Stock Mice Research Articles

Cryoconservation—archiving for the future

Mouse genetics is set to play a pivotal role in the key post-genome challenge-the study of mammalian gene function. Addressing this challenge will involve the development and application of systematic mutagenesis approaches. The expanding mouse mutant resource that will result threatens to overwhelm the currently available animal facility space. Cryopreservation of both mouse embryos and spermatozoa is currently widely employed for the efficient archiving of mouse stocks. Distribution and dissemination of new and existing mouse strains is simplified by the availability of extensive frozen archives. Also, the availability of archives of frozen spermatozoa provides a potential powerful route for the production of backcross progeny for rapid genetic mapping. Moreover, frozen oocytes and ovaries may offer a valuable addition to the current cryopreservation approaches. Comprehensive mouse mutant archives will provide an essential resource for mammalian genetics throughout the 21(st) century.

Phenotypic and Genotypic Relationships Between Ethanol Tolerance and Sensitivity in Mice Selectively Bred for Initial Sensitivity to Ethanol (SS and LS) or Development of Acute Tolerance (HAFT and LAFT)

Genetically based risk for development of alcoholism in humans seems to be related to initial sensitivity and/or acute tolerance to ethanol. The genetic basis for the development of tolerance has received less attention than other ethanol-related behaviors. We have selected lines of mice, according to genetics, which are differentially sensitive to the initial hypnotic effect of ethanol (Short Sleep and Long Sleep, SS and LS) and other lines that differentially develop acute functional tolerance to ethanol (High and Low Acute Functional Tolerance, HAFT and LAFT). We review reports of the relationship between initial sensitivity and two forms of tolerance as measured using different behavioral measures and different time scales. The goal of the study was to investigate alcohol tolerance as measured by different behavioral tests conducted over different time periods and relate these variables to hypnotic sensitivity. We investigated the phenotypic and genotypic relationships between different measures of tolerance to ethanol in the SS and LS mice. We used two measures of tolerance: (a) The time an animal can remain on a stationary dowel or roto-rod at 5-min intervals up to 30 minutes after a single low dose of ethanol (Acute Single Dose Tolerance, ASDT-dowel or ASDT-roto-rod); and (b) The difference in blood ethanol levels taken when a mouse could repeatedly regain balance on a stationary dowel or roto-rod after successive doses of ethanol (Acute Functional tolerance, AFT-dowel or AFT-roto-rod). The time course in AFT was much longer, up to 2 hours. We carried out the same studies on the High and Low Acute Functional Tolerance (HAFT and LAFT) mice. SS and LS mice differ in hypnotic sensitivity as measured by sleep time, and they differ in all forms of acute tolerance that were measured except in AFT-dowel. Although there were phenotypic correlations between AFT-dowel and ASDT-roto-rod in the Heterogeneous Stock (HS) of mice, provisional Quantitative Trait Loci (determined with Recombinant Inbred mice from a SS X LS cross) for the two phenotypes did not overlap, which indicated that there was little or no genetic correlation between the measures. HAFT and LAFT mice do not differ in hypnotic sensitivity as measured by sleep time measurements nor in ataxic sensitivity as measured on the dowel. The HAFT and LAFT mice both developed tolerance when tested in the 30-minute time frame, but the differences between the lines was largely in the rate of development of tolerance and not the amount developed. On the other hand, when tolerance was measured over 2 hr on the dowel or roto-rod, the HAFT and LAFT animals developed different levels of tolerance. We concluded that measures of tolerance depended on both the time of ethanol's action and the behavioral task used. It seemed that the measures of tolerance used in this study had different genetic bases in mice. Presumably, tolerance will also vary in humans depending on the behavioral measure, and tolerance will also have different genetic bases for the different behavioral measures in humans.

Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions.

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic beta cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic beta cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4alphabeta Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted beta cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.

Alopecia mice derived from albino ddY mice: breeding, histological and ultrastructural studies.

Sparse-haired (SH) mice arose spontaneously in a stock of sib-mated albino ddY mice. Hair loss in the skin of SH mice begins at 3 weeks of age and progresses with age. On the basis of breeding studies between SH mice and albino ddY mice, the phenotype of SH mice is identified as autosomal recessive inheritance. Histological examination shows that numerous pilary canals begin to widen and the outer root sheath becomes thin at 1 week of age. The inner root sheath and the hair were separated. Ultrastructural examination reveals that electron dense materials which formed coalesced and irregularly coiled chains are seen at the level of firm attachment of the hair to its inner root sheath. At the level of hair and inner root sheath separation, linear materials with high electron density are observed in the exocuticle of the hair cuticle. These results suggest that the appearance of electron dense materials is closely related to the mechanism of hair loss in SH mice and SH mice may be useful as an animal model in human hypotrichosis.

X chromosome inactivation revealed by the X-linked lacZ transgene activity in periimplantation mouse embryos.

Using H253 mouse stock harboring X-linked HMG-lacZ transgene, we examined X chromosome inactivation patterns in sectioned early female embryos. X-gal staining patterns were generally consistent with the paternal X inactivation in the trophectoderm and the primitive endoderm cell lineages and random inactivation in the epiblast lineages. The occurrence of embryonic visceral endoderm cells apparently at variance with the paternal X chromosome inactivation in 7.5 dpc embryos was explained by the replacement of visceral endoderm cells with cells of epiblast origin. The frequency of cells negative for X-gal staining in 4.5-5.5 dpc XmXp* embryos fluctuated considerably especially in the extraembryonic ectoderm and the primitive endoderm, whereas it was less variable in the embryonic ectoderm. We could not, however, determine whether it is a normal phenomenon revealed for the first time by the use of HMG-lacZ transgene or an abnormality caused by the multicopy transgene.

Mouse (Mus musculus) stocks derived from tropical islands: new models for genetic analysis of life-history traits.

Founder effects, together with access to unoccupied ecological niches, may allow rodent populations on isolated islands to evolve constellations of life‐history traits that distinguish them from their mainland relatives, for example in body size, litter size, and longevity. In particular, low intrinsic mortality risks on islands with reduced predator numbers and not subject to harsh winter climates may in principle support the development of stocks with extended longevity. Conversely, the conditions under which laboratory rodents are typically bred are thought to select for genotypes that produce large, rapidly maturing races with high early reproductive rates but diminished longevity. To test these ideas, and to generate new mouse stocks suitable for genetic and molecular analysis of the processes that time life‐history events, we have developed specific pathogen‐free stocks from mice trapped from three distinct populations: the U.S. mainland (Idaho) and the tropical Pacific islands Majuro and Pohnpei. Mice from all three locations were found to be shorter and lighter, to have smaller litters, and to have higher faecal corticosterone levels than mice of a genetically heterogeneous stock derived from four common laboratory inbred strains. Among the wild‐derived stocks, mice from Pohnpei and Majuro were significantly lighter and shorter than Idaho‐derived animals, even in populations kept from birth under identical housing conditions. Litter size and reproductive success rates did not differ significantly among the three wild‐derived stocks. Although further work will be needed to see if, as predicted, the wild‐derived stocks differ from one another and from laboratory mice in longevity, these stocks provide useful tools for genetic dissection of factors that regulate body size and reproductive success.

Mouse (Mus musculus) stocks derived from tropical islands: new models for genetic analysis of life-history traits

Mouse (Mus musculus) stocks derived from tropical islands: new models for genetic analysis of life-history traits

Absence or pharmacological blocking of placental P-glycoprotein profoundly increases fetal drug exposure.

It was recently shown that naturally occurring Mdr1a mutant fetuses of the CF-1 outbred mouse stock have no placental Mdr1a P-glycoprotein (P-gp) and that this absence is associated with increased sensitivity to avermectin, a teratogenic pesticide. To further define the role of placental drug-transporting P-gp in toxicological protection of the fetus, we used mice with a targeted disruption of the Mdr1a and Mdr1b genes. Mdr1a(+/-)/1b(+/-) females were mated with Mdr1a(+/-)/1b(+/-) males to obtain fetuses of 3 genotypes (Mdr1a(+/+)/1b(+/+), Mdr1a(+/-)/1b(+/-), and Mdr 1a(-/-)/1b(-/-)) in a single mother. Intravenous administration of the P-gp substrate drugs [(3)H]digoxin, [(14)C]saquinavir, or paclitaxel to pregnant dams revealed that 2.4-, 7-, or 16-fold more drug, respectively, entered the Mdr1a(-/-)/1b(-/-) fetuses than entered wild-type fetuses. Furthermore, placental P-gp activity could be completely inhibited by oral administration of the P-gp blockers PSC833 or GG918 to heterozygous mothers. Our findings imply that the placental drug-transporting P-gp is of great importance in limiting the fetal penetration of various potentially harmful or therapeutic compounds and demonstrate that this P-gp function can be abolished by pharmacological means. The latter principle could be applied clinically to improve pharmacotherapy of the unborn child.

Announcement: four-way cross mouse stocks: a new, genetically heterogeneous resource for aging research.

Announcement: four-way cross mouse stocks: a new, genetically heterogeneous resource for aging research.

Inbred lines of mice derived from long-term divergent selection on fat content and body weight.

For identification of the genetic and physiological basis of growth and fatness, the establishment of inbred lines derived from divergently selected lines is an important tool (Lander and Botstein 1989; Festing 1997). It is necessary, first, to develop highly divergent selection lines for the traits of interest, and then to derive inbred lines from them. In this paper the origins and characteristics are described of two pairs of such inbreds derived from long-term selected lines for high and low body weight and fatness. The primary aim of selection experiments in mice is to increase the understanding of the genetics of quantitative traits. Once the divergence between selected lines is of sufficient magnitude, however, they become a valuable resource for studies in other disciplines of biology. Selection lines can be used to investigate underlying physiological or behavioral changes and to elucidate which components have changed and contributed to the observed selection response. Such investigations can focus on all levels of the organism—gene, hormones, enzymes, cell, tissue, organ, whole animal. For example, body weights and body composition have been extensively studied in mice as model mammalian quantitative traits (for example, Eisen 1989; Falconer 1992). However, the number, location, and effects of individual genes contributing to variation in those traits are mostly unknown. A primary goal of QTL mapping studies is to identify these genes (Falconer and Mackay 1996; Lynch and Walsh 1998). Some of the variation in general growth is caused by genes with major effects. A few mutations (hg, lit, dw, dw, pg, cmpt) have been found in mice that have major effects on growth altering the growth process in different ways and to different degrees. Whereas the hg mutation can increase weight gain (from 21 to 42 days) by 30–50%, other mutations decrease body weight by up to 75%. A dependence of gene effects on the genetic background can be expected, but has not yet been well investigated. Some of the variation in fat aggregation is caused by genes with major effects (for example, Fisler and Warden 1997; Pomp 1997). Five single gene mutations of major effects at fatness-related loci have been mapped in mice (A locus with two alleles A and A, Cpe, Tub, Lep, Lepr), altering fatness in different ways. The resulting obesity differs in extent, age of onset, and progression to obesity, and depends on genetic background. In some cases, excessive deposition of adipose tissue can lead to a twofold increase of body weight (Nobentrauth et al. 1996). Body weight is a composite trait aggregating both fat and non-fat tissue. Some genes may affect only fat or lean, whereas others may affect both; thus, if body composition is unknown, it is difficult to disentangle the underlying genes. It is certainly an advantage to have lines differing in body weight but not in fat percentage, and to have lines differing in fat percentage but not in the underlying fat-free body mass. Derivation of (outbred) selection lines. Selection lines were initiated in this laboratory in Edinburgh from a 3-way cross (two inbred [CBA, JU] and one outbred line [CFLP]) base (Sharp et al. 1984). Lines (P, or protein lines) were divergently selected for 20 generations for high (EPH) and low (EPL) lean mass, estimated from an index of body weight and gonadal fat pad weight in males. In subsequent generations, the selection criterion was body weight in both sexes at 10 weeks of age. From the same base population, lines were selected for increased and decreased fat content, resulting in fat (EF) and lean (EL) lines, with selection for the first 20 generations based on the ratio of gonadal fat pad weight to body weight of 10-week-old males. Subsequently, selection was on dry matter content of males at 14 weeks, which is strongly correlated with fat content (Hastings and Hill 1989). For the first 20 generations, three replicates were maintained per line, after which they were crossed to form one replicate. Selection was suspended from generation 53 to 58 (EPH, EPL) or 59 (EF, EL), while all mouse stocks were transferred to a new mouse house by embryo transfer.

Nackt (nkt), a new hair loss mutation of the mouse with associated CD4 deficiency.

A spontaneous recessive mutation named nackt (symbol: nkt) affecting hair growth and T-cell development was discovered in a moderately inbred stock of mice. Skin lesions were characterized by sparse rough coat, bare patches around the eyes and neck, and a scratching behavior throughout life. Fluorescence-activated cell sorter analysis indicated a deficiency in the CD4(+) 8(-) T-cell subset in the thymus and a marked decrease in CD4(+) T cells in peripheral lymphoid organs. Linkage analysis using a set of molecular markers and an F2 intersubspecific cross indicated that the mutation maps to the central region of mouse chromosome 13, in a region homologous to human chromosome 5q22-q35.

Nesting material and number of females per cage: effects on mouse productivity in BALB/c, C57BL/6J, DBA/2 and NIH/S mice

Two different materials-aspen wood-wool and paper towel-were compared as nesting material for three inbred mouse strains (BALB/c, C57BL/6J and DBA/2) housed in barrier conditions. In addition, the effect of varying the number of females per cage (one to three per cage) of these three strains and with NIH/S outbred mouse stock was studied. The number of litters, litter size and neonatal mortality were determined, as well as age, sex and weight of weanlings. The type of nesting material did not affect the characteristics monitored. In all strains, the number of weanlings per female was greatest in singly-housed females. In terms of the number of weanlings per cage, two females per cage gave the best result. In DBA/2 mice, neonatal mortality increased when several females were caged together.

Sensitivity to cocaine and amphetamine among mice selectively bred for differential cocaine sensitivity.

Selective breeding of mice for differences in response to a drug offers a powerful means for testing hypotheses regarding underlying mechanisms and relationships between drug-induced behaviors. Starting from a heterogeneous stock of mice, we have selectively bred lines of mice for extreme differences in their locomotor response to 10 mg/kg cocaine HCl. Selection pressure has been maintained for 12 generations and has resulted in two cocaine sensitive (CAHI) and two cocaine insensitive (CALO) lines. Across the generations of selection, the CAHI lines showed progressively greater amounts of cocaine-induced locomotion, with mice from the S12 generation traveling over 21,000 cm/30 min. following 10 mg/kg cocaine. The CALO lines, in contrast, did not substantially diverge from control values until the S8 generation. By generation 12, however, the LO lines traveled no further following 10 mg/kg cocaine (7000 cm/30 min), than they did following an initial saline injection. Cocaine and amphetamine dose-response analyses were conducted on drug-naive mice from the tenth generation. The CAHI lines were extremely sensitive to the locomotor activating effects of all doses of cocaine, displaying from 2- to 6-fold greater amounts of cocaine-induced locomotion than the CALO lines. The CALO lines, in contrast, were completely insensitive to the psychomotor stimulant effects of cocaine. The CAHI lines were also more sensitive to the locomotor activating effects of amphetamine. Both lines showed dose-dependent amphetamine-induced locomotion that peaked at 3 mg/kg. However, at all doses, the CAHI lines showed a 2- to 4-fold greater amount of locomotion than CALO lines. Thus, the sensitivity to cocaine developed through selection using a single dose of cocaine has generalized to a range of doses of cocaine and to at least one other psychostimulant.

Marker Loci Associated With Life Span in Genetically Heterogeneous Mice

Little is known about the number or chromosomal location of genetic loci that might identify individuals destined to have a long life span. Analysis of gene/life span associations in mice, which are short-lived compared to humans, might provide guidance for an analysis of the genetic basis of life span in humans. A group of 144 genetically heterogeneous mice, produced by a four-way cross between two F1 hybrid mouse stocks, was genotyped at 82 loci, and the mice were allowed to live either until their natural deaths or until they became extremely ill. Each mouse was also necropsied to determine the probable cause of death. An analysis of variance was used to seek relationships between life span (dependent variable) and the independent variables sex and allele for each marker locus. Five markers on different chromosomes were associated with differential longevity in male mice, and two other markers were associated with longevity in female mice. Post hoc probabilities were suggestive but not definitive, reaching p < .003 for the four strongest effects. Associations between marker loci and life span were sex-specific in almost all cases, affecting either males or females but not both. The strongest effects led to differences in mean survival of about 20% in the affected sex. The survival curves are consistent with the idea that the markers are linked to loci that influence the mortality risks of the longest-lived animals in the cohort. Associations between markers and life span did not appear to reflect associations of these markers to specific diseases in these mice. Associations between genetic markers and life span in mice bred by using a four-way cross are strong enough to deserve further analysis and seem to be sex-specific in their effects.

Mortality and biomarkers of aging in heterogeneous stock (HS) mice.

A longitudinal study was undertaken to evaluate the relationships among a battery of aging biomarkers and subsequent survival time in 319 genetically heterogenous stock (HS) mice. The biomarker variables chosen were selected from the broad domains of behavior, homeostatic physiology, oxidative defense, and immune function; biomarkers were measured at 45, 90, 360, 630, and 900 days of age. Sex differences were found in the survivor and mortality functions, with a mortality rate crossover occurring at about 525 days and a survival curve crossover at about 750 days of age. Females experienced lower initial mortality but had more sharply increasing mortality with age than did males. Survival analysis using Gompertz parametric models with biomarkers as time-varying covariates yielded significant biomarkers from each domain. Following backward elimination procedures, the final set of independent mortality predictors included headpokes in the File activity apparatus, maximum cord drop time, weight, hematocrit, urine concentration, natural killer cell activity, and concanavalin A response.

Deer Mice As Laboratory Animals.

Although laboratory mice (Mus) and rats (Rattus) are the most widely used research rodents, deer mice (Peromyscus maniculatus) and their congeneric species are favored as nontraditional alternatives for some purposes. Mice of the native genus Peromyscus are the most abundant and widely distributed rodents in North America. They occur in a great diversity of habitats and play a significant role in natural ecosystems. Because of their abundance, peromyscines are commonly hosts for larva of ticks that transmit Lyme disease bacteria, and they are implicated in several other vector-borne diseases. Deer mice also are the principal carriers of the virus that causes hantaviral pulmonary syndrome, or "Four Corners disease." Deer mice are useful as laboratory models for a variety of other types of pure and applied research. They are easily maintained and bred in captivity using the husbandry protocols developed for other small laboratory rodent species. The Peromyscus Genetic Stock Center at the University of South Carolina maintains more than 50 laboratory-bred, well-characterized stocks of deer mice and other peromyscine species for research and educational use.

Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase.

A transgenic mouse strain with early and uniform expression of the Cre site-specific recombinase is described. In this strain, PGK-Crem, Cre is driven by the early acting PGK-1 promoter, but, probably due to cis effects at the integration site, the recombinase is under dominant maternal control. When Cre is transmitted by PGK-Crem females mated to males that carry a reporter transgene flanked by loxP sites, even offspring that do not inherit PGK-Cre delete the target gene. It follows that in the PGK-Crem female Cre activity commences in the diploid phase of oogenesis. In PGK-Crem crosses complete recombination was observed in all organs, including testis and ovary. We prepared a mouse stock that is homozygous for PGK-Crem and at the albino (c) locus. This strain will be useful for the early and uniform induction of ectopic and dominant negative mutations, for the in vivo removal of selective elements from targeted mutations and in connection with the manipulation of targeted loci in 'knock in' and related technologies.

Subacute Pb Exposure During Development and Unbaited Tunnel Maze Performance in Mice

Although research has linked chronic, low-level Pb exposure to behavioral and cognitive changes in humans and animals, far less is known about the effects of transient, subchronic Pb exposure during early postnatal development. The need to understand such effects is underscored by the possibility that subchronic Pb exposure may not produce chronically elevated blood–Pb levels, but may produce long-term behavioral changes. To test this hypothesis, we investigated the effect of low-level Pb exposure on unbaited tunnel maze performance in Binghamton Heterogeneous Stock mice. Mice were either nontreated or given subchronic sodium acetate, 5, 10, or 25 mg/kg Pb acetate intragastrically on postnatal (PN) days 6, 9, 12, 15, and 18. No further Pb exposures were given after postnatal day 18. Blood–Pb measurements were taken from selected mice on PN 18, 19, 23, 28, and 38. On PN 38–42, all mice were individually tested in an unbaited tunnel maze under nondeprived conditions. Locomotor activity, exploration, and experience-dependent changes in cul-de-sac entries were recorded. Although Pb did not affect bodyweight and blood–Pb levels were below 10 μg/dl at the time of behavioral testing, a history of low-level preweaning Pb exposure caused a dose-dependent increase in cul-de-sac entries. This behavioral change was dissociable from changes in bodyweight, degree of exploration or an a priori bias to enter cul-de-sacs. The current results support the hypothesis that brief, subchronic Pb exposure during development produces behavioral changes that last well beyond the exposure period, even when blood–Pb declines to within “acceptable” levels (10 μg/dl).

Variation and its implications for the design of experiments in toxicological research

The controlled experiment is one of the most powerful tools available in biomedical research, and thousands of such experiments are conducted annually. However, the basic principles of designing such experiments do not seem to be widely understood. If the experimental material is heterogeneous, then the treated and control groups may not be identical at the start of the experiment, even if large sample sizes are used. Yet genetically heterogeneous ‘outbred’ stocks of laboratory mice and rats are widely used in the pharmaceutical industry in spite of the availability of more homogeneous inbred strains. The use of such stocks is justified on two grounds: that humans are also heterogeneous, and that such stocks offer a wide range of phenotypes for toxicological screening. Both arguments are spurious. It is not necessary for atoxicological model of humans to be a goodgenetic model. Models do not have to resemble their target in every respect. Were they to do so they would no longer be models. The genetic heterogeneity within an outbred stock simply creates ‘noise’ when comparing treated and control groups, and prevents more exact matching of groups before the experiment starts. Phenotypic heterogeneity can be achieved without this disadvantage using a factorial experimental design involving more than one strain, with groups exactly matched genetically, but without increasing total numbers.

Association of a murine chromosome 9 locus (Psl1) with susceptibility to mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

It has been known for many years that there are dramatic differences in the susceptibility of mouse stocks and strains to two-stage skin carcinogenesis and that these differences are due the animals' responsiveness to tumor-promoting agents. In earlier studies using several inbred mouse strains, we found that susceptibility to skin tumor promotion by phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) is a multigenic trait. To extend this work, we conducted a genome scan of (C57BL/6 x DBA/2)F1 x C57BL/6 mice previously scored for sensitivity to skin tumor promotion by TPA. As a result of this scan, we now report an association of increased TPA promotion susceptibility with inheritance of the DBA/2 alleles of markers on the distal portion of mouse chromosome 9. Additional linkage analyses using (C57BL/6 x DBA/2)F2 and B x D recombinant inbred mice confirmed this association and suggested that a TPA promotion susceptibility locus maps near D9Mit51 (LODw = 4.1). We designated this locus promotion susceptibility locus 1 (Ps/1).