Genomics from the Perspective of the Laboratory Mouse

Overview “Muromics”: Genomics from the Perspective of the Laboratory Mouse

Stephen W. Barthold, DVM, PhD

The laboratory mouse has emerged as the preeminent model though mice are genetically well characterized, they also add an system for mammalian genomics research, and it continues its additional depth of complexity because of their genetic back- importance as a valuable model system for hypothesis-driven re- ground. By various counts, there are approximately 3,000 inbred, search. As scientific enterprise moves from the genomics era to the congenic, coisogenic, consomic, recombinant inbred, mutant, and post-genomics era, a rich lexicon of “-omics” has arisen, including other strains of mice. Genetically altered mice are swelling these phenomics, physiomics, proteomics, glycomics, dramanomics, numbers to unprecedented levels. The following overview illus- metabolomics, pharmacogenomics, and toxicogenomics. What is trates how the inherent value of these incredibly powerful re- missing is “muromics,” a rhetorical term used here to emphasize search models is diluted by genetic mismanagement or naivete. the focus of this review: understanding genomics and phenomics in the context of the complex biology of the laboratory mouse (the What is a laboratory mouse? murome). A proper definition of the modern laboratory mouse requires The inbred nature of laboratory mice is their greatest quality, an understanding of its history and origins. What is known as allowing control of important genetic variables and precise in- the laboratory mouse evolved at the beginning of the past cen- vestigation of specific gene alterations or gene function. Despite tury, during the American scientific movement through the im- this ability to control these variables, the mouse is a complex portation of “fancy mice” into the laboratory. Long before that biologic organism with complex biologic responses. When we time, mice had been selectively bred for coat color and other (the human species) attempt to explain gene function, we tend “phenotypes” for thousands of years. The first historical record to simplify and think in terms of linear, predictable outcomes. In of color-variant mice goes back to 1100 B.C. in China. Waltzing fact, biological systems, including mouse phenotypes, are non- mice were described in Japan in 80 B.C. (1, 2). The mouse was linear and complex, but not random. Nearly half of gene alter- brought into the laboratory with the rediscovery of Mendel’s ations result in no detectable phenotype, and nearly half of gene laws of heredity, and application of those principles to mamma- alterations result in unexpected phenotypes. These statistics lian inheritance. Early work by Castle (who coined the term “ge- are only roughly accurate, as few mice are examined compre- netics”) focused principally on inheritance of coat color and hensively. Furthermore, “forward genomics,” in which an out- other external characteristics because of the plethora of visible come (phenotype) is predicted from the alteration of a known variants among fancy mice, but interest rapidly turned to the gene, is fraught with investigator bias due to wishful thinking genetics of cancer. for a desired phenotype. “Reverse genomics,” such as that which Because of the complexity of inheritance, Little started in- involves random chemical mutagenesis, overcomes this bias, breeding mice to obtain “homogeneity of genetic constitution” to but requires screening processes that can be seriously disrupted effectively investigate the genetic basis of cancer (1). His first by misinterpretation of phenotype or comprehensiveness of the inbred mouse was the dilute brown non-agouti (DBA), which screening battery. was created in the early 1900s. This strain was nearly wiped out A glimpse at the “Trans-NIH Mouse Initiative” web site from an outbreak of “paratyphoid” while at Cold Spring Harbor (www.nih.gov/science/models/mouse/), which depicts an image Laboratory, portending the continuing threat of infectious dis- of laboratory rats rather than mice, illustrates the naivete con- ease among mice with uniform genetic susceptibility. This cata- cerning phenotype at the highest levels of the scientific commu- strophic event had the beneficial effect of stimulating creation nity. More than a few mouse pathologists must face scientists of other inbred strains of mice. During the 1920s and 1930s, the with the stark reality of abscesses, rather than transplantable efforts of Little, Strong, MacDowell, Dunn, Furth, Bagg, and tumors; lymph nodes, rather than mammary neoplasms; or several others resulted in the creation of nearly all of the inbred seminal vesicles, rather than uterine horns. Few scientists, and strains of mice that are used in contemporary biomedical re- in particular, few molecular biologists are fully cognizant of the search. The AKR mouse strain was developed by Furth because complexity of the multi-systemic mammalian organism, and al- MacDowell was unwilling to share his cancer-prone C58 mice, which were the key to his research funding by private benefac- Received: 12/11/01. Revision requested: 1/24/02. Accepted: 3/15/02. Professor and Director, Center for Comparative Medicine, University of Califor- tors. This early example of proprietary control of mouse strains nia at Davis, One Shields Avenue, Davis, California 95616. pales in comparison with today’s legal constraints placed on ge- Supported by grant RR-14905 “University of California Davis Mutant Mouse Regional Resource Center” from the National Center for Research Resources, netically engineered mice with perceived commercial value, National Institutes of Health. such as Onco-mice and Cre-Lox mice. 206 “Muromics”

Most of the breeding stock for these early lines of mice were velop amyloidosis (except for tumor-related amyloid, which is derived from fancy mouse stocks, and often from common common). Most BALB/c mice do not have a corpus callosum, and sources, such as Miss Abbie Lathrop’s Granby Mouse Farm in have progressive hearing loss associated with a recessive gene Massachusetts. The alphabet soup of letters and numbers that that differs from that of B6 mice (6). BALB/c mice also develop represent many inbred strains of mice are derived from various high prevalence of pulmonary adenomas, salivary myoepithelio- line and subline laboratory designations of these early efforts to mas, mammary tumors, generalized lymphoproliferative disease inbreed. There are exceptions, such as mice developed by Lynch, (lymphoma), and glomerulonephritis. The C3H strain was origi- who imported her own mice directly from Switzerland, giving nally derived by Strong for its susceptibility to mammary cancer, rise to “Swiss” mice and their inbred and outbred derivatives. but this strain also is prone to hepatocellular cancer. They are a Another is BALB/c (Bagg albino) mice, whose progenitors origi- notably docile mouse, because they are totally blind at weaning nated from a dealer in Ohio (1). A recent geneology chart of in- age due to retinal degeneration. Because these three strains of bred mouse strains, which is based on historical data and recent mice have been commonly used in toxicology and aging research, new data, has been compiled (2). the pathologist is fortunate to benefit from a large body of litera- Fancy mice have been traded throughout Asia, Europe, and ture involving their background pathology (phenotype) (7-9). North America for centuries. Thus, the progenitors of the labora- Genomics research has also increased the use of 129 and FVB tory mouse strains that are used today are a hybrid of various spe- mice, and a potpourri of other strains. The 129 strain of mice is cies or subspecies of the Mus musculus genospecies complex. Most extensively used in genomics research as a source of embryonic inbred strains of mice have an undefined mix of European and stem (ES) cells. This strain did not enjoy much prominence as Asian genes/alleles contributed by M. musculus, M. domesticus, M. research animals except for its tendency to develop testicular ter- castaneus, M. molossinus (which is a natural mix of M. castaneus atomas (a.k.a. teratocarcinomas, embryonal carcinomas). These and M. musculus), and possibly other members of the M. musculus mice were developed by Dunn from English fancy mice and chin- genospecies complex, including M. spretus (3, 4). chilla stock provided by Castle. The derivation of stem cells from Various inbred strains of mice, because of their diverse back- embryos was a natural extension of considerable earlier effort grounds, are genetic apples and oranges, and therefore, each based on teratocarcinoma stem cells (which were of 129 strain with their own distinct strain characteristics. A valuable re- origin). Substrains of 129 mice (formerly named 129/Sv and 129/ source for information on general inbred mouse strain charac- Sv-ter), with enhanced rates of teratoma development, were thus teristics that is periodically updated is available from Festing: produced (10). Although ES cells can be derived from other (http://www.informatics.jax.org/external/festing/mouse/ mouse strains, most targeted mutations are created from well STRAINS.shtml). established 129 ES cell lines, and often backcrossed onto a more desirable or better standardized strain, such as B6 mice. Like The big five and a sprinkling of others BALB/c mice, many 129 mice (and therefore, mice derived from Among the numerous mouse strains available to the scientific 129 stem cells) do not have a corpus callosum. Aside from their community, few are used for genomics research. The three most propensity to develop teratocarcinomas, there is only limited in- commonly used mice for biomedical research in general are formation on the background pathology (phenotype) of this C57BL/6 (B6), BALB/c and C3H mice, plus FVB and 129 mice for strain (11, 12). A novel syndrome that is seen in mice derived genomics research. Other strains or substrains of mice are also from 129 ES cell chimeras is midline teratomas (13). used in genomics, such as DBA/2, C57BL/10, CBA, but generally, FVB mice have gained favor as a way to circumvent the need as parental partners for creating hybrids for transgenesis and for using hybrid mice in creating transgenic mice. Micro-injec- chemical mutagenesis, or for other specific purposes. Examina- tion of DNA is facilitated by large pronuclei and robust zygotes tion of the family tree of laboratory mice indicates that Swiss to survive the trauma. This was initially performed in hybrid mice are probably the closest relatives to pure M. domesticus, mice, which favored these characteristics. FVB mice were rap- whereas the other inbred strains are generally Mus genetic hy- idly adopted for this purpose as an inbred mouse with the large brids. This point is made because of the common misconception pronuclei and the robust characteristics of Swiss mice (14). FVB that outbred Swiss mice somehow represent wild-type versions of mice originated from general purpose outbred NIH Swiss mice inbred mice. Furthermore, their outbred nature is only relative. that were inbred for pertussis research. A subline was discov- None of these strains was developed specifically for modern sci- ered to carry the Fv1b allele, which rendered them of interest to ence, and indeed, they were developed for totally unrelated pur- retrovirus (RV) research. “FVB” designation refers to Fv (Friend poses. The C57BL strain was developed by Little as a low-tumor, virus) B tropism. Swiss mice, including FVB mice, are blind due long-lived strain (in contrast to C58BL high-leukemia mice, which to retinal degeneration and, like B6 mice, are prone to late-on- were developed by MacDowell from the same progenitor sire). set amyloidosis. As an inadvertent outcome of this selection and inbreeding Furthermore, FVB mice are prone to seizures and neuro- process, B6 mice have, as ancillary strain characteristics, a ten- behavioral abnormalities (15). They also are prone to develop- dency for hydrocephalus, ocular malformations, progressive hear- ment of prolactin-secreting pituitary adenomas and hyperplasia, ing loss due to degeneration of the organ of Corti, and late-onset with failure of mammary involution following lactation (16). No- amyloidosis, among other traits. A large number of hearing and tably, much of the research on mammary neoplasia involves use vestibular defects are common among inbred mouse strains, of FVB transgenic mice. which are genetically determined by approximately 60 mutant loci (5). The BALB/c strain was originally selected by Bagg for Embryonic stem cells are mice too behavioral studies. Their male aggression characteristic is re- ES cell lines contribute even more complexity to genetic nowned. In contrast to B6 mice, BALB/c mice rarely if ever de- variation. It appears that a number of laboratories, through 207 Vol 52, No 3 Comparative Medicine June 2002 well-intentioned efforts to make the less-than-optimal breeding being mutated, truncated, defective, extensively methylated, al- performance or teratocarcinoma incidence in 129 mice better, tered by genetic integrations or deletions, or repressed by other have purposely added outside genetic material to their lines. genes. Retroviruses, although representing genetic acquisitions Others have inadvertently contaminated 129 mice with spuri- that are millions of years old, are promiscuous and continue to ous genetic material (mice have a way of getting around). A sur- actively recombine and transpose themselves within the ge- vey of random loci among several 129 mouse lines revealed at nome. This contributes substantially to ongoing genetic subline least one polymorphism among 43% of randomly selected loci divergence (23). tested. Unlike other inbred mouse sublines, which are suffi- More than 85% of the murine genome can be mapped with ciently related so as to accept reciprocal skin grafts, various retroviral integrations, using only three nonecotropic proviral lines of 129 mice reject skin transplants among one another (17, oligonucleotide probes (24). Various retroviral elements prob- 18). This is far from subline divergence, and underscores the ably were acquired by infection with exogenous RVs in the an- mongrelism of this “strain.” The remarkable genetic diversity of cient evolutionary history of the Mus genus; it is notable that 129 mouse lines has recently resulted in new nomenclature spe- xenotropic and polytropic proviruses are most widely dispersed cific to the 129 mouse (19), but scientists are often unaware that among progenitor wild mice (therefore, more ancient), whereas there is equal genetic diversity among their ES cell lines that ecotropic proviruses appear to be more recent (relatively speak- were derived from these various types of 129 mice. ing) acquisitions among mice of Asian origin (M. castaneus and For these reasons, it is important that the parental genotype M. molossinus) (25). Thus, the genetic contributions of Euro- of the 129 ES cell line be isogenic with the mouse genomic DNA pean and Asian Mus species to various inbred strains of mice library that is used, since important differences in recombina- can be traced through these retroviral contributions. tion efficiency (up to 25-fold) have been noted because of such Endogenous RVs (proviruses) and RV sequences are inher- genetic mismatching (20-22). Furthermore, the 129 lines that ited as Mendelian traits and are given gene designations (i.e., gave rise to 129 ES lines may now be extinct or, at the very Akv-1, Mtv-2, Fv-4). Because of their retained infectivity for least, have genetically changed due to subline divergence or ge- mouse cells, the historically more recent ecotropic endogenous netic contamination. Backcrossing 129 ES cell germline mice retroviruses receive the most attention. The content and inte- with a transgene onto 129 mice is one way of creating geneti- gration sites of endogenous ecotropic retroviruses were charac- cally homogeneous mice (termed quasi-co-isogenic), but this terized for 54 inbred strains of mice, were found to be located in must be done in isogenic mice to preclude inadvertent introduc- at least 29 distinct integration sites, and most were related to tion of genetic variables. the AKR ecotropic virus (26). The AKR-type ecotropic proviruses have homologues among AKR, C57, C58, C3H, BALB mice Old mice and retroviruses (among others examined) and Japanese mice (M. molossinus), Retroviruses and RV-like elements are scattered in literally emphasizing the contribution of Asian mouse genes to the ge- thousands of copies throughout the genome of the laboratory netic makeup of laboratory mice (27). mouse. They represent an evolutionary history of the mouse Mixing mouse strains literally reshuffles the cards for not only and contribute substantially to strain characteristics of all labo- unexpected genetic effects, but also retroviral effects. Cross- ratory mice. Thus, “muromics” requires an appreciation of, and breeding among inbred strains allows enhanced opportunity for respect for these factors. The mouse genome encodes exogenous expression and integration of proviruses (23). For example, 75% and endogenous murine leukemia viruses (MuLVs), mouse of SWR/J-RF/J hybrid progeny were documented to carry novel mammary tumor viruses (MMTVs), and more ancient replica- germline ecotropic proviral integrations (28). Retroviruses also tion-incompetent MuLVs, MMTVs, and RV-like elements, in- contribute to subline divergence. Among AKR mice, ecotropic cluding intracisternal A particles (IAP), virus-like 30S (VL30) proviral acquisitions, as well as losses, with every 50 to 100 gen- RNA sequences, murine retrovirus-related (MuRRS) DNA se- erations of inbreeding has been reported, and germline move- quences, (tRNA glutathione-like (GLN) sequences, murine re- ment of other RV-like elements, although less frequent, also is peated virus sequences on the Y chromosome (MuRVY), and continuous (23). early transposon (ETn) elements. Retrovirus-like elements are often swept aside as “junk DNA,” but they are transposable Retroviruses have phenotypes too (retrotransposons) and still active within the genome. Exog- Many of the basic characteristics of inbred strains can be at- enous (conventionally transmissible) MuLVs and MMTVs have tributed to RVs. The propensity of C3H mice to develop mam- been eliminated by rederivation and husbandry practices, but mary neoplasia early in life was found to be associated with continue to be purposely maintained as experimental tools in exogenous MMTV (Bittner agent), whereas late-onset mam- the form of Friend, Moloney, and Rauscher MuLVs (among oth- mary tumors in this strain are associated with endogenous ers) and MMTV-S (the Bittner agent). Genomics research also MMTV. The high-leukemia AKR and C58 strains have high lev- takes advantage of RVs as a means of assisting with transgenic els of ecotropic viral expression, whereas the leukemia-resistant retroviral vectors or tissue-specific promotion, using the long C57BL mouse expresses only low levels of ecotropic virus. terminal repeat (LTR) regions of MuLV and MMTV. Expression of ecotropic virus is insufficient for neoplasia. For Replication-competent endogenous RVs can be genetically and example, a number of mouse strains have and express AKR-type biologically clustered into ecotropic (capable of infecting mouse provirus genes without consequence, but AKR mice consistently cells), xenotropic (capable of infecting cells of other species, but develop thymic lymphoma as a predictable strain characteristic not mouse), and polytropic (capable of infecting cells of a variety (29). It took decades to decipher, but AKR thymic lymphomas de- of species). Most other retroviral sequences, particularly xenotro- velop due to the complex sequential expression and fortuitous pic proviruses and RV-like elements, are replication incompetent, recombination (for science, not the mouse) of endogenous 208 “Muromics”

ecotropic and xenotropic RVs. Susceptibility to transformation by with age, associated with high levels of endogenous ecotropic oncogenic RVs was found to be genetically determined by an ar- and polytropic MuLV expression and melanocyte dysfunction in ray of resistance or blocking factors that include other endog- affected areas of skin resulting from pre- or early postnatal enous provirus gene expression. One such factor is FV-4, a retroviral integrations (42). When B6 Nf1 null mutant mice defective endogenous RV envelope gene (Fv-4) that is expressed (which do not manifest leukemia) are backcrossed onto BXH-2 on the cell surface and blocks entry of ecotropic RV. Another re- background mice (which carry an expressed ecotropic RV), then sistance factor, Fv-1, regulates intracellular RV integration and are allowed to age, some of the N3 Nf1 heterozygous mice de- replication. Two alleles of the Fv-1 gene are Fv-1b and Fv-ln, velop myelogenous leukemia. Tumors derived from these mice which determine B (BALB) or N (NIH Swiss) cell tropism of some have somatic retroviral integration into the Epi1 locus, which RVs (30-32). Thus, although a particular strain of mouse may results in truncation of the functional Nf1 allele, and dysfunc- express a B or N ecotropic virus, infection and integration re- tion of both alleles and phenotype (43). quire the compatible Fv-1 and Fv-4 determinants. These factors co-evolved in wild mice as checks and balances, but when strains Genetic approaches can create genetic of mice were selectively bred for tumor phenotype in the labora- variables tory, the checks and balances were perturbed. Although scientists often refer to their particular mouse The leukemia/mammary tumor virus designation is mislead- strain as B6, C3H, BALB, or other, the importance of subline ing, as MMTVs can be associated with leukemia, and exogenous divergence and substrains is increasingly overlooked. Breeding MuLVs can be transmitted through milk (33). The MMTVs in- schemes to minimize subline divergence are often not used in fect and are expressed by lymphoid cells, which serve as a maintaining colonies of mutant mice, and the small population mechanism for trafficking to and infecting mammary gland. size of mutant mouse colonies makes them particularly vulner- The T-cell thymic lymphomas that appear in GR mice contain able to this effect. There can be substantial difference among MMTV proviral integrations (34). SJL mice develop germinal sublines of the same strain resulting from mutations, RV inte- center B-cell lymphoid tumors that express an endogenous grations, RV excisions, residual heterozygosity, and genetic con- MMTV provirus (Mtv-29) superantigen (vSAg), which stimu- tamination. An obvious example is C3H/HeJ versus C3H/HeN lates T cells to produce cytokines that stimulate B-cell tumor mice and C57BL/10ScCr versus C57BL/10Sc mice. More than growth (35). 30 years ago, C3H/HeJ and C57BL/10ScCr mice acquired spon- Retroviruses are generally known for their oncogenic proper- taneous mutations that resulted in unresponsiveness to bacte- ties and their contribution to the strain-related characteristics of rial lipopolysaccharide. Both substrains are now known to have tumor-prone mouse strains. However, they also determine a wide mutations in the toll-like receptor 4 (Tlr4) gene (44). Many array of other characteristics in mice. Xenotropic provirus-re- other subline differences have not been defined, but have appre- lated envelope glycoproteins (gp70s) are polymorphic products of ciable effects on experimental variables. Different sublines of a multi-gene family, some of which contribute to tissue differen- inbred FVB mice vary in their prevalence of pituitary hyperpla- tiation, with expression on various cells and tissues during the sia and adenomas, with differences in postlactational mammary stages of development (36). The NZB mice develop autoimmune gland involution (45). disease (a model for systemic lupus erythematosis), character- Although outbred Swiss mice are often considered to be “wild ized by high titer of autoantibody to endogenous retroviral gp70 type’ in terms of their genetic heterozygosity, they are far from proteins that are associated with immune complex glomerulone- outbred. Considering the fact that all Swiss mice were derived phritis (37). Non-obese diabetic (NOD) mice develop immune- from a handful of progenitors brought from Switzerland by mediated destruction of pancreatic islets, with a correlation Lynch, and different stocks of Swiss mice were subsequently between the presence of RV expression in pancreatic b cells and derived from finite subpopulations, there is considerable genetic development of insulitis and diabetes mellitus (38). homozygosity as well as subline divergence among various Swiss stocks. As with inbred strains, different lines of Swiss Retroviruses are Mother Nature’s mice have their own unique patterns of stock-specific diseases. transgenes Outbred CD-1, CF-1, and NIH Swiss stocks are different from Retroviruses can act as endogenous transgenes that integrate one another and should really be considered “not quite” inbred. into various regions of the genome, resulting in null mutations As a result, their value to biomedical research is limited in that and other genetic alterations. These effects contribute to strain they are not what they are “cracked up to be” (outbred), plus characteristics, subline divergence, and phenotypically new mu- they don’t offer the advantage of being genetically defined. If tants. The dilute, brown non-agouti phenotype of DBA mice is a hybrid vigor is sought, it is better to use F1 hybrid mice to result of random insertion of an ecotropic RV into the Myo5a achieve maximal, but predictable heterozygosity. locus, resulting in a pigment defect in melanocytes. This is a natural gene knockout whose phenotype stems back to Asian Blame it all on the parents ancestry of fancy mice (39, 40). Another is the hairless mouse, Nearly all commonly used ES cell lines are “male,” and thus, with a retroviral insertion in a locus pigment that was discov- XY genotype. Male ES cells produce a higher proportion of male ered in a natural mutant mouse from a London aviary in the chimeras, and thus, male chimeras can be bred more rapidly to 1930s (41). test for germline transmission. When XY ES cells are inserted Retroviral integrations into the genome are not only retained in into female recipient embryos, fertile male progeny often are the germline of mice, but can also take place postnatally in somatic produced. Thus, there is considerable sex bias in genomics re- cells, with phenotypic effects. Various strains of C57BL/10 H-2 search. Although female ES cell lines are just as easily created, congenic mice were found to have different patterns of graying their XX status is reputedly unstable (resulting in XO sterile 209 Vol 52, No 3 Comparative Medicine June 2002 genotype), and they are less efficient in selecting for germline ground strain genetics. Null mutations of the p53 gene result in transmission. Sex conversion of male embryos by XX ES cells increased tumor prevalence, but the array of tumor types that does not occur as readily as the converse, and may result in in- develop vary vastly with background strain genetics (49, 50). fertile hermaphrodites or gynandromorphs (46). The TGF-1 null mice also have marked variation among differ- Aside from the effects of the X chromosome, a seldom consid- ent parental genotypes, with embryo lethality due to three de- ered issue is the effect of the maternal genome. In the field of velopmental phenomena in different strains of mice (51). mouse biology and genomics, it is common practice to transfer Although phenotype pattern may be similar among different embryos of one genotype into the uteri of foster dams of another genetic strains, penetrance of a gene alteration can vary with genotype, as well as to create multi-genotypic chimeras through background strain. The TGF-b1 null mutants develop progres- blastocyst injection or morula aggregation. In the mouse, mater- sively severe multisystemic inflammatory lesions during early nal cells readily populate fetal bone marrow during mid-gesta- life, but with marked background strain variation in severity tion; thus, pups derived from such approaches are fetal-maternal and distribution of the inflammatory lesions (52, 53). In general, chimeras to variable degrees. Although these maternal cells do the identity or function of these modifier genes has not been not contribute to the germ line, it is not known whether such defined, but they can vary from immune response genes, as is blood chimerisms pass beyond the first generation through ma- the case of TGF-b1 null mutants (54), to strain-specific differ- ternal lineages. Maternally derived chimeric cells can poten- ences in transgene methylation (55). tially express endogenous RVs that could find a receptive home The effect of background strain genetics is particularly well il- in their new recipient environment. In addition, mitochondria lustrated among 129 strain mice. Serotonin-1B receptor null (with their own genome and background strain characteristics) mutant mice were initially created with 129/SvPas ES cells, bred are maternal in origin, and must be a consideration in breeding into129/Sv-ter mice, and later crossed with 129/SvTac mice. The schemes. In other words, AxB F1 mice are not genetically the original mutants consumed twice as much alcohol as did their same as BxA F1 mice (47). Furthermore, when mice are back- wild-type counterparts. Several years later, these original find- crossed onto a particular background strain, mutant males are ings could not be reproduced by the same laboratory due to generally selected for breeding to females. Unless there is at changes in the genetic background contributed by different 129 least backcross from mutant female to male, the Y chromosome mouse lines (56). Anterior commissure closure defects, with ab- of the background strain will not be represented. sence of a corpus callosum, are common (up to 70%) in BALB/c Parental gene imprinting is a major epigenetic factor that is and 129 mice (including 129/J, 129/Sv, and 129/Ola) (57-59). As seldom considered in mouse biology. The male and female paren- a result, 129 mice are poor test subjects for learning behavior tal genomes have appreciable effects on imprinting the fetal ge- (60). Forebrain commissure (corpus callosum) defects, which nome. Thus, AxB mice are likely to express parental (B) male have been attributed to alterations in specific genes, are thus imprinting effects, whereas BxA mice will be influenced by pa- substantially influenced by the 129 phenotype (61). This under- rental (A) male imprinting effects (and the converse for female scores the need for complete backcrossing away from 129 mice for parental genomes). These effects are minimized by use of geneti- such studies. The combined complications for neurobehavioral re- cally homozygous inbred mice, but can be substantial in hybrids search related to 129 genetic diversity, 129 strain characteristics, and crosses of different sublines within a strain type. Thus, the issues relating to backcrossing, and misinterpretation of pheno- commonly held impression that F1 mice are genetically identical type have been reviewed (62). is true, but they may vary in phenotype, due to parental imprint- Even in fully backcrossed mutants that have achieved congenic ing, depending on whether they are AxB or BxA crosses. status, functional modifier genes can flank the target site. The region surrounding a transgenic insertion generally contains about Background genetics and genomics ± 10 centimorgans of flanking DNA. This can occur with over Expression of a particular gene as a phenotype is substantially 1,000 backcross generations, influencing gene expression and influenced by modifier and compensatory genes within the strain phenotype. Thus, lines of mice with the same, precisely targeted background genome. Gene alterations result in expression of a mutation may differ genetically and phenotypically, even if they phenotype that is the coordinated expression, through modifying are congenic and fully backcrossed. Congenic mice are not co- or compensatory genes, of the other 99.9967% of the 30,000 to isogenic (62). 35,000 genes that make up the mouse genome. Insulin-depen- Reliance on databases to provide strain-related background dent diabetes mellitus in the mouse, for example, is the result of pathology is not an appropriate control for genomic studies. Use 17 or more interacting genes on 13 chromosomes (47). of parental strains, F1 hybrids, N2 backcrosses, or F2 inter- There are many examples of the concept that specific gene crosses as controls also is a common, but inappropriate practice. alterations have different phenotypes, depending on back- Another common approach is to partially backcross mice, stop ground genetics of the mouse. A commonly cited example is epi- at N4 or so, then start intercrossing the partially backcrossed thelial growth factor receptor (EGFR) knockout mice. The line. This essentially creates unique recombinant inbred (or in- EGFR null mice on CF-1 (outbred Swiss) background die in completely inbred) strains, and the contributions of the parental utero, with degeneration of the inner cell mass, during the peri- lines are essentially undefined. This poses problems when at- implantation period. In contrast, mice with the EGFR null muta- tempting to relate gene function across a common currency of tion on a 129/Sv background die during mid-gestation due to background genetics among laboratories, or performing experi- placental defects, and null mutants on CD-1 background (out- ments with genetically identical controls. Thus, literature bred Swiss) live to weaning, but die due to multiple organ abnor- abounds with descriptions of phenotypes that represent ge- malities (48). Although they are outbred, the marked differences nomic mutants with background strain genetics that are still between CF-1 and CD-1 mice emphasize the importance of back- segregating or are undefined. Ideally, mutants should be main- 210 “Muromics”

tained on a fully inbred strain background as heterozygotes, and is genetically distinct from its littermate because transgenes and phenotype should be evaluated in homozygous, heterozy- are randomly inserted into each genome, often as tandem re- gous, and wild-type littermates. This is seldom done due to cost. peats and sometimes in multiple locations. Thus, transgenes can not only result in a phenotype due to expression of the transgene, Convergence of background strain but also due to disruption of one or more other genes from ran- genetics, genomic approaches, and dom insertion of the transgene. In targeted mutations, each gene expression founder pup is also genetically distinct, but for other reasons. With the contemporary focus on the genome, the importance Expression (transcription) of any transgene (random or ho- of “homogeneity of genetic constitution” advocated by Little can mologous integration) is controlled by genetic and epigenetic be lost in the genomics shuffle. Genetic engineering is accom- processes. The site of integration can determine the level and plished by use of various methods, each relying on different ge- tissue of transgene expression due largely to effects of the sur- netic strains of mice, and various laboratories use different rounding chromatin. Transgene expression is not usually uni- sublines of some strains. There are a number of reviews and form. Furthermore, if transgene integration takes place after texts on the subject of genomic techniques (63, 64), so the cur- the two-cell stage, transgenic mosaicism can result. Even when rent discussion emphasizes the experimental variables that can introduced at the optimal zygote stage, transgene expression arise from these different approaches. tends to vary at the cellular or tissue level, which in turn, results Although the term “transgenic” technically applies to any in variation at the whole-animal level. Transgene expression and mouse created by insertion of foreign genetic material, either by silencing depend on the components of the transgene (promoters, homologous (targeted) or random integration, the term tends to tissue specificity), the number of transgene copies (high tandem apply to random integration. Many transgenic mice are created copy numbers have a tendency to be low expressors), and the in- from hybrids of various types, as well as outbred and inbred tegration site of the transgene (68, 69). Swiss mice. Hybrid and outbred mice created in this way have Although concern is often focused on under-expression of limited value as research models because of their genetic het- transgenes, over-expression can also result in unexpected effects. erozygosity. Thus, backcrossing such mice onto a standard ge- For example, transgenic mice that over-express MHC class-II netic background may be required, but technical proficiency can genes in pancreatic cells develop non-autoimmune diabetes mel- allow micro-injection in inbred mouse zygotes. Alternatively, in- litus due to islet cell dysfunction (70). Fortunately, the degree of bred Swiss FVB mice have been heavily used, as they offer the gene silencing, variegation, or expression tends to be stable robust advantages of Swiss mice, but they are inbred (14). within a transgenic mouse line (71). Unfortunately, expression of In contrast, targeted mutations (knockouts and knockins) are the transgene can be influenced by age of the mouse (72), and generally developed by homologous recombination of genes in- transgene expression in random mutants, as well as gene-tar- serted into 129 ES cell lines, then selected ES cells are injected geted mutant mice, has been documented to be substantially in- into the inner cell mass of blastocysts of a recipient mouse em- fluenced by genetic background (68, 73, 74). bryo. If the 129 line goes “germline,” then the altered gene is se- Transgene expression is typically promoted by a variety of el- lected while mice are backcrossed onto B6 or some other inbred ements within the construct. These include promoter elements strain of mice. The B6 embryos are highly compatible hosts for of SV40 virus, herpes simplex virus, human cytomegalovirus 129 ES cells, thereby facilitating chimerism and achieving (HCMV), Rous sarcoma virus, and the LTR promoter elements germline transmission. As a result, B6 mice are becoming the of MMTV. Most of these viral enhancer/promoter elements have “gold standard” background genotype for most targeted muta- been used to drive expression of a variety of genes, but they may tions. The ES cell lines vary considerably in their potential con- not be ubiquitous in their activity. Those of HCMV and MMTV, tribution to chimerism (46). Although they are more difficult to for example, result in organ- and cell-type specificity that corre- develop, there is a growing tendency for creation of ES cell lines, lates with biological behavior of the virus. There are also a num- including, but not limited to B6 and BALB/c (65, 66). Albino B6 ber of mammalian endogenous “housekeeping” gene promoters, mice or albino B6 ES cell lines offer solutions to visually select- including translation elongation factor-1a promoter, ubiquitin C ing likely germline chimeras by coat color. These are desirable promoter and others. These promoters tend to be less powerful directions for the future, in that they preclude the necessity, in exerting transcription, compared with viral-based promoters cost, and ambiguity of backcrossing 129 mice onto B6 mice. (75). These systems, as mentioned previously, are not always Complete backcrossing requires 10 generations to make a line ubiquitous or tissue specific, and rarely has true tissue distribu- congenic at the locus of interest. Phenotypes can literally come tion been defined; this is likely to vary with genetic background. and go as backcrossing proceeds and congenic status is achieved. Promoters can influence the transgene of interest, and result- The influence of each background genome, with a random mix of ing phenotype in unexpected ways. The MTV LTR promoter tar- gene segregation still ongoing, renders the reproducibility of the gets mammary tissue. Mammary tumors under the influence of phenotype in partially backcrossed mice questionable. “Speed the same MTV LTR promoter, but associated with ras, myc, or congenics,” in which genetic markers (micro-satellites or single neu transgenes have predictable, but different phenotypes (76). nucleotide polymorphisms) of the desired background genotype Although the MTV LTR promoter targets mammary glands, it are selected along with the mutated gene, can accelerate the also targets salivary and lacrimal glands. As with mammary process of backcrossing to nearly half the time, but this process tumors, the phenotype of the salivary gland tumor under the tends to underestimate achievement of congenic status by up to influence of the same MTV LTR promoter varies with the two generations (67). transgene, with different tumor morphologies arising from ras, Regardless of background strain, every pup in a founder litter ras plus myc, wnt-1, wnt-3, fgf-8, neu, or SV40 Tag. Further- created by random transgenesis is hemizygous for the transgene, more, the salivary gland (submandibular, parotid, or sublingual) 211 Vol 52, No 3 Comparative Medicine June 2002 specificity also varies with these different transgenes under the transgenes. In addition, highly precise point mutations can be influence of the identical MTV LTR promoter (77). introduced into genes and functional domains thereof, with the Although the mammary and salivary gland specificity of the creation of null, truncated, or minimally mutated genes. The MTV LTR promoter is well known, unexpected outcomes, such growing sophistication of tissue-specific promoters and induc- as Harderian gland tumors, can arise in strains of mice that are ible expression systems lends enormous future potential to prone to develop this neoplasm. More precise tissue-specific pro- genomics research (84). moters are being used, including neuron-specific enolase pro- These binary systems, although elegant in terms of genomic moter and the cardiac specific a-myosin heavy chain promoter, manipulation, are not without genetic complications. The Cre-LoxP among others (75, 78). The specificity of such promoters may not mice, for example, have specific genes that are targeted and be that well-defined, resulting in unexpected phenotypes. flanked by homologous recombination in 129 ES cells, then Unexpected outcomes can result from various other components placed in recipient (usually B6) blastocysts to create chimeras, of the transgene construct. Two examples, involving the b-globin selected for germline transmission, then backcrossed onto B6 gene illustrate this concept. The MuLV LTRs allow efficient inte- mice. These mice (often only partially backcrossed) are then gration of the construct, but their presence can severely inhibit crossed with Cre recombinase transgenic mice (therefore, FVB, transgene expression (79). Targeted deletion of the 5’ DNase hy- hybrids of various inbred strains, and sometimes 129 mice back- persensitive site 2 of the b-globin locus control region resulted in crossed onto B6 mice) to create the Cre-LoxP mouse. The Cre-LoxP profound (and expected) phenotypic effects on all of the genes in mice can be a genetic mix of 129 mice (with their checkered ge- the locus, but when the PGKneo selection cassette was removed netic history), backcrossed (often incompletely) onto B6 mice, from the construct, globin gene expression was (unexpectedly) then crossed with Cre transgenic FVB or hybrid mice. Thus, normal (80). This example underscores misinterpretation of phe- there is the potential for four (or more) background genomes to notype when only the target gene is considered. influence the gene alteration. The coat color variation among Inducible gene expression is offering many advantages over some Cre-LoxP mice is a sight to behold, and illustrates the rich traditional transgenic approaches, and can overcome difficulties diversity of their background genetics. with embryonic lethal effects. Expression of transgenes can be The expression of Cre, and thus the efficiency of recombina- regulated conditionally in mice with inducible promoters, such as tion of the target gene, can vary considerably, thereby substan- the glucocorticoid-responsive MMTV LTR, the interferon-induc- tially affecting phenotype. The Cre recombination is improved ible Mx1 promoter, and heavy metal inducible metallothionein by including mammalian Kozak consensus sequences, but pro- promoters. These systems can have toxic effects or influence re- moters vary considerably in their transcriptional activity, and productive performance and organogenesis, expression levels of such activity can vary considerably in different tissues. As with the desired gene may be variable, and in some instances, random transgenic approaches, LoxP plasmids can insert them- nonrepressible transgene expression can occur in the absence of selves randomly in multiple copies and in random orientations induction. Most of these systems also lack tissue specificity (78, throughout the genome, with unintentional consequences, in- 81, 82). Tetracycline-induced gene expression systems are bi- cluding loss of chromosomes during Cre excision (85). functional, and have been modified to act either as a “Tet-on” or Mutagenesis, chemical and radiation induced, is now being “Tet-off” switch (75, 78). These systems can have variable out- used in large-scale efforts to acquire variant phenotypes, using comes, in that if tetracycline or doxycycline are given orally, ef- high through-put screening assays. In contrast to the aforemen- fect depends on the less than optimal intestinal absorption of tioned forward genomics, or gene-driven, approaches, mutagen- these drugs by the mouse (83), the differential organ distribu- esis relies on reverse genomics, or phenotype-driven approaches tion of these drugs, including the blood-brain barrier, and mouse (86). Thus, discovery of novel phenotypes relies on the accuracy strain differences in drug metabolism. and thoroughness of observation. There are a number of ap- These approaches are being creatively combined to increase proaches, depending on whether the goal is detecting dominant precise control of the temporal and spatial, on- and off-functions or recessive mutations, or mutations within specific loci. Unlike of genes. These “binary” approaches require two (or more) genetic transgenic insertions, mutagenesis tends to produce precise manipulations that are integrated independently in separate mutations, with the advantage of creating co-isogenic mutant transgenic mouse lines, then combined by cross-breeding the lines. Background strain becomes a critical determinant in ex- lines (78). Site-specific recombinases, including phage-derived pression and interpretation of phenotype. The commonly used Cre recombinase and the yeast-derived Frt recombinase, allow chemical mutagen N-ethyl-N-nitrosourea (ENU) is toxic, and development of conditional knockouts that lack a gene in a par- there are marked genetic strain differences in tolerance to the ticular tissue or at a specific stage of development. The Cre and drug. Thus, F1 hybrid mice are often studied. Considering the Frt site-specific recombinases target LoxP or flp sites, respec- importance of background strain in discovery of phenotype and tively. These target sites can be inserted by homologous recombi- the desire to have precise co-isogenic lines, use of inbred mice is nation to flank a target gene or segments of the genome of desirable. BTBR/N inbred mice are particularly resistant to the interest. The system can be manipulated to translocate, delete, or toxic effects of ENU, but other strains that have been used in- invert large segments of DNA, and manipulate the genome in a clude A/J, C57BL/6J, BALB/cJ, and several C3H substrains. highly precise and timed manner. Thus, a gene functions nor- Furthermore, screens for recessive phenotypes often involve use mally until recombination (with resulting intervening gene dele- of crosses with other mouse strains, including FVB (87). Thus, tion) occurs. like forward-genomic approaches that lack a genetic standard, Variations on this approach can also be used to induce pro- reverse genomic approaches may have genetic uniformity moters to activate silent transgenes that would otherwise be within a laboratory or program, but are acquiring genetic diver- deleterious during earlier stages of development, or inactivate sity within the overall scientific community. 212 “Muromics”

High through-put phenotyping yields far more mutants than Are mouse phenotypes valid models of can be fully characterized, and these approaches require large human disease? populations of mice. Thus, demand for gamete cryopreservation It may seem obvious, but despite the homology of the mouse is growing (88). Spermatozoa cryopreservation, if optimized, genome to that of humans, there are differences. Thus, valida- will allow efficient archiving of mutant mice, thus minimizing tion of phenotype as a model for human disease may be impor- the number of live mice that must be maintained. It also offers tant if the model is to be used as such. Although breast cancer an efficient means of transport, receipt, and rederivation of biology was, and continues to be a major impetus for creating mice, thereby controlling infectious disease risk. However, at- and studying inbred strains of mice, naturally acquired mouse tention must be paid to issues relating to matching recipient mammary tumors do not morphologically or biologically re- strains and sublines. As international traffic increases in this semble human breast cancer. However, transgenesis allows cre- regard, there must be consistency in a common currency of ge- ation of models that more faithfully mimic the human disease, netic background. This is true for receipt and breeding of live and that respond to chemotherapeutic drugs in a similar way mice as well. (76, 94, 95). More often, the phenotype of a particular gene alteration is the expression of the mouse genome in a way that the Mutations of the same gene in the same mouse genome is prone to express (strain characteristics). This strain may not result in the same is especially true for null mutations, in which a functional for- phenotype eign transgene has not been introduced. This does not necessar- With the increasing sophistication of genomics technology, as ily negate the validity of the mouse model. For example, well as the growing number of chemically mutagenized strains, GM-CSF knockout mice, created in 129 ES cells backcrossed collections of mice with different mutations of the same gene or onto B6 mice, are a well recognized model for human progres- locus are expanding. This is an exciting new dimension for sive pulmonary proteinosis (96). A similar disease is seen B6 genomics research, but adds complexity as profound differences background NADPH oxidase-deficient p47phox (97) and gp91 in phenotype can be attributed to a single gene, depending on phox knockout (98) mice, CYP1A2 knockout mice (12), and IL- nature of the construct, nature of the mutation, and the genetic 13 knockout mice (99). The disease induced by these mutations background. The Myo5a mutation of DBA mice, which arose is equivalent to the B6 “motheaten” mutation (100). These naturally from an RV integration event into the noncoding re- seemingly disparate factors merge with the reality of this lesion gion of the Myo5a locus, results in a benign change in coat color. being a late-onset, age-related lesion in the lungs and other or- However, more substantial mutations, unrelated to RV integra- gans of B6 (the background strain common to all) and 129 mice tions, have occurred repeatedly in the same locus, resulting in (the ES cell strain common to the knockout mice) (12, 98, 101). lethal or severe neurologic phenotypes, but all with the dilute Genetic manipulation, either by gene targeting or mutation, ac- coat color (89, 90). An example arising from genomic manipula- celerates the onset and severity of this strain-specific disease. Is tion is targeted mutation of the p53 gene. The p53 null mutants it a valid phenotype? Perhaps, but the phenotype would not have well known susceptibility to tumor formation (49, 50). happen, or not happen as robustly, if it were not for the strain However, p53 mutations with a truncated, but functional p53 characteristics of the B6 or 129 mouse. gene have enhanced tumor resistance (rather than susceptibility) and age prematurely, with development of osteoporosis, di- Infectious disease: Case of the minished stress tolerance, and generalized organ atrophy (91). disappearing phenotype The converse also can happen. Mice with a point mutation in One of the first erroneous phenotypes in a mutant mouse asso- the cardiac a-myosin heavy chain gene develop severe cardi- ciated with infectious disease was the “nude” mutation. The omyopathy, whereas mice with the complete null mutation do “pleiotropic” effects of the nude gene were, in retrospect, the re- not (92). More commonly, single nucleotide differences, muta- sult of chronic mouse hepatitis virus (MHV) infection in athymic tions in noncoding regulatory regions, or partial null mutations mice with T-cell deficiency at a time when T cells were not well lead to different phenotypes due to partial disruption of gene understood (102). It is ironic that MHV, which was initially de- function. These “hypomorphs” thus add complexity to the phe- scribed in 1949 (103), continues as one of the most common infec- notype and phenotypic characterization of mice. The opportu- tive agents in mouse populations today. This author has worked nity for modifying and compensatory gene activity and the with MHV for decades, and heretofore thought that he had seen effects of parental genotype increase. So does the necessity of every possible ramification of MHV infection, until the recent Little’s concept of “homogeneity of genetic constitution.” advent of genetically engineered mice. Mice with various severe Finally, phenotypes resulting from specific gene alterations immune deficiencies, such as severe combined immunodeficient must now be viewed in terms of the temporal kinetics of their (SCID), nude, and Rag1 knockout, develop predictable patterns transcription or lack thereof. New genetic variables are intro- of MHV infection. However, more subtle gene alterations in spe- duced by the ontogeny of gene expression within various stages cific interleukin genes have resulted in totally new manifesta- of development. For example, tetracycline induction of a mutations of this old disease. tionally activated b-isoform of protein kinase C in the develop- Interferon-g knockout mice, created independently in Japan, ing neonate is lethal, whereas activation of the same gene in Australia, and the United States, develop a unique granuloma- adult mice results in only modest ventricular hypertrophy (93). tous polyserositis in association with natural MHV infection The phenotype of mice resulting from a null mutation, in which (104, 105). Laboratory mice are subject to more than 60 infec- mice develop with selection for strong compensatory pathways, tive pathogens, including viruses, bacteria, protozoa, fungi, hel- compared with mice that have gene transcription or the gene minths, and arthropods (16). They have been introduced into product itself ablated as adults, can be profoundly different. laboratory mouse populations, and into the genome in the case of 213 Vol 52, No 3 Comparative Medicine June 2002

RVs, from the diverse origins of mice that contributed to the cre- the risk of human exposure is accentuated (114). ation of the laboratory mouse. Most of these agents were originally The profound differences in clinical presentation of naturally natural pathogens of wild mice, with tendency toward commensal- acquired infectious diseases in mice has lead to considerable ism, and therefore, are benign in outbred and enzootically infected misunderstanding about their importance. Even more impor- populations. Appreciable differences exist in the clinical disease tantly, infective agents that would otherwise be dismissed as profiles between enzootic and epizootic infections with the same unimportant or otherwise overlooked in other outbred species of agent. This is especially true for viruses. For example, highly con- animal are highly relevant in the context of mouse biology. The tagious enterotropic MHV, when initially introduced to a naïve intrinsic value of the mouse as a research tool is based on con- breeding population of mice results in 100% mortality among in- trol of genetic and microbial variables. When intercurrent infec- fants, whereas once the virus becomes enzootic within the colony, tious diseases, even subclinical infections, are introduced as the only evidence of infection is seroconversion (106). This is due to untoward variables, there can be a broad array of substantial protection of mice during infancy by maternally derived immunity. consequences, including immunomodulation, modified tumor Infant mice are immunodeficient at birth, with evolution of innate kinetics, numerous effects on cell and tissue function/response, and acquired immune competence over the course of several and altered patterns of expression of strain-specific pathology. weeks. The rate of maturation varies with the various components Many of the effects on mouse-based research that are known of the immune response, and depends on strain of mouse (16). have been reviewed (115, 116). Unfortunately, these effects are These differences in biologic behavior of infective agents leads to unpredictable and not all known, as they depend on the experi- misunderstanding of their nature and importance. mental variable, the assay, the agent, the agent strain (viru- Selective inbreeding of mouse strains for different characteris- lence, tropism), mouse genetics, mouse age, mouse immune tics has resulted in unanticipated consequences in immune re- competence, epizootiology, and (now) genomic manipulation. sponsiveness, such as natural killer cell deficiency in SJL mice Infective agents can interact with one another and, when (107) or C5 complement deficiency in strain A, AKR, DBA/2, coupled with the appropriate mouse genetic background or gene SWR, and other strains of mice (108). In other instances, more alteration, result in remarkably different outcomes. Pneumocystis overt immune deficiency syndromes have arisen as spontaneous carinii has minimal if any effect on immunocompetent mice, but mutations, such as athymic nude and SCID mice. As knowledge can be a substantial cause of pulmonary pathology in severely im- of immunology increases, there are growing numbers of geneti- munodeficient mice. The growing number of marginally immuno- cally engineered immune mutants. In addition, unexpected im- deficient strains of mice may also develop atypical mild forms of munologic perturbations can result from genomic manipulation. P. carinii-induced pulmonary disease. When such mice are co-in- This is not surprising, considering the many pathways and ele- fected with Pasteurella pneumotropica, a normally innocuous or ments of the innate and acquired immune response. Such effects at best opportunistic bacterium, mice can develop florid bacterial can result from transgene insertions, or from unexplained effects. bronchopneumonia (117). The AKR and C58 mice express high In addition to these factors, the mouse contributes substan- titer of endogenous ecotropic RV, which infects neurons during tially to the complexity of infectious disease. The concept of ge- embryogenesis. Effects are not seen from such events unless netic susceptibility to infectious disease, which can be expressed these strains of mice later become infected with lactate dehydro- at the cellular (intrinsic resistance) or at the organism (extrinsic genase-elevating virus (LDEV), which will attack the retrovirus- resistance) level was established by Bang (109), using inbred infected motor neurons, resulting in paralytic disease. In the strains of mice and MHV. The near catastrophic loss of inbred absence of these genetic and retroviral components, LDEV virus DBA mice at Cold Spring Harbor due to a “paratyphoid” out- causes subclinical infections (118). break was a classic example of the consequences of uniform ge- In phenotypes with autoimmune features, such as TGF-b1 netic susceptibility of inbred mice to infectious disease. DBA null mutants, the influence of adventitious infective agents mice also suffer high mortality during outbreaks of Sendai virus, must be considered. For example, the effects of P. carinii on the and susceptibility has been mapped to several genetic loci (110, pulmonary inflammatory lesions in these mice has not been 111). The classic studies of Fenner on genetic susceptibility of thoroughly investigated. In rats, infection with coronaviruses mice to ectromelia virus (112) ring true during natural outbreaks has been documented to stimulate graft-versus-host reactions of mousepox, when animal rooms with mixed populations of vari- in allogeneic bone marrow chimeras that were otherwise dis- ous inbred strains experience the full spectrum of subclinical to ease free prior to infection. Localized graft-versus-host lesions lethal infections that are faithful to genetic background (113). developed subsequent to infection and clearance of virus in lac- The numerous and diverse infective agents indigenous to, rimal and salivary glands (119). Differences in distribution and coupled with the refined genetic nature of the mouse, have offered severity of autoimmune lesions among different laboratories themselves as useful research model systems. These include, but and strains of mice with null mutations of TGF-b1 could easily are not limited to lymphocytic choriomeningitis virus, MHV, be skewed by infective factors, such as P. carinii, and under- polyoma virus, Salmonella, Citrobacter, and Cryptosporidium spp., scores the importance of interpretation of data between labora- and others. In addition, the mouse is susceptible to a number of tories with different conditions (52, 53). other infective agents of non-murine origin. Nearly all such models With chemical mutagenesis programs, a number of high have a genetic basis of susceptibility. An unfortunate outcome of through-put screens involve assessment of litter size, size of using these mouse pathogens as experimental models has been pups, and physical condition of pups. Infective agents, particularly iatrogenic re-introductions of mouse pathogens into naïve viruses, can influence these parameters when first introduced to a mouse populations (16). When zoonotic viruses, such as lympho- mouse population. Neurobehavioral phenotyping screens can be cytic choriomeningitis virus, are iatrogenically introduced to, hampered by sporadic infectious disease syndromes that are not then transmitted among immunodeficient mouse populations, likely to be verified as genetic, but certainly will waste time and 214 “Muromics”

effort. Mice are prone to bacterial otitis media and interna, with in searches for immunologic mutants (130, 131), underscoring vestibular signs, including head tilt, circling, and ataxia. Out- the importance of infectious disease control in such colonies. breaks of bacterial vestibular syndrome can follow outbreaks of otherwise subclinical respiratory virus infections. Mice of many Domino effect of infectious disease on strains are susceptible to sporadic segmental vasculitis that may phenotype result in vestibular signs. Direct infection of the central nervous Infectious diseases can indirectly influence strain character- system, with highly varied clinical presentations, can be associated istics and phenotype. Perhaps the best example is renal glom- with MHV, mouse encephalomyelitis virus, RV/LDEV, and others erular disease, which is common among inbred strains of mice, (16). All of these syndromes can be influenced by genetic (strain), but for different reasons. The B6 and Swiss (including FVB) as well as genomic variables. mice, for example, are prone to late-onset glomerular amyloido- Helicobacter infections are a good example of the disappearing sis, and BALB/c and 129 mice, which tend not to develop amyloi- phenotype phenomenon. Several immunologic null mutants were dosis, are prone to development of glomerulonephritis (11, 16). initially reported to develop inflammatory lesions of the small Both syndromes are exacerbated by chronic inflammatory dis- intestine. These included null mutants of IL-2 (120), IL-10 (121), ease, particularly bacterial infections, which are enhanced by T-cell receptor (Tcr)-a, Tcr-b, Tcr-b/d, and M.C. class II (122). The immunodeficiency, and this can be an unanticipated effect of ge- infective cause (Helicobacter sp.) of these syndromes was not nomic alteration. Aryl-hydrocarbon (Ahr) knockout mice (B6 known at the time. These mutants, as well as other mouse mu- background) have defective DNA repair. They develop associated tants, are often presented as models of human “inflammatory immunodeficiency (an unanticipated, but logical consequence), bowel disease” (IBD) that are purportedly due to immune with chronic skin and gastrointestinal and genitourinary tract dysregulation, and often without mention of Helicobacter sp. infections. They also manifest an unexpected phenotype of cardi- (123). It is now well established that Helicobacter spp. are associ- omegaly and congestive heart failure (132). Cardiac disease ated with proliferative bowel lesions in immune deficient as well could easily be construed as a phenotype of the gene alteration, as some strains of immunocompetent mice (124), and although particularly when it does not develop in age- and sex-matched immune dysregulation can exacerbate disease, disease also de- congenic controls. However, the immunodeficiency leads to op- velops in globally immunodeficient mice (so much for the autoim- portunistic bacterial infections, which in turn can accelerate the mune theory). Rederivation (elimination of Helicobacter spp.) onset of multi-systemic amyloidosis. Amyloidosis typically de- results in elimination of the phenotype (125). Furthermore, velops as a late-onset disease in B6 mice, but chronic infections Helicobacter colitis is not a valid model of human IBD, which is a in the null mutation can accelerate its evolution. Renal glom- term reserved by human pathologists for ulcerative colitis and erular amyloidosis can predispose these mice to coagulopathy, Crohn’s disease, neither of which resemble Helicobacter colitis in which can lead to atrial (auricular) thrombosis, cardiomegaly, mice. It is, however, an interesting disease that is worth studying and heart failure (16). Cardiomegaly and heart failure have also as a model of intestinal inflammation. been described in FVB mice expressing green fluorescence protein In some instances, the enzootic Helicobacter infection of an ex- in the heart. A figure illustrating the enlarged heart depicted what perimental mouse colony is featured as an essential component appeared to be atrial thrombosis. Because the “phenotype” was ob- of the phenotype. Combined glutathione peroxidase Gpx1/GPx2 viously in the heart, lungs, and liver, other tissues (including kid- knockout mice have accentuated early-onset colitis. This is pos- ney) were not examined (133). Similar to B6 mice, FVB mice are sible because mice in the colony were purposely maintained with prone to amyloidosis. Although BALB/c mice do not develop renal H. hepaticus, H. typhlonius, and H. rodentium as part of their amyloidosis, they are also prone to atrial thrombosis due to their microflora (126). Despite awareness of most scientists about the propensity for developing glomerulonephritis. The driving forces role of Helicobacter spp. in colitis, there is generally little aware- for these immune-related disorders (amyloidosis and glomerulone- ness of other infective agents known to influence mucosal hyper- phritis) can be subtle. Ectoparasitism by fur mites is common but plasia in mice, including Citrobacter rodentium, Clostridium often unrecognized among laboratory mice, largely because these spp., Escherichia coli, and MHV (16). Mucosal proliferation, in- organisms are innocuous. However, some strains of mice, including duced by any number of these agents alone, in combination, or B6 mice, are especially prone to hypersensitivity to Myobia mus- in concert with genetic or experimental manipulation of the host culi, which, in combination with their grooming behavior, makes can influence experimental outcome. Mice infected with C. them prone to ulcerative dermatitis, accelerating the aforemen- rodentium, for example, have enhanced sensitivity to chemical tioned amyloid-renal disease-coagulopathy syndrome (16). carcinogenesis (16). These examples are reasonably easy to explain and under- Immunomodulation is a common effect of naturally acquired stand, but others are less so. In the previously discussed study murine infections. Sendai virus is known among immunologists involving H-2 congenic C57BL/10 lines, premature grayness for its immunomodulating effects (127). MHV and parvoviruses was found to be associated with expression of RV in some of the are common and often cause subclinical infections, yet they have congenic lines. The reproducible phenotypes that appeared in appreciable effects on immune responsiveness (128, 129). The ef- specific congenic lines disappeared on rederivation of the mice fects on immune response can be varied, resulting from direct to a pathogen-free barrier. The reversion of phenotype was lymphocytotropism (e.g., viruses such as MHV, minute virus of found to be due to failure of the pathogen-free mice to express mice [MVM], and mouse parvovirus [MPV]), indirect effects on the ecotropic MuLV until later in life (134). immune response (e.g., viruses such as Sendai virus), macrophage infection (e.g, LDEV or MHV), B-cell mitogenic effects (e.g., Re-emerging and emerging infectious Mycoplasma pulmonis), or the effects of T-cell superantigens diseases (e.g., MMTV) (16). The ENU mutagenesis programs are engaged In the past decade, numbers of genetically altered mice have 215 Vol 52, No 3 Comparative Medicine June 2002 burgeoned, and are being distributed directly among investiga- found to be infected with Helicobacter sp. as a result of foster- tors and among institutions, much like what occurred in the nursing on infected dams provided by a commercial vendor. early part of the past century. As such unregulated traffic increases, opportunities for infectious disease re-emergence and The other “-omic”: Economics spread are staggering. The infectious disease status of the Having made a case for the importance of the myriad of infec- nation’s mouse colonies is changing progressively. Diseases are tious variables in mouse biology, another factor is the increasing re-appearing that have not been seen in many decades. A report cost of infectious disease surveillance, diagnosis, and control. of Demodex musculi infestation in a specific-pathogen-free Prior to the molecular revolution and the rapid increase in ge- transgenic mouse colony in 1999 (135) exemplifies this, since netically engineered mice, most research mice were produced by this parasite had not been detected in laboratory mice since a small number of commercial sources. For a brief period, there 1917. Animal care programs are faced with the dilemma of was consensus between producers of mice and research institu- lengthening, rather than shortening, the lists of agents for diag- tions, so that there was growing uniformity in infectious disease nostic surveillance, and such testing must be increased and ever quality control. more vigilant due to more frequent importations, illicit importa- The mouse research world is in an upward spiral of increasing tions, traffic, crowding, and dense housing conditions. cost. As awareness increases over the need for infectious disease Not only are infective agents re-emerging in mouse populations, control in animal facilities, mouse housing, such as micro-isolator but new agents are still being discovered. Genomics research has caging and individually ventilated caging, becomes more sophis- contributed to this phenomenon. In recent years, the discovery of ticated (and expensive) and infectious disease surveillance be- novel pathogens has been the result of development of more sensi- comes more difficult, requiring more testing (and expense) to tive serodiagnostic assays, such as that for MPV (129), or increas- ensure infectious disease quality control. Samples obtained from ing and parallel awareness of similar infective agents in other immunodeficient strains of mice or mice with autoimmune disor- species, such as Helicobacter spp. (136, 137). Mouse parvovirus was ders, such as MRL-lpr or NZB mice, may give spurious results on found to have more appreciable immune perturbations on research serologic assays, and the degree of seroconversion can vary with than does the other well known parvovirus of mice (MVM), and mouse strain. The most common strain of mouse, B6, may not awareness of the “first” Helicobacter species, H. hepaticus, led to readily seroconvert to viruses like MPV because of their genetic subsequent discovery of H. bilis, H. rodentium, H. muridarum, resistance, and sentinel mice may not acquire MPV infection H. typhlonius, and possibly others yet to be named (138). The re- readily from resistant B6 mice (146). These issues require extra cent discovery of Hanta-like viruses in laboratory rats and mice effort and cost to ensure infectious disease control, not to men- (139) is another example of this “emergence” phenomenon. Sub- tion the cost of controlling and eliminating actual infectious dis- clinical Leptospira infection is widespread in wild mouse popula- eases through quarantine, depopulation, and re-derivation. tions, yet testing for this zoonotic agent isn’t performed in These economic pressures are exacerbated when institutions, laboratory mouse programs. which face many financial demands, do not consider the health The line between relevant and irrelevant pathogens contin- and welfare of laboratory animals and the quality of the science ues to move as genomics research discovers unique infectious derived from those animals as institutional responsibilities. syndromes in genetically altered mice. Pulmonary abscesses Thus, scientists are expected to carry the burden of animal care due to infection with Paecilomyces variotii were reported in costs, and naturally seek to reduce them. Diagnostic and infectious NADPH oxidase-deficient gp91phox null mice (98). This oppor- disease surveillance programs are the first to be cut or attenuated tunistic human pathogen, which was commonly isolated from at a time when there has never been a greater need (147). the respiratory tract of rodents, was predicted to become a patho- Furthermore, it is difficult to decide which agents to include on gen of laboratory animal relevance (140). Another new infectious the list of “specific” pathogens when defining a population as syndrome in immunodeficient mice is granulomatous inflamma- SPF, and which agents qualify for quarantine or destruction of tion of the tail associated with Mycobacterium chelonae, an op- mouse colonies when infections occur. Commercial vendors are portunistic human pathogen (141). faced with difficult decisions over how exclusive their lists of spe- Biological products derived from mice, including transplant- cific pathogens must be, and how open they should be about the able tumors, tumor cell lines, cell lines, tissues, and serum, have presence of marginally pathogenic agents in their mice. It is a long been known as a source of infection in mouse populations, major financial decision to break down a production area due to and as a source of agents, such as lymphocytic choriomeningitis introduction of an infective agent, and this decision becomes dif- virus (142), an important zoonotic risk. Caution should extend ficult when the agent is considered marginally relevant. On the to mouse ES cells. The cells are of mouse origin, involve use of basis of tradition more than fact, some vendors might be more mouse feeder cells, and the medium contains mouse products apt to eliminate nearly innocuous agents, such as reovirus, before (143). Mouse ES cells are susceptible to MHV infection, with more relevant agents, such as Helicobacter sp. minimal cytopathic effects (144). Unrestricted world trade of In some instances, agents that are not pathogenic, even in mouse serum, and unrestricted introduction of such biological globally immunodeficient mice, are included (by tradition) on material into laboratory mice at the recipient institutions re- lists for surveillance and diagnosis. Examples include Entam- cently resulted in multiple outbreaks of mouse pox throughout eba muris and Tritrichomonas species. Klebsiella species are of- the United States (145). The growing complexity of infectious ten included on screening lists and SPF profiles; the entire disease between vendor and institution can result in interesting genus has been condemned because of a report of endometritis outcomes. This author recalls a colony of pathogen-free mice due to opportunistic infection with Klebsiella oxytoca among which became infected with ectromelia virus. After considerable aged mice with cystic endometrial hyperplasia (148). Surveil- time, effort, and cost, the successfully re-derived mice were lance for normal microflora and microfauna that have never been 216 “Muromics”

documented to cause disease under any circumstances contrib- of stereotypy, without environmental enrichment, also is stress- ute to unnecessary costs, and occasionally result in unnecessary ful (154, 157). Isolated mice will adjust, but not without appre- rederivation or interference with research by institutional offi- ciable changes in behavioral testing paradigms (158). In contrast, cials. Vendors may be forced to include such agents on their spe- crowding of mice in a cage also can be stressful, with proven ef- cific pathogen lists due to demand by naïve clients. If a vendor fects on immune responsiveness, adrenocortical activity, and sus- reported high prevalence of Lactobacillus sp. from the intestine ceptibility to infectious disease (159, 160). of mice, some users would not choose that source of mice. Behavioral phenotyping of individual mice, therefore, can be substantially affected by these social issues. Strain-related pat- Mice pay a high price too terns of behavior and dominance vary at the cage and indi- The need for pathogen-free mice has created a demand for spe- vidual levels. Thus, although identically sized groups of mice cialized caging, including micro-isolator cages, individually venti- may consist of the same inbred strain, microbial status, sex, and lated cages, ventilated racks, change stations, and isolators. age, each mouse from each cage has established itself within its Public Health Service and AAALAC standards are becoming in- deme’s social order, which in turn appreciably affects behavior creasingly rigorous, but many of the recommended guidelines and reproductive physiology of the individual. Even the intrau- (which essentially become rules) are anthropocentric, not scien- terine position of the fetus has life-long effects on physiology, be- tifically based, and are not designed for the species. With species havior, and social stature (161). In a recent multi-site study and subspecies genetic variations on the theme, M. musculus is a involving three geographically separate laboratories, apparatus, nocturnal, burrowing, highly social, pheromone-driven animal test protocols, and many environmental variables were rigor- that builds complex nests with shared maternal responsibilities ously equated, and several inbred strains and one null mutant (149). The dietary intake of the mouse consists of one third feces, were tested simultaneously. Significant site-related differences which is nutritionally required for vitamin acquisition. Although were found in nearly all variables tested. The study indicated frequent cage changes may be mandated by human standards, that strong strain-related behavioral characteristics were ro- such practices are quite stressful to mice. The chaos created bust and detectable, but more subtle behavioral phenotypes, within a mouse room after cage changing is profound. Mice are such as those likely to be sought in mutagenesis and genomic generally observed during the day, when they appear content manipulations, were variably detected (162). and asleep. If viewed during their nocturnal activity periods, a Aside from behavioral phenotypes, strain-specific behavioral different picture emerges, with substantial stereotypic behav- patterns can affect other phenotypes. The hair plucking and ioral anomalies. barbering that occurs among maladjusted B6 mice gives rise to Human standards for mouse husbandry and disease control chronic dermatitis, which can lead to opportunistic bacterial (sta- can be contrary to what mice prefer. In two controlled studies, phylococcal) ulcerative dermatitis. These chronic lesions acceler- one with B6 and another with BALB/c mice, with measurement ate the onset and prevalence of amyloidosis, to which this strain of a number of parameters including mortality, adrenal gland of mouse is prone, thereby limiting lifespan from amyloid-related weight, plasma glucocorticoid concentration, and selected im- protein-losing enteropathy, glomerulopathy, or heart failure mune measures, mice actually benefitted from less space than through coagulopathy (secondary to renal disease). Male and fe- National Research Council recommended standards (150, 151). male mice have a number of sexual dimorphisms. One such di- Given a choice, mice vacate individually ventilated cages, with a morphism is relative granulocytosis among adult males, which preference for more stagnant environments. If forced to live in is influenced by social interactions (fighting). This can become individually ventilated cages, mice will build baffles out of bed- an important factor when interpreting the phenotype of mice, ding material to block air flow (152). Many of the guidelines for such as mice with targeted disruption of the CD18 gene. The mouse husbandry standards are generic, and are based on spe- CD18 disruption is hypomorphic, and results in granulocytosis cies other than the mouse, particularly rats (153). Thus, these (163), but this phenotype is not apparent in young mice with guidelines, which essentially have become law of the land, may this mutation (author’s personal experience). not be in the best interests of laboratory mice. Background genetics and neurobehavioral No rewards for bad behavior phenotypes: Deaf, dumb, or blind The basic social unit of the mouse is the deme, with pro- Inbred strains of mice have clear phenotypic differences in nounced social dominance tiering among males and females. various behavioral testing modalities. Similar to other issues Dominance-associated barbering, trichotillomania, and pugi- discussed, background strain is a major consideration when per- lism, plus stereotypic behavior, such as bar chewing, rolling, forming behavioral research (164). Behavioral research is par- flipping, and head tilting, are common manifestations of malad- ticularly prone to environmental influences, with known effects justed mice. Barbering and trichotillomania (the Delila effect) of prenatal, postnatal, preweaning, nutritional, husbandry, and are dominance-associated vices with strong genetic predisposi- physical environment. The effects on behavioral phenotypes in- tion, such as that in B6 and A2G mice. Learning and environ- duced by the stress of shipping mice, compared with “home rear- ment also contribute to these syndromes, and appear to be a ing,” can be not only relevant, but also permanent (164). form of coping with inappropriate housing and social interac- It is daunting to see large-scale, chemical mutagenesis and tions (154, 155). Stereotypies, such as head tilt or circling, can high through-put phenotyping efforts being directed at some types persist when individual mice are assessed. Separation and iso- of mice. What’s your choice for high through-put neurobehavioral lation of pugilistically inclined or dominant barberers from the phenotyping…“deaf, dumb, or blind”? Incomplete penetrance of group destabilizes the social heirarchy, and isolation of the per- several of these strain characteristics, such as corpus callosal de- petrators is stressful to the individual mouse (156). Prevention fects, seizures, hydrocephalus, and microophalmia, complicate 217 Vol 52, No 3 Comparative Medicine June 2002 this effort. In neurobehavioral studies involving comparison be- fecundity and large litters tend to be most resistant to the ef- tween C3H and B6 mice, C3H mice were found to have appre- fects of estrogen (180). Standard commercial laboratory diet has ciable deficiencies in learning, on the basis of results of maze been documented to protect against MMTV-induced mammary assays (165). Such findings could easily be skewed by the fact and endometrial tumors in mice (181). that C3H mice (as well as Swiss mice, including FVB) are blind. Commercial diets can also lead to increased amounts of DNA Although B6 mice are not blind due to retinal degeneration, they adducts, with associated oxidative damage to DNA and poten- frequently manifest a number of other neurologic defects with in- tial impact on susceptibility to experimentally induced carcino- complete penetrance, including ocular defects (166, 167), hydro- genesis/mutagenesis (182). Other dietary factors that are cephalus, and progressive sensorineural hearing loss (168, 169). largely undefined in rodent diets include nitrosamines, heavy metals, aflatoxins, and insecticides, as well as xenobiotic agents Shedding light on environmental in bedding (171). Uncharacterized factors in various commercial variables mouse diets also have been found to significantly affect intesti- It is well established that husbandry factors, including infec- nal mucosal proliferative kinetics, with influence on bacteria- tious disease status, caging environment, bedding, and diet, induced colitis (183). Specific examples of these variables on substantially influence age- and strain-related patterns of dis- genetically engineered mice are not cited, but the likelihood of ease, as well as lifespan (170). In addition to behavioral and in- effects is clear. fectious disease variables, laboratory mice are subjected to a Special notice is made of drinking water. Aside from its intrin- number of other environmental variables that can potentially sic qualities and contaminants, it is common practice to acidify or affect phenotype. Environmental variables, including thus cag- chlorinate rodent drinking water. This practice was originally ing, diet, temperature, humidity, ventilation, light, noise, trans- implemented as a means of controlling Pseudomonas aeruginosa portation, chemicals in feed and bedding, air quality, water transmission via water bottle sipping tubes. Pseudomonas treatment, drugs, and other factors, can influence or complicate aeruginosa is a common (if not ubiquitous) environmental bacte- animal research (153, 171). rium that can be an opportunistic pathogen if mice are neutro- Photoperiod, light, intensity, and wave-length are well known penic, such as when irradiated or treated with cyclophosphamide factors that influence physiologic responses and pathology in (16). Although these procedures are seldom performed on mice or mice. Reproductive physiology and circadian rhythms are appre- only on specific groups of mice, the practice of water treatment ciably influenced by photoperiod (171). It is not surprising that is universal. This not only adds to the cost of animal care, but various strains and ages of mice, given the opportunity to choose, also has deleterious effects on mice that far exceed the adverse have clear yet different preferences for illumination intensity effects of P. aeruginosa. Aside from being unpalatable to mice, and photoperiod (172). Albino strains, such as BALB mice, do not hyperchlorinated water can have effects on immune response have genetically determined retinal degeneration, but can un- (184, 185), and acidified water can influence intestinal microflora dergo light-associated retinal degeneration, the severity of which (186). Acidified water has been associated with severe dental car- varies with proximity of the mice to the room light source (173). ies in mice (187). Addition of tetracycline to drinking water is a Cage shelf level, presumably due to light intensity, has also been growing practice in genomics research, but it is not without ad- reported to significantly influence prevalence and time of onset verse effects. Water treated with tetracycline can induce signifi- of several types of naturally and experimentally induced tumors cant effects on immune response in mice (185). in mice (174). The spectral quality of light has been found to have Genetically determined disease patterns vary substantially significant effects on body and organ weight in mice (175). in different environments. It is impossible to compare disease Diet is an uncontrolled and important variable in mouse-re- prevalence data between various studies of the same mouse lated research. Laboratory mice tend to over-eat in the absence of strain, emphasizing the requirement for simultaneously main- other environmental stimuli. In a study in which B6C3F1, tained controls for every study. An example is a study on spon- C57BL6, and B6D2F1 mice, compared with ad libitum-fed mice, taneous neoplasia, non-neoplastic lesions, and lifespan among were subjected to 40% food restriction, the food-restricted mice three groups of genetically similar (in this case outbred) CD-1 had significantly (by a third) lengthened lifespan. Longevity was mice that originated from different, geographically separate in part due to reduction in the rate of neoplasia, with near aboli- production facilities of the same vendor. The mice were main- tion of pituitary tumors and other endocrine tumors among all tained at a single location for the study (188). The investigators groups fed restricted diets (176). Glaucoma and other eye lesions concluded that origin of the mice was the critical variable (which also are attenuated in aging food-restricted DBA/2N mice (177). is possibly due to subline divergence), but failed to recognize Most mouse studies are performed with mice fed commercially that, although the groups of mice were maintained at a single lo- available open- or closed-formula diets composed of natural in- cation, they were maintained at different times over a four-year gredients that are rich in phytoestrogens. Even with the defined period. Variation in disease profiles also is seen among the same composition of open-formula diets, the amount of phytoestrogens genetic types of mice maintained in different facilities (189). can vary with time of harvest, geographic location, processing, and other factors (178). Lest this be under-emphasized, serum Don’t despair, it isn’t all that bad concentrations of isoflavones in mice fed some commonly used The preceding discussion should not be construed as an ex- commercial diets exceed the animal’s endogenous estrogen con- haustive review of mouse biology, but rather an illustration of centration by 30,000- to 60,000-fold, with appreciable maternal- important principles in mouse-related research. The central fetal transfer (179). It should be no surprise that susceptibility message is that mice are not merely reagents, but rather com- to endocrine disruption by estrogen and phytoestrogens is plex, multi-systemic organisms whose genome and phenome are strain-related. Strains of mice that have been selected for high influenced not only by genomic manipulation, but also by back- 218 “Muromics”

ground genetics and environment. The value of the mouse as a 13. Blackshear, P. J., Mahler, L. M. Bennet, K. A. McAllister, D. model system is the opportunity to control many genetic vari- Forsythe, and B. J. Davis. 1999. Extragonadal teratocarcinoma ables, and that opportunity should not be missed. This is par- in chimeric mice. Vet. Pathol. 36:457-460. 14. Taketo, M., A. C. Schroeder, L. E. Mobraaten, K. B. Gun- ticularly true when evaluating polygenic traits, performing ning, G. Hanten, R. R. Fox, T. H. Roderick, C. L. Stewart, F. quantitative trait analyses, and examining age-related pheno- Lilly, C. T. Hansen, and P. A. Overbeek. 1991. FVB/N: an in- types. Otherwise, genetically mixed mice pose insurmountable bred mouse strain preferable for transgenic analyses. Proc. Natl. problems for good genomics research. With that said, an illus- Acad. Sci. USA 88:2065-2069. 15. Goelz, M. F., J. Mahler, J. Harry, P. Meyers, J. Clark, J. E. tration of other variables should not be ignored, but can be Thigpen, and D. B. Forsythe. 1998. Neuropathologic findings minimized by appropriate husbandry practices and proper ex- associated with seizures in FVB mice. Lab. Anim. Sci. 48:34-37. perimental design. 16. Percy, D. H., and S. W. Barthold. 2000. Pathology of laboratory Good science requires simultaneous genetic-, age-, sex- and rodents and rabbits, 2nd ed. Iowa State University Press, Ames, environment-matched controls in the experimental design. Ide- Iowa. 17. Simpson, E. M., C. C. Linder, E. E. Sargent, M. T. Davisson, ally, studies should be performed in homozygous, heterozygous, L. E. Mobraaten, and J. J. Sharp. 1997. Genetic variation and wild-type littermates. Economic pressures should not su- among 129 substrains and its importance for targeted mutagen- percede good science. If these admonitions are heeded, the out- esis in mice. Nat. Genet. 16:19-27. come of genomic manipulation can be effectively deciphered. It 18. Threadgill, D. W., D. Yee, A. Matin, J. H. Nadeau, and T. Magnuson. 1997. Genealogy of the 129 inbred strains: 129/SvJ should also be emphasized that “it takes a village” to create, is a contaminated inbred strain. Mamm. Genome 8:390-393. maintain, characterize, understand, validate, and use mouse 19. Festing, M. F. W., E. M. Simpson, M. T. Davisson, and L. E. models for biomedical research. Interdisciplinary research has Mobraaten. 1999. Revised nomenclature for strain 129 mice. never been more vibrant and exciting. Mamm. Genome 10:836. “It all started with a mouse” (Walt Disney). 20. Deng, C., and M. R. Capecchi. 1992. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell Biol. 12:3365-3371. Acknowledgment 21. teRiele, H., E. R. Maandag, and A. Berns. 1992. Highly effi- Portions of this article were presented in the Wallace P. Rowe Lec- cient gene targeting in embryonic stem cells through homolo- ture at the 52nd AALAS National Meeting in Baltimore, Maryland gous recombination with isogenic DNA constructs. Proc. Natl. on October 23, 2001. Acad. Sci. USA 89:5128-5132. 22. vanDeursen, J., and R. Wieringa. 1992. Targeting of the cre- atine kinase M gene in embryonic stem cells using isogenic and nonisogenic vector. Nucleic Acids Res. 20:3815-3820. References 23. Coffin, J. M., S. H. Hughes, and H. E. Varmus. 1997. The in- 1. Morse, H .C. III. 1981. The laboratory mouse—a historical per- teractions of retroviruses and their hosts, p. 343-435. In J. M. spective, p. 1-16. In H. L. Foster, J. D. Small, and J. G. Fox (ed.), Coffin, S. H. Hughes, and H. E. Varmus (ed.), Retroviruses. Cold The mouse in biomedical research. Academic Press, New York. Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2. Beck, J. A., S. Lloyd, M. Hafezparast, M. Lennon-Pierce, J. 24. Frankel, W. N., J. P. Stoye, B. A. Taylor, and J. M. Coffin. T. Eppig, M. F. Festing, and E. M. Fisher. 2000. Geneologies 1990. A genetic linkage map of endogenous murine leukemia vi- of mouse inbred strains. Nat. Genet. 24:23-25. ruses. Genetics 124:221-236. 3. Rikke, B. A., Y. Zhao, L. P. Daggett, R. Reyes, and S. C. 25. Kozak, C. A., and R. R. O’Neill. 1987. Diverse wild mouse ori- Hardies. 1995. Mus spretus LINE-1 sequences detected in the gins of xenotropic, mink cell focus-forming, and two types of Mus musculus inbred strain C57BL/6J using LINE-1 DNA probes. ecotropic proviral genes. J. Virol. 61:3082-3088. Genetics 139:901-906. 26. Jenkins, N. A., N. G. Copeland, B. A. Taylor, and B. K. Lee. 4. Tucker, P. K., B. K. Lee, B. L. Lundrigan, and E. M. Eicher. 1982. Organization, distribution, and stability of endogenous 1992. Geographic origin of the Y chromosomes in “old” inbred ecotropic murine leukemia virus DNA sequences in chromosomes strains of mice. Mamm. Genome 3:254-261. of Mus musculus. J. Virol. 43:26-36. 5. Steel, K. P. 1995. Inherited hearing defects in mice. Ann. Rev. 27. Steffen, D. L., S. Bird, and R. A. Weinberg. 1980. Evidence for Genet. 29:675-701. the Asiatic origin of endogenous AKR type murine leukemia provi- 6. Willot, J. F., L. C. Erway, J. R. Archer, and D. E. Harrison. ruses. J. Virol. 35:824-835. 1995. Genetics of age-related hearing loss in mice. II. Strain dif- 28. Jenkins, N. A., and N. G. Copeland. 1985. High frequency ferences and effects of caloric restriction on cochlear pathology germline acquisition of ecotropic MuLV proviruses in SWR/J-RF/J and evoked response thresholds. Hear. Res. 88:143-155. hybrid mice. Cell 43:811-819. 7. Frith, C. H., and J. M. Ward. 1988. Color atlas of neoplastic 29. Thomas, C. Y., and J. M. Coffin. 1982. Genetic alterations of and non-neoplastic lesions in aging mice. Elsevier, New York. RNA leukemia viruses associated with the development of spon- 8. Maronpot, R. R., G. A. Boorman, and B. W. Gaul. 1999. Pa- taneous thymic leukemia in AKR/J mice. J. Virol. 43:416-426. thology of the mouse. Cache River Press, Vienna, Ill. 30. Lilly, F., and A. Mayer. 1980. Genetic aspects of murine type-C 9. Mohr, U., D. L. Dungworth, C. C. Capen, W. W. Carlton, J. P. viruses and their hosts in oncogenesis, p. 89-108. In G. Klein (ed.), Sundberg, and J. M. Ward. 1996. Pathobiology of the aging Viral oncology. Raven Press, New York. mouse, vol. 1 and 2. ILSI Press, Washington, D.C. 31. Ikeda, H., and T. Odaka. 1983. Cellular expression of murine 10. Stevens, L. C. 1983. Teratocarcinoma stem cells, p. 23-36. In L. leukemia virus gp70 related antigen on thymocytes of uninfected M. Silver, G. R. Martin, and S. Strickland (ed.), Cold Spring Har- mice correlates with FV-4 gene controlled resistance to Friend bor Conferences on cell proliferation. Cold Spring Harbor Labo- leukemia virus infection. Virology 128:127-139. ratory Press, Cold Spring Harbor, N.Y. 32. Gardner, M. B., C. A. Kozak, and S. J. O’Brien. 1991. The 11. Haines, D. C., S. Chattopadhyay, and J. M. Ward. 2001. Pa- lake Casitas wild mouse: genetic resistance to retroviral disease. thology of aging B6;129 mice. Toxicol. Pathol. 29:653-661. Trends Genet. 7:22-27. 12. Ward, J. M., M. Yoon, M. R. Anver, D. C. Haines, G. Kudo, F. 33. Law, L. W., and J. B. Moloney. 1961. Studies of congenital trans- J. Gonzalez, and S. Kimura. 2001. Hyalinosis and Ym1/Ym2 mission of a leukemia virus in mice. Proc. Soc. Exp. Biol. Med. gene expression in the stomach and respiratory tract of 129S4Jae 108:715-723. and wild-type and CYP1A2-null B6,129 mice. Am. J. Pathol. 158:323-332. 219 Vol 52, No 3 Comparative Medicine June 2002

34. Michalides, R., E. Wagenaar, J. Hilkens, J. Hilgers, B. 52. Boivin, G. P., B. A. O’Toole, I. E. Ormsby, R. J. Diebold, M. J. Groner, and N. E. Hynes. 1982. Acquisition of proviral DNA of Eis, T. Doetschman, and A. B. Kier. 1995. Onset and progres- mouse mammary tumor virus in thymic leukemia cells from GR sion of pathological lesions in transforming growth factor-beta mice. J. Virol. 43:819-829. 1-deficient mice. Am. J. Pathol. 146:276-288. 35. Zhang, D. J., V. K. Tsiagbe, C. Huang, and G. J. Thorbecke. 53. Shull, M. M., I. Ormsby, A. B. Kier, S. Pawlowski, R. J. 1996. Control of endogenous mouse mammary tumor virus Diebold, M. Yin, R. Allen, C. Sidman, G. Proetzel, D. Calvin, superantigen expression in SJL lymphomas by a promoter within N. Annuniziata, and T. Doetschman. 1992. Targeted disrup- the env region. J. Immunol. 157:3510-3517. tion of the mouse transforming growth factor-beta 1 gene results 36. Elder, J. H., F. C. Jensen, M. L. Bryant, and R. A. Lerner. in multifocal inflammatory disease. Nat. Genet. 359:693-699. 1977. Polymorphism of the major envelope glycoprotein (gp70) of 54. Diebold, R. J., M. J. Eis, M. Yin, I. Ormsby, G. P. Boivin, B. J. murine C-type viruses: virion associated and differentiation an- Darrow, J. E. Saffitz, and T. Doetschman. 1995. Early-onset tigens encoded by a multigene family. Nature 267:23-28. multifocal inflammation in the transforming growth factor beta 37. Izui, S., P. J. McConahey, A. N. Theofilopoulous, and F. J. 1-null mouse is lymphocyte mediated. Proc. Natl. Acad. Sci. USA Dixon. 1979 Association of circulating retroviral gp70- and anti- 92:12215-12219. gp70 immune complexes with murine systemic lupus erythematosis. 55. Weng, A., T. Magnuson, and U. Storb. 1995. Strain-specific J. Exp. Med. 149:1099-1116. transgene methylation occurs early in mouse development and 38. Gaskins, H. R., M. Prochazka, K. Hamaguchi, D. V. Serreze, can be recapitulated in embryonic stem cells. Development and E. H. Leiter. 1992. Beta cell expression of endogenous xe- 121:2853-2859. notropic retrovirus distinguishes diabetes-susceptible NOD/Lt 56. Phillips, T. J., R. Hen, and J. C. Crabbe. 1999. Complications from resistant NON/Lt mice. J. Clin. Invest. 90:2220-2227. associated with background effects in research using knockout 39. Jenkins, N. A., N. G. Copeland, B. A. Taylor, and B. K. Lee. mice. Psychopharmacology 147:5-7. 1981. Dilute (d) coat colour mutation of DBA/2J mice is associ- 57. Wahlsten, D. 1974. Heritable aspects of anomalous myelinated ated with the site of integration of an ecotropic MuLV genome. fibre tracts in the forebrain of the laboratory mouse. Brain Res. Nature 293:70-374. 68:1-18. 40. Copeland, N. G., K. W. Hutchison, and N. A. Jenkins. 1983. 58. Wahlsten, D., J. C. Crabbe, and B. C. Dudek. 2001. Behavioural Excision of the DBA ecotropic provirus in dilute coat-color rever- testing of standard inbred and 5HT(1B) knockout mice: implica- tants of mice occurs by homologous recombination involving the tions of absent corpus callosum. Behav. Brain Res. 125:23-32. viral LTRs. Cell 33:379-387. 59. Livy, D. V., and D. Wahlsten. 1997. Retarded formation of the 41. Stoye, J. P., S. Fenner, G. E. Greenoak, C. Moran, and J. M. hippocampal commisure in embryos from mouse strains lacking Coffin. 1988. Role of endogenous retroviruses as mutagens: the a corpus callosum. Hippocampus 7:2-14. hairless mutation of mice. Cell 54:383-391. 60. Lipp, H.-P., M. Stagliar-Bozizevic, and D. P. Wolfer. 1995. 42. Morse, H. C. III, R. A. Yetter, J. H. Stimpling, O. M. Pitts, T. N. Analysis of behavior in large numbers of knockout mice: results, Fredrickson, and J. W. Hartley. 1985. Greying with age in mice: caveats, and perspectives. Behav. Genet. 25:275. relation to expression of murine leukemia virus. Cell 41:439-448. 61. Magara, F., U. Muller, Z. W. Li, H. P. Lipp, C. Weisman, M. 43. Blaydes, S. M., S. C. Kogan, B.-T. Truong, D. J. Gilbert, N. A. Staglar, and D. P. Wolfer. 1999. Genetic background changes Jenkins, N. G. Copeland, D. A. Largaespada, and C. I. the pattern of forebrain commissure defects in transgenic mice Brannan. 2001. Retroviral integration at the Epi1 locus cooper- underexpressing the beta-amyloid-precursor protein. Proc. Natl. ates with Nf1 gene loss in the progression to acute myeloid leu- Acad. Sci. USA 96:4656-4661. kemia. J. Virol. 75:9427-9434. 62. Gerlai, R. 1996. Gene-targeting studies of mammalian behav- 44. Poltorak, A., Z. X. He, I. Smirnova, M. Y. Lieu, C. V. Haffel, ior: is it the mutation or the background genotype? Trends X. Du, D. Birdwell, E. Alejos, M. Silva, C. Galanos, M. Neurosci. 19:177-181. Freudenberg, P. Ricciardi-Castagnoli, B. Layton, and B. 63. Tymms, M. J., and I. Kola (ed.). 2001. Gene knockout proto- Beutler. 1998. Defective LPS signaling in C3H/HeJ and cols, vol. 158. Humana Press, Totowa, N.J. C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085- 64. Jackson, I. J., and C. M. Abbot. 2000. Mouse genetics and 2088. transgenics: a practical approach. In B. D. Hames (ed.), The prac- 45. Cardiff, R. D. 2002. Personal communication. tical approach series. Oxford University Press, Oxford. 46. Papaioannou, V., and R. Johnson. 1993. Production of chi- 65. Ledermann, B., and K. Burki. 1991. Establishment of a germ- meras and genetically defined offspring from targeted ES cells, line competent C57BL/6 embryonic stem cell line. Exp. Cell Res. p. 107-146. In A. L. Joyner (ed.), Gene targeting: a practical ap- 197:254-258. proach. IRL Press at Oxford University Press, Oxford. 66. Noben-Trauth, N., G. Kohler, K. Burki, and B. Ledermann. 47. Brownstein, D. G. 1998. Genetically engineered mice: the holes 1996. Efficient targeting of the IL4 gene in a BALB/c embryonic in the sum of the parts. Lab. Anim. Sci. 48:121-122. stem cell line. Transgenic Res. 5:487-491. 48. Threadgill, D. W., A. A. Dlugosz, L. A. Hanson, T. Tennenbaum, 67. Wakeland, E., L. Morel, K. Achey, M. Yui, and J. Longmate. U. Lichti, D. Yee, C. LaMantia, T. Mourton, K. Herrup, R. C. 1997. Speed congenics: a classic technique in the fast lane (rela- Harris, J. A. Barnard, S. H. Yuspa, R. J. Coffey, and T. tively speaking). Immunol. Today 18:472-477. Magnuson. 1995. Targeted disruption of mouse EGF receptor: ef- 68. Martin, D. I. K., and E. Whitelaw. 1996. The vagaries of varie- fect of genetic background on mutant phenotype. Science 269:230- gating transgenes. Bio Essays 18:919-923. 234. 69. Dobie, K., M. Mehtali, and R. L. M McClenaghan. 1997. Var- 49. Donehower, L. A., M. Harvey, H. Vogel, M. J. McArthur, C. iegated gene expression in mice. Trends Genet. 13:56-59. A. Montgomery, Jr., S. H. Park, T. Thompson, R. J. Ford, 70. Lo, D., L. C. Burkly, G. Widera, C. Cowing, R. A. Flavell, R. and A. Bradley. 1995. Effects of genetic background on tumori- D. Palmiter, and R. L. Brinster. 1988. Diabetes and tolerance genesis in p53-deficient mice. Mol. Carcinog. 14:16-22. in transgenic mice expressing class II M.C. molecules in pancre- 50. Harvey, M., M. J. McArthur, C. A. Montgomery, Jr., A. Bra- atic beta cells. Cell 53:159-168. dley, and L. A. Donehower. 1993. Genetic background alters 71. Robertson, G., D. Garrick, W. Wu, M. Kearns, D. Martin, the spectrum of tumors that develop in p53-deficient mice. FASEB and E. Whitelaw. 1995. Position-dependent variegation of globin J. 7:938-943. transgene expression in mice. Proc. Natl. Acad. Sci. USA 92:5371- 51. Bonyadi, M., S. A. Rusholme, F. M. Cousins, H. C. Su, C. A. 5375. Biron, M. Farrall, and R. J. Akhurst. 1997. Mapping of a ma- 72. Robertson, G., D. Garrick, M. Wilson, D. I. Martin and E. jor genetic modifier of embryonic lethality in TGF beta 1 knock- Whitelaw. 1996. Age-dependent silencing of globin transgenes out mice. Nat. Genet. 15:207-211. in the mouse. Nucleic Acids Res. 24:1465-1471. 73. Allen, N. D., M. L. Norris, and M. A. Surani. 1990. Epigenetic control of transgene expression and imprinting by genotype-specific modifiers. Cell 61:853-861.

220 “Muromics”

74. Erickson, R. P. 1996. Mouse models of human genetic disease: 96. Zsengeller, Z. K., J. A. Reed, C. J. Bachurski, A. M. Levine, which mouse is more like a man? Bio Essays 18:993-997. S. Forry-Schaudies, R. Hirsch, and J. A. Whitsett. 1998. Ad- 75. Ristevski, S. 2001. Transgenic studies in the mouse: improving enovirus-mediated granulocyte-macrophage colony-stimulating the technology towards a conditional temporal and spatial ap- factor improves lung pathology of pulmonary alveolar proteinosis proach, p. 319-334. In M. J. Tymms and I. Kola (ed.), Methods in in granulocyte-macrophage colony-stimulating factor-deficient molecular biology: gene knockout protocols. Humana Press, mice. Hum. Gene Ther. 9:2101-2109. Totowa, N.J. 97. Harbord, M., M. Novelli, B. Canas, D. Power, C. Davis, J. 76. Cardiff, R. D., E. Sinn, W. Muller, and P. Leder. 1991. Godovac-Zimmerman, J. Roes, and A. W. Segal. 2002. Ym1 Transgenic oncogen mice. Tumor phenotype predicts genotype. is a neutrophil granule protein that crystallizes in p47phox defi- Am. J. Pathol. 139:495-501. cient mice. J. Biol. Chem. 277:5468-5475. 77. Dardick, I., J. Ho, M. Paulus, P. L. Mellon, and L. Mirels. 98. France, M. P., and D. Muir. 2000. An outbreak of pulmonary 2000. Submandibular gland adenocarcinoma of intercalated duct mycosis in respiratory burst-deficient (gp91phox -/-) mice with origin in Smgb-Tag mice. Lab. Invest. 80:1657-1670. concurrent acidophilic macrophage pneumonia. J. Comp. Pathol. 78. Fishman, G. I. 2002. Timing is everything in life. Conditional 123:190-194. transgene expression in the cardiovascular system. Circ. Res. 99. Zhu, Z., R. J. Homer, Z. Wang, Q. Chen, G. P. Geba, J. Wang, 82:837-844. Y. Zhang, and J. A. Elias. 1999. Pulmonary expression of 79. McCune, S. L., and T. M. Townes. 1994. Retroviral vector se- interleukin-13 causes inflammation, mucus hypersecretion, sub- quences inhibit human beta-globin gene expression in transgenic epithelial fibrosis, physiologic abnormalities, and cotaxin produc- mice. Nucleic Acids Res. 22:4477-4481. tion. J. Clin. Invest. 103:779-788. 80. Fiering, S., E. Epner, K. Robinson, Y. Zuang, A. Telling, M. 100. Ward, J. M. 1978. Pulmonary pathology of the motheaten mouse. Hu, D. I. Martin, T. Enver, T. J. Lev, and M. Groudine. 1995. Vet. Pathol. 15:170-178. Targeted deletion of the 5'HS2 of the murine beta-globin LCR 101. Murray, A. B., and A. Luz. 1990. Acidophilic macrophage pneu- reveals that it is not essential for proper regulation of the beta- monia in laboratory mice. Vet. Pathol. 27:274-281. globin locus. Genes Dev. 9:2203-2213. 102. Flanagan, S. P. 1966. “Nude,” a new hairless gene with pleiotro- 81. Kuhn, R., F. Schwenk, M. Aguet, and K. Rajewsky. 1995. In- pic effects in the mouse. Genet. Res. 8:295-309. ducible gene targeting in mice. Science 269:1427-1429. 103. Cheever, F. S., J. B. Daniels, A. M. Pappenheimer, and O. T. 82. Fishman, G. I. 1995. Conditional transgenics. Trends Cardiovasc. Bailey. 1949. A murine virus (JHM) causing disseminated en- Med. 5:211-217. cephalomyelitis with extensive destruction of myelin: I. Isolation 83. Moody, K. D., R. L. Adams, and S. W. Barthold. 1994. Effec- and biologic properties. J. Exp. Med. 90:181-194. tiveness of antimicrobial treatment against Borrelia burgdorferi 104. France, M. P., A. L. Smith, R. Stevenson, and S. W. Barthold. infection in mice. Antimicrob. Agents Chemother. 38:1567-1572. 1999. Granulomatous peritonitis and pleuritis in interferon- 84. Wilson, T. J., and I. Kola. 2001. The LoxP/CRE system and ge- gamma gene knockout mice naturally infected with mouse hepa- nome modification, p. 83-94. In M. J. Tymms and I. Kola (ed.), titis virus. Aust. Vet. J. 77:600-604. Methods in molecular biology: gene knockout protocols. Humana 105. Kyuwa, S., Y. Tagawa, K. Machii, S. Shibata, K. Doi, K. Press, Totowa, N.J. Fujiwara, and Y. Iwakura. 1998. MHV-induced fatal peritoni- 85. Lewandoski, M., and G. R. Martin. 1997. Cre-mediated chro- tis in mice lacking IFN-gamma. Adv. Exp. Med. Biol. 440:445- mosome loss in mice. Nat. Genet. 17:223-225. 450. 86. Balling, R. 2001. ENU mutagenesis: analyzing gene function in 106. Barthold, S. W., A. L. Smith, P. F. S. Lord, P. N. Bhatt, R. O. mice. Ann. Rev. Genomics Hum. Genet. 2:463-492. Jacoby, and A. J. Main. 1982. Epizootic coronaviral typhlocolitis 87. Herron, B. J., W. Lu, C. Rao, S. Liu, H. Peters, R. T. Bronson, in suckling mice. Lab. Anim. Sci. 32:376-383. M. J. Justice, J. D. McDonald, and D. R. Beier. 2002. Effi- 107. Kaminsky, S. B., V. Milisauskas, P. B. Chen, and I. cient generation and mapping of recessive developmental muta- Nakamura. 1987. Defective differentiation of natural killer cells tions using ENU mutagenesis. Nat. Genet. 30:185-189. in SJL mice. J. Immunol. 138:1020-1025. 88. Marschall, S., and H. deAngelis. 1999. Cryopreservation of 108. Bockenstedt, L. K., S. W. Barthold, K. Deponte, N. mouse spermatozoa–double your mouse space. Trends Genet. Marcantonio, and F. S. Kantor. 1993. Borrelia burgdorferi in- 15:128-131. fection and immunity in mice deficient in the fifth component of 89. Sweet, H. O. 1993. Remutations at The Jackson Laboratory. complement. Infect. Immun. 61:2104-2107. Mouse Genome 1993:862-865. 109. Bang, F. B., and A. Warwick. 1960. Mouse macrophages as host 90. Searle, A. G. 1952. A lethal allele of dilute in the house mouse. cells for the mouse hepatitis virus and the genetic basis of their Heredity 6:395-401. susceptibility. Proc. Natl. Acad. Sci. USA 46:1065-1075. 91. Tyner, S. D., S. Venkatachalam, J. Choi, S. Jones, N. 110. Brownstein, D. G., and S. Winkler. 1987. Genetic determinants Ghebranious, H. Ingelmann, X. Lu, G. Soron, B. Cooper, C. of lung virus titers and resistance to lethal Sendai virus pneu- Brayton, S. Hee Park, T. Thompson, G. Karsenty, A. Brad- monia. Arch. Virol. 96:201-214. ley, and L. A. Donehower. 2002. p53 mutant mice that display 111. Brownstein, D. G. 1987. Resistance/susceptibility to lethal early ageing-associated phenotypes. Nature 415:45-53. Sendai virus infection genetically linked to a mucociliary trans- 92. Geisterfer-Lowrance, A. A., M. Christe, D. A. Conner, J. S. port polymorphism. J. Virol. 61:1670-1671. Ingwall, F. J. Schoen, C. E. Seidman, and J. G. Seidman. 112. Fenner, F. 1949. Mouse-pox (infectious ectromelia of mice): a re- 1996. A mouse model of familial hypertropic cardiomyopathy. view. J. Immunol. 63:341-373. Science 272:731-734. 113. Fenner, F. 1982. Mousepox, p. 209-230. In H. L. Foster, J. D. Small, 93. Bowman, J. C., S. F. Steinberg, T. Jiang, D. L. Geenen, G. I. and J. G. Fox (ed.), The mouse in biomedical research. Academic Fishman, and P. M. Buttrick. 1997. Expression of protein ki- Press, New York. nase C beta in the heart causes hypertrophy in adult mice and 114. Dykewicz, C. A., V. M. Dato, S. P. Fisher-Hoch, M. W. sudden death in neonates. J. Clin. Invest. 100:2189-2195. Howarth, G. I. Perez- Oronoz, S. M. Ostroff, H. Gary, Jr, L. 94. Cardiff, R. D., and S. R. Wellings. 1999. The comparative pa- B. Schonberger, and J. B. McCormick. 1992. Lymphocytic thology of human and mouse mammary glands. J. Mammary choriomeningitis outbreak associated with nude mice in a re- Gland Biol. Neoplasia 4:105-122. search institute. J.A.M.A. 267:1349-1353. 95. Cardiff, R. D., M. R. Anver, B. A. Gusterson, L. Hennighausen, 115. Lindsey, J. R., G. A. Boorman, M. J. Collins, Jr., C.-K. Hse, R. A. Jensen, M. J. Merino, S. Rehm, J. Russo, F. A. Tavassoli, G. L. VanHoosier, Jr., and J. E. Wagner. 1991. Infectious dis- L. M. Wakefield, J. M. Ward, and J. E. Green. 2000. The mam- eases of mice and rats. ILAR/NRC, National Academy Press, mary pathology of genetically engineered mice: the consensus re- Washington, D.C. port and recommendations from the Annapolis meeting. Oncogene 116. Bhatt, P. N., R. O. Jacoby, H. C. Morse III, A. E. New. 1986. 19:968-988. Viral and mycoplasmal infections of laboratory rodents: effects on biomedical research. Academic Press, Orlando, Fla.

221 Vol 52, No 3 Comparative Medicine June 2002

117. Macy, J. D., Jr., E. C. Weir, S. R. Compton, M. J. Shlomchik, 136. Ward, J. M., J. G. Fox, M. R. Anver, D. C. Haines, C. V. George, and D. G. Brownstein. 2000. Dual infection with Pneumocystis M. J. Collins, Jr., P. L. Gorelick, K. Nagashima, M. A. Gonda, carinii and Pasteurella pneumotropica in B cell-deficient mice: R. V. Gilden, J. G. Tully, R. J. Russell, R. E. Beneveniste, B. diagnosis and therapy. Comp. Med. 50:49-55. J. Paster, F. E. Dewhirst, J. C. Donovan, L. M. Anderson, 118. Anderson, G. W., and P. G. W. Plagemann. 1995. Expression of and J. M. Rice. 1994. Chronic active hepatitis and associated ecotropic murine leukemia virus in the brains of C58/M, DBA2/J, liver tumours in mice caused by persistent bacterial infection and in utero-infected CE/J mice. J. Virol. 69:8089-8095. with a Helicobacter species. J. Natl. Cancer Inst. 86:1222-1227. 119. Rossie, K. M., J. F. Sheridan, S. W. Barthold, and P. J. 137. Ward, J. M., M. R. Anver, D. C. Haines, and R. E. Beneveniste. Tutschka. 1988. Graft-versus-host disease and sialoadenitis vi- 1994. Chronic active hepatitis in mice caused by Helicobacter ral infection in bone marrow transplanted rats. Transplantation hepaticus. Am. J. Pathol. 145:959-968. 45:1012-1016. 138. Feng, S., E. Hodzic, L. V. Kendall, A. Smith, K. Freet, and S. 120. Sadlack, B., H. Merz, H. Schorle, A. Schimpl, A. C. Feller, W. Barthold. 2002. An immunoreactive protein for Helicobacter and I. Horak. 1993. Ulcerative colitis-like disease in mice with bilis serodiagnosis. Clin. Diagn. Lab. Immunol. In press. a disrupted interleukin-2 gene. Cell 75:253-361. 139. Riley, L. K. 2002. Personal communication. 121. Kuhn, R., J. Lohler, D. Rennick, K. Rajewsky, and W. Muller. 140. Kunstyr, I., F. Jelinek, U. Bitzenhofer, and W. Pittermann. 1993. Interleukin-10-deficient mice develop chronic enterocoli- 1997. Fungus Paecilomyces: a new agent in laboratory animals. tis. Cell 75:263-274. Lab. Anim. 31:45-51. 122. Mombaerts, P., E. Mizoguchi, M. J. Grusby, L. H. Glimcher, 141. Mahler, M., and F. Jelinek. 2000. Granulomatous inflamma- A. K. Bhan, and S. Tonegawa. 1993. Spontaneous development tion in the tails of mice associated with Mycobacterium chelonae of inflammatory bowel disease in T cell receptor mutant mice. infection. Lab. Anim. 34:212-216. Cell 75:275-282. 142. Collins, M. J., and J. C. Parker. 1972. Murine virus contami- 123. Cahill, R. J., C. J. Foltz, and J. G. Fox. 1997. Inflammatory nation of leukemia viruses and transplantable tumors. J. Natl. bowel disease: an immunity-mediated condition triggered by bac- Cancer Inst. 49:1139-1143. terial infection with Helicobacter hepaticus. Infect. Immun. 143. Nicklas, W., and J. Weiss. 2000. Survey of embryonic stem cells 65:3126-3131. for murine infective agents. Comp. Med. 50:410-411. 124. Foltz, C. J., J. G. Fox, R. Cahill, J. C. Murphy, L. Yan, B. 144. Kyuwa, S. 1997. Replication of murine coronaviruses in mouse Shames, and D. B. Schauer. 1998. Spontaneous inflammatory embryonic stem cell lines. Exp. Anim. 46:311-313. bowel disease in multiple mutant mouse lines: association with 145. Lipman, N. S., S. Perkins, H. Nguyen, M. Pfeffer, and H. colonization by Helicobacter hepaticus. Helicobacter 3:69-78. Meyer. 2000. Mousepox resulting from use of ectromelia virus- 125. Kullberg, M. C., J. M. Ward, P. L. Gorelick, P. Caspar, S. contaminated, imported mouse serum. Comp. Med. 50:426-435. Hieny, A. Cheever, D. Jankovic, and A. Sher. 1998. 146. Besselsen, D. G., A. M. Wagner, and J. K. Loganbill. 2000. Helicobacter hepaticus triggers colitis in specific-pathogen-free Effect of mouse strain and age on detection of mouse parvovirus interleukin-10 (IL-10)-deficient mice through an IL-12- and 1 by use of serologic testing and polymerase chain reaction as- gamma interferon-dependent mechanism. Infect. Immun. says. Comp. Med. 50:498-502. 66:5157-5166. 147. Barthold, S. W. 1998. Opportunistic infections in research rodents: 126. Esworthy, R. S., R. Arand, M. G. Martin, J. H. Doroshow, S. the challenges are great and the hour is late. ILAR J. 39:316-321. W. Binder, and F. F. Chu. 2001. Mice with combined disruption 148. Davis, J. K., D. J. Gaertner, N. R Cox, J. R. Lindsey, G. H. of Gpx1 and Gpx2 genes have colitis. Am. J. Physiol. Gastrointest. Cassell, M. K. Davidson, K. C. Kervin, and G. N. Rao. 1987. Liver Physiol. 281:G848-G855. The role of Klebsiella oxytoca in utero-ovarian infection of B6C3F1 127. Brownstein, D. G., and E. C. Weir. 1987. Immunostimulation mice. Lab. Anim. Sci. 37:159-166. in mice infected with Sendai virus. Am. J. Vet. Res. 48:1692-1696. 149. Whittingham, D. G., and M. J. Wood. 1976. Reproductive physi- 128. Barthold, S. W. 1986. Mouse hepatitis virus: biology and epi- ology, p. 137-164. In H. L. Foster, J. D. Small, and J. G. Fox (ed.). zootiology, p. 571-601. In P. N. Bhatt, R. O. Jacoby, H. C. Morse The mouse in biomedical research. III. Normative biology, im- III, and A. E. New (ed.), Viral and mycoplasmal infections of labo- munology, and husbandry. Academic Press, New York. ratory rodents: effects on biomedical research. Academic Press, 150. Fullwood, S., T. A. Hicks, J. C. Brown, R. L. Norman and J. J. Orlando, Fla. McGlone. 1998. Floor space needs for laboratory mice: C57BL/6 129. Jacoby, R. O., L. J. Ball-Goodrich, D. G. Besselsen, M. D. males in solid-bottom cages with bedding. ILAR J. 39:29-36. McKisic, L. K. Riley, and A. L. Smith. 1996. Rodent parvovirus 151. McGlone, J. J., D. L. Anderson, and R. L. Norman. 2001. Floor infections. Lab. Anim. Sci. 46:370-380. space needs for laboratory mice: BALB/cJ males or females in 130. Nelms, K. A., and C. C. Goodnow. 2001. Genome-wide ENU solid-bottom cages with bedding. Contemp. Top. Lab. Anim. Sci. mutagenesis to reveal immune regulators. Immunity 15:409-418. 40:21-25. 131. Flaswinkel, H., F. Alessandrini, B. Rathkolb, T. Decker, E. 152. Baumans, V., F. Schlingmann, M. Vonck, and H. A. VanLith. Kremmer, A. Servatius, T. Jakob, D. Soewarto, S. Marschall, 2002. Individually ventilated cages: Beneficial for mice and men? C. Fella, H. Behrendt, J. Ring, E. Wolf, R. Balling, M. Hrabe Contemp. Topics Lab. Anim. Sci. 41:13-19. de Angelis, and K. Pfeffer. 2000. Identification of immunologi- 153. NRC/ILAR. 1996. Guide for the care and use of laboratory ani- cal relevant phenotypes in ENU mutagenized mice. Mamm. Ge- mals. National Academy Press, Washington, D.C. nome 11:526-527. 154. Wurbel, H., R. Chapman, and C. Rutland. 1998. Effect of feed 132. Fernandez-Salguero, P. M., J. M. Ward, J. P. Sundberg, and and environmental enrichment on development of steotypic wire- F. J. Gonzalez. 1997. Lesions of aryl-hydrocarbon receptor-defi- gnawing in laboratory mice. Appl. Behav. Sci. 60:69-81. cient mice. Vet. Pathol. 34:605-614. 155. Samna, J. R., R. H. Dyck, and I. Q. Whish. 2000. The Dalla 133. Huang, W.-Y., J. Aramburu, P. S. Douglas, and S. Izumo. 2000. effect: C57BL6 mice barber whiskers by plucking. Behav. Brain Transgenic expression of green fluorescence protein cause dilated Res. 108:39-45. cardiomyopathy. Nat. Med. 6:482-483. 156. Yamaha, K., H. Ohki-Hamazaki, and K. Wada. 2000. Differen- 134. Morse, H.C. III. 1986. Unexpected biologic consequences of tial effects of social isolation upon body weight, food consumption, rederivation of mouse strains, p. 689-699, In P. N. Bhatt, R. O. Jacoby, and responsiveness to novel and social environment in bombesin H. C. Morse III, A. E. New (ed.), Viral and mycoplasmal infections of receptor subtype-3 (BRS-3) deficient mice. Physiol. Behav. 68:555- laboratory rodents: effects on biomedical research. Academic Press, 561. Orlando, Fla. 157. Wurbel, H., and M. Stauffacher. 1996. Prevention of stereotypy 135. Hill, L. R., P. S. Kille, D. A. Weiss, T. M. Craig, and L. G. in laboratory mice: effects on stress physiology and behaviour. Coghlan. 1999. Demodex musculi in the skin of transgenic mice. Physiol. Behav. 59:1163-1170. Contemp. Top. Lab. Anim. Sci. 38(6):13-18.

222 “Muromics”

158. Hilakivi, L. A., M. Ota, and R. G. Lister. 1989. Effect of isola- 174. Greenman, D., R. Kodell, and W. Sheldon. 1984. Association tion on brain monoamines and the behavior of mice in tests of between cage shelf level and spontaneous and induced neoplasms exploration, locomotion, anxiety and behavioral “despair.” in mice. J. Natl. Cancer Inst. 73:107-113. Pharmacol. Biochem. Behav. 33:371-374. 175. Saltarelli, C., and C. Coppola. 1979. Influence of visible light 159. Edwards, E., and L. Dean. 1977. Effects of crowding of mice on on organ weights in mice. Lab. Anim. Sci. 29:319-322. humoral antibody formation and protection to lethal antigenic 176. Sheldon, W. G., T. J. Bucci, B. Blackwell, and A. Turturro. challenge. Psychosom. Med. 39:19-24. 1996. Effect of ad libitum feeding and 40% feed restriction on 160. Ortiz, R., A. Armario, J. M. Castellanos, and J. Balash. 1985. body weight, longevity, and neoplasms in B6C3F1, C57BL6, and Post weaning crowding induces corticoadrenal hyperactivity in B6D2F1 mice, p. 21-44. In U. Mohr, D. L. Dungworth, C. C. Capen, male mice. Physiol. Behav. 34:857-860. W. W. Carlton, J. P. Sundberg, and J. M. Ward (ed.), Pathobiology 161. vomSaal, F. 1984. The intrauterine position phenomenon: effects of the aging mouse, vol. 1. ILSI Press, Washington, D.C. on physiology, aggressive behavior and population dynamics in 177. Sheldon, W., A. R. Warbritton, T. J. Bucci, and A Turturro. house mice, p. 135-179. In K. Flannelly, R. Blanchard, and D. 1995. Glaucoma in food-restricted and ad libitum-fed DBA/2NNia Blanchard (ed.), Biological perspectives in aggression: progress mice. Lab. Anim. Sci. 45:508-518. in clinical biological research. 178. Thigpen, J. E., K. D. R. Setchell, K. B. Ahlmark, J. Locklear, 162. Crabbe, J. C., D. Wahlsten, and B. C. Dudek. 1999. Genetics T. Spahr, G. F. Caviness, M. F. Goelz, J. K. Haseman, R. R. of mouse behavior: interactions with laboratory environment. Newbold, and D. B. Forsythe. 1999. Phytoestrogen content of Science 284:1670-1672. purified, open- and closed-formula laboratory animal diets. Lab. 163. Wilson, R. W., C. M. Ballantyne, C. W. Smith, C. Montgom- Anim. Sci. 49:530-536. ery, A. Bradley, W. E. O’Brien, and A. L. Beaudet. 1993. Gene 179. Brown, N. M., and K. D. Setchell. 2001. Animal models im- targeting yields a CD18-mutant mouse for study of inflamma- pacted by phytoestrogens in commercial chow: implications for tion. J. Immunol. 151:1571-1578. pathways influenced by hormones. Lab. Invest. 81:735-747. 164. Crawley, J. N., J. K. Belknap, A. Collins, J. C. Crabbe, W. 180. Spearow, J. L., P. Doemeny, R. Sera, R. Leffler, and M. Frankel, N. Henderson, R .J. Hitzeman, S. C. Maxson, L. L. Barkley. 1999. Genetic variation in susceptibility to endocrine Miner, A. J. Silva, J. M. Wehner, A. Wynshaw-Boris, and R. disruption by estrogen in mice. Science 285:1259- 1261. Paylor. 1997. Behavioral phenotypes of inbred mouse strains: 181. Malloy, V. L., H. L. Bradlow, and N. Orentreich. 1997. Inter- implications and recommendations for molecular studies. Psy- actions between a semisynthetic diet and indole-3-carbinol on chopharmacology 132:107-124. mammary tumor incidence in Balb/cfC3H mice. Anticancer Res. 165. Ammassari-Teule, M., H. H. J. Hoffman, and C. Rossi- 17:4333-4337. Arnaud. 1993. Learning in inbred mice: strain-specific abilities 182. Zhou, G. D., N. S. Hernandez, E. Randerath, and K. across three radial maze problems. Behav. Genet. 23:405-412. Randerath. 2000. Effects of different diets and dietary restric- 166. Robinson, M. L., A. Holmgren, and M. J. Dewey. 1993. Ge- tion on perinatal endogenous DNA adducts. Time dependence of netic control of ocular morphogenesis: defective lens development oxidative and presumptive non-oxidative lesions. Mutat. Res. associated with ocular anomalies in C57BL/6 mice. Exp. Eye Res. 447:137-147. 56:7-16. 183. Barthold, S., G. W. Osbaldiston, and A. M. Jonas. 1977. Di- 167. Smith, R. S., T. H. Roderick, and J. P. Sundberg. 1994. Mi- etary, bacterial, and host genetic interactions in the pathogen- crophthalmia and associated abnormalities in inbred black mice. esis of transmissible murine colonic hyperplasia. Lab. Anim. Sci. Lab. Anim. Sci. 44:551-560. 27:938-945. 168.Hulcrantz, M. and H. S. Li. 1993. Inner ear morphology of 184. Fidler, I. 1977. Depression of macrophages in mice drinking CBA/Ca and C57Bl/6J mice in relationship to noise, age and phe- hyperchlorinated water. Nature 270:735-736. notype. Eur. Arch. Ororhinolaryngol. 250:257-264. 185. Herman, L., W. White, and C. Lang. 1982. Prolonged exposure 169. Walton, J. P., R. D. Frisina, and L. R. Meierhans. 1995. Sen- to acid, chlorine, or tetracycline in drinking water: effects on de- sorineural hearing loss alters recovery from short-term adapta- layed-type hypersensitivity, hemagglutination titer, and reticu- tion in the C57BL/6 mouse. Hearing Res. 88:19-26. loendothelial clearance rates in mice. Lab. Anim. Sci. 32:603-608. 170. Lipman, R. D., E. T. Gaillard, D. E. Harrison, R. T. Bronson. 186. Hall, J., W. White, and C. Lang. 1980. Acidification of drinking 1993. Husbandry factors and the prevalence of age-related amy- water: its effects on selected biologic phenomena in male mice. loidosis in mice. Lab. Anim. Sci. 43:439-444. Lab. Anim. Sci. 30:643-651. 171. Pakes, S., Y.-S. Lu, and P. C. Meunier. 1984. Factors that com- 187. Brayton, C. 2002. Personal communication. plicate animal research, p. 649-665. In J. Fox, B. Cohen, and F. 188. Engelhardt, J. A., C. L. Gries, and G. G. Long. 1993. Inci- Loew (ed.), Laboratory animal medicine. Academic Press, New dence of spontaneous neoplastic and nonneoplastic lesions in York. Charles River CD-1 mice varies with breeding origin. Toxicol. 172. Wax, T. 1977. Effects of age, strain, and illumination intensity Pathol. 21:538-541. on activity and self- selection of light-dark schedules in mice. J. 189. Everett, R. 1984. Factors affecting spontaneous tumor incidence Comp. Physiol. Psychol. 91:51-62. rates in mice: a literature review. Crit. Rev. Toxicol. 13:235-251. 173. Greenman, D. L., P. Bryant, R. L. Kodell, and W. Sheldon. 1982. Influence of cage shelf on retinal atrophy in mice. Lab. Anim. Sci. 32:353-356.

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