), and non-agouti (a). In 1918, Little accepted a position at the Cold Spring Harbor Laboratory, and with colleagues that followed — including Leonell Strong, L. and E. C. MacDowell,— developed the most famous early inbred lines including B6, B10, C3H, CBA, and BALB/c. Although an original rationale for their development was to demonstrate the genetic basis for various forms of cancer, these inbred lines have played a crucial role in all areas of mouse genetics by allowing independent researchers to perform experiments on the same genetic material, which in turn allows results obtained in Japan to be compared directly with those obtained halfway around the world in Italy. A second, and more important, contribution of Little to mouse genetics was the role that he played in founding the Jackson Laboratory in Bar Harbor, Maine, and acting as its first director (Russell, 1978). The laboratory was inaugurated in 1929 — as "the natural heir to the Bussey" (Snell and Reed, 1993) — with eight researchers and numerous boxes of the original inbred strains.

With the demonstration in the mouse of genetic factors that impact upon cancer, millions upon millions of animals were used to elucidate the roles of these factors in more detail. However, for the most part, these biomedical researchers did not breed their own animals. Rather, they bought ready-made, off-the-shelf, specialized strains from suppliers like Taconic Farms, Charles River Laboratories, and the Jackson Laboratory (addresses of these and other suppliers are provided in appendix A). The strong focus of mouse research in the direction of cancer can be seen clearly in the Table of contents from the first edition of the landmark book Biology of the Laboratory Mouse published in 1941: five of thirteen chapters are devoted to cancer biology, with only two chapters devoted to other aspects of genetic analysis (Snell, 1941).

Until the last decade, the community of geneticists that actually performed their own in-house breeding studies on the mouse was rather small. For the most part, individual mouse geneticists worked in isolation at various institutions around the world. Typically, each of these researchers focused on a single locus or well-defined experimental problem that was amenable to analysis within a small breeding colony. Members of the mouse community kept track of each other’s comings and goings through a publication called The Mouse Newsletter. In its heyday during the 1960s, more than sixty institutions would routinely contribute "a note" to this effect. These contributed notes served the additional purpose of providing researchers with a means for announcing and reading about the various strains and mutations that were being bred around the world. A characteristic of the genetics community, during this period, was the openness with which researchers freely traded specialized mouse stocks — not available from suppliers — back and forth to each other.

Apart from this cottage industry style of conducting mouse genetics were three institutions where major commitments to the field had been made in terms of personnel and breeding facilities. These three institutions were the Oak Ridge National Laboratory in Oak Ridge, Tennessee, the Atomic Energy Research Establishment in Harwell, England, and the Jackson Laboratory (JAX) in Bar Harbor, Maine. The genetics programs at both Harwell and Oak Ridge were initiated at the end of the second world war with the task of defining the effects of radiation on mice as a model for understanding the consequences of nuclear fallout on human beings. Luckily, researchers at both of these institutions — prominently including Bill and Lee Russell at Oak Ridge and T. C. Carter, Mary Lyon and Bruce Cattanach at Harwell — appreciated the incredible usefulness of the animals produced as byproducts of these large-scale mutagenesis studies in providing tools to investigate fundamental problems in mammalian genetics (see section 6.1).

The third major center of mouse genetics — the Jackson Laboratory — has always had, and continues to maintain, a unique place in this field. It is the only non-profit institution ever set up with a dedication to basic research on the genetics of mammals as a primary objective. Although the JAX originally bred many different species (including dogs, rabbits, guinea pigs and others), it has evolved into an institution that is almost entirely directed toward the mouse.  Genetic mapping and descriptions of newly uncovered mutations and variants have been a focus of research at the laboratory since its inception in 1929. But in addition to its own in-house research, the JAX serves the worldwide community of mouse geneticists in three other capacities. The first is in the maintenance and distribution of hundreds of special strains and mutant stocks. The second is as a central database resource. The third is in the realm of education in mouse genetics and related fields with various programs for non-scientists and high school and college students, as well as summer courses and conferences for established investigators.

Even with the three centers of mouse research and the cottage industry described above, genetic investigations of the mouse were greatly overshadowed during the first eighty years of the twentieth century by studies in other species, most prominently, the fruit fly Drosophila melanogaster. The reasons for this are readily apparent. Individual flies are exceedingly small, they reproduce rapidly with large numbers of offspring, and they are highly amenable to mutagenesis studies. In comparison to the mouse, the fruit fly can be bred more quickly and more cheaply, both by many orders of magnitude. Until the 1970s, Drosophila provided the most tractable system for analysis of the genetic control of development and differentiation. In the 1970s, a competitor to Drosophila appeared in the form of the nematode Caenorhabditis elegans. which is even more tractable to the genetic analysis of development as well as neurobiology. So why study the mouse at all?

The answer is that a significant portion of biological research is aimed at understanding ourselves as human beings. Although many features of human biology at the cell and molecular levels are shared across the spectrum of life on earth, our more advanced organismal-based characteristics are shared in a more limited fashion with other species. At one extreme are a small number of human characteristics — mostly concerned with brain function and behavior — that are shared by no other species or only by primates, but at a step below are a whole host of characteristics that are shared in common only with mammals. In this vein, the importance of mice in genetic studies was first recognized in the intertwined biomedical fields of immunology and cancer research, for which a mammalian model was essential. Although it has long been obvious that many other aspects of human biology and development should be amenable to mouse models, until recently, the tools just did not exist to allow for a genetic dissection of these systems.

The movement of mouse genetics from a backwater field of study to the forefront of modern biomedical research was catalyzed by the recombinant DNA revolution, which began 20 years ago and has been accelerating in pace ever since. With the ability to isolate cloned copies of genes and to compare DNA sequences from different organisms came the realization that mice and humans (as well as all other placental mammals) are even more similar genetically than they were thought to be previously. An astounding finding has been that all human genes have counterparts in the mouse genome which can almost always be recognized by cross-species hybridization. Thus, the cloning of a human gene leads directly to the cloning of a mouse homolog which can be used for genetic, molecular, and biochemical studies that can then be extrapolated back to an understanding of the function of the human gene. In only a subset of cases are mammalian genes conserved within the genomes of Drosophila or C. elegans.

This result should not be surprising in light of current estimates for the time of divergence of mice, flies and nematodes from the evolutionary line leading to humans. In general, three types of information have been used to build phylogenetic trees for distantly related members of the animal kingdom — paleontological data based on radiodated fossil remains, sequence comparisons of highly conserved proteins, and direct comparisons of the most highly conserved genomic sequences, namely the ribosomal genes. Unfortunately, flies (Drosophila) and nematodes (C. elegans) diverged apart from the line leading to mammals prior to the time of the earliest fossil records in the Cambrian period which occurred 500-600 million years ago. Nevertheless, sequence data together with taxonomic considerations indicate a distant point of departure of C. elegans and vertebrates from a common ancestor that lived on the order of one billion years ago (Field et al., 1988). Drosophila diverged apart from the vertebrate line at a somewhat later period approximately 700 million years ago (Dayhoff, 1978). The divergence of mice and people occurred relatively recently at 60 million years before present (see section 2.2.1). These numbers are presented graphically in figure 1.3 where a quick glance serves to drive home the fact that humans and mice are ten-times more closely related to each other than either is to flies or nematodes.

Although the haploid chromosome number associated with different mammalian species varies tremendously, the haploid content of mammalian DNA remains constant at approximately three billion basepairs. It is not only the size of the genome that has remained constant among mammals; the underlying genomic organization (discussed in chapter 5) has also remained the same as well. Large genomic segments — on average, ten to twenty million basepairs — have been conserved intact between mice, humans, and other mammals as well. In fact, the available data suggest that a rough replica of the human genome could be built by simply breaking the mouse genome into 130-170 pieces and pasting them back together again in a new order (Nadeau, 1984; Copeland et al., 1993). Although all mammals are remarkably similar in their overall body plan, there are some differences in the details of both development and metabolism, and occasionally these differences can prevent the extrapolation of mouse data to humans and vice versa (Erickson, 1989). Nevertheless, the mouse has proven itself over and over again as being the model experimental animal par excellence for studies of nearly all aspects of human genetics.

Among mammals, the mouse is ideally suited for genetic analysis. First, it is among the smallest mammals known with adult weights in the range of 25 to 40 grams, 2,000-3,000-fold lighter than the average human adult. Second, it has a short generation time — on the order of 10 weeks from being born to giving birth. Third, females breed prolifically in the lab with an average of 5—10 pups per litter and an immediate postpartum estrus. Fourth, an often forgotten advantage is the fact that fathers do not harm their young, and thus breeding pairs can be maintained together after litters are born. Fifth, for developmental studies, the deposition of a vaginal plug allows an investigator to time all pregnancies without actually witnessing the act of copulation and, once again, without removing males from the breeding cage. Finally, most laboratory-bred strains are relatively docile and easy to handle.

The high resolution genetic studies to be discussed later in this book require the analysis of large numbers of offspring from each of the crosses under analysis. Thus, a critical quotient in choosing an organism can be expressed as the number of animals bred per square meter of animal facility space per year. For mice, this number can be as high as 3,000 pups/m2 including the actual space for racks (five shelves high) as well as the inter-rack space as well. All of the reasons listed here make the mouse an excellent species for genetic analysis and have helped to make it the major model for the study of human disease and normative biology.

The close correspondence discovered between the genomes of mice and humans would not, in and of itself, have been sufficient to drive workers into mouse genetics without the simultaneous development, during the last decade, of increasingly more sophisticated tools to study and manipulate the embryonic genome. Today, genetic material from any source (natural, synthetic or a combination of the two) can be injected directly into the nuclei of fertilized eggs; two or more cleavage-stage embryos can be teased apart into component cells and put back together again in new "chimeric" combinations; nuclei can be switched back and forth among different embryonic cytoplasma; embryonic cells can be placed into tissue culture, where targeted manipulation of individual genes can be accomplished before these cells are returned to the embryo proper. Genetically-altered live animals can be obtained subsequent to all of these procedures, and these animals can transmit their altered genetic material to their offspring. The protocols involved in all of these manipulations of embryos and genomes have become well-established and cookbook manuals (Joyner, 1993; Wassarman and DePamphilis, 1993; Hogan et al., 1994) as well as a video guide to the protocols involved (Pedersen et al., 1993) have been published.

While it is likely that none of these manipulations has yet been applied to human embryos and genomes, it is ethical, rather than technical, roadblocks that impede progress in this direction. The mental image invoked is of a far more sophisticated technology than the so-called futuristic scenario of embryo farms described in Huxley’s Brave New World. (1932).

Progress has also been made at the level of molecular analysis within the developing embryo. With the polymerase chain reaction (PCR) protocol, DNA and RNA sequences from single cells can be characterized, and enhanced versions of the somewhat older techniques of in situ hybridization and immuno-staining allow investigators to follow the patterns of individual gene expression through the four dimensions of space and time (Wassarman and DePamphilis, 1993; Hogan et al., 1994). In addition, with the omnipresent micro-techniques developed across the field of biochemistry, the traditional requirement for large research animals like the rat, rabbit, or guinea pig has all but evaporated.

Finally, with the automation and simplification of molecular assays that has occurred over the last several years, it has become possible to determine chromosomal map positions to a very high degree of resolution. Genetic studies of this type are relying increasingly on extremely polymorphic microsatellite loci (section 8.3) to produce anchored linkage maps (chapter 9), and large insert cloning vectors — such as YACs — to move from the observation of a phenotype, to a map of the loci that cause the phenotype, to clones of the loci themselves (section 10.3). Thus, many of the advantages that were once uniquely available to investigators studying lower organisms, such as flies and worms, can now be applied to the mouse through the three-way marriage of genetics, molecular biology, and embryology represented in figure 1.4. It is the intention of this book to provide the conceptual framework and practical basis for the new mouse genetics.