Rflps for Somatotropic Genes Identify Quantitative Trait Loci for Growth in Mice

Restriction fragment length polymorphisms for somatotropic genes were tested for associations with body weight and postweaning growth rate in mice. Polymorphisms for growth hormone (GH) and insulin-like growth factor 2 (IGF-2) genes were identified in stock population lines which had been subjected to long-term selection for high 42-day body weight (H lines) or randomly mated (FP and C lines). Two F2 populations of mice (5F2 and MF2) were generated from crosses between a single H line of mice and two unselected control lines and subsequently, two divergently weight selected sublines were generated from each F2 population. The GHh allele which had originally been fixed in three of four H lines and absent from all FP and C lines was found to have a significant (P less than 0.01) effect on 42-day weight and postweaning growth rate in the F2 populations. However, GHh was associated with lower 42-day weight in the F2 populations, suggesting that the positive association between GHh and weight in the stock population was unique to the high weight selected genetic background of those lines. In agreement with this, the frequency of GHh increased in sublines selected for high 42-day weight and decreased in sublines selected for low 42-day weight. The IGF-2H5 allele was associated with higher weights in a sex-dependent manner in 5F2. In the high selected subline derived from 5F2, a significant increase in the frequency of IGF-2H5 was observed. Therefore this allele, in contrast to GHh, appears to be a positive indicator of growth irrespective of genetic background.     

Longer life spans and delayed maturation in wild-derived mice

Nearly all the experimental mice used in aging research are derived from lineages that have been selected for many generations for adaptation to laboratory breeding conditions and are subsequently inbred. To see if inbreeding and laboratory adaptation might have altered the frequencies of genes that influence life span, we have developed three lines of mice (Idaho [Id], Pohnpei [Po], and Majuro [Ma]) from wild-trapped progenitors, and have compared them with a genetically heterogeneous mouse stock (DC) representative of the laboratory-adapted gene pool. Mean life span of the Id stock exceeded that of the DC stock by 24% (P < 0.00002), and maximal life span, estimated as mean longevity of the longest-lived 10% of the mice, was also increased by 16% (P < 0.003). Mice of the Ma stock also had a significantly longer maximal longevity than DC mice (9%, P = 0.04). The longest-lived Id mouse died at the age of 1450 days, which appears to exceed the previous longevity record for fully fed, non-mutant mice. The life table of the Po mice resembled that of the DC controls. Ma and Id mice differ from DC mice in several respects: both are shorter and lighter, and females of both stocks, particularly Id, are much slower to reach sexual maturity. As young adults, Id mice have lower levels of insulin-like growth factor 1 (IGF-I), leptin, and glycosylated hemoglobin compared with DC controls, implicating several biochemical pathways as potential longevity mediators. The results support the idea that inadvertent selection for rapid maturation and large body size during the adaptation of the common stocks of laboratory mice may have forced the loss of natural alleles that retard the aging process. Genes present in the Id and Ma stocks may be valuable tools for the analysis of the physiology and biochemistry of aging in mice.     

Alcohol withdrawal reactions in mouse strains selectively bred for long or short sleep times.

Two lines of mice that differ in their sensitivity to acute hypnotic effects of ethanol were tested for alcohol withdrawal reactions after a standard 3-day exposure to ethanol. The “long-sleep” strain showed a much milder withdrawal reaction than did the “short-sleep” strain. The two strains did not differ in sensitivity to convulsions elicited by pentylenetetrazol. Sleep times and withdrawal reactions did not correlate in individual mice of the genetically heterogeneous stock from which the two lines were derived.     

Quantitative Genetic Analysis of Temperature Regulation in MUS MUSCULUS. I. Partitioning of Variance

Heritabilities (from parent-offspring regression) and intraclass correlations of full sibs for a variety of traits were estimated from 225 litters of a heterogeneous stock (HS/Ibg) of laboratory mice. Initial variance partitioning suggested different adaptive functions for physiological, morphological and behavioral adjustments with respect to their thermoregulatory significance. Metabolic heat-production mechanisms appear to have reached their genetic limits, with little additive genetic variance remaining. This study provided no genetic evidence that body size has a close directional association with fitness in cold environments, since heritability estimates for weight gain and adult weight were similar and high, whether or not the animals were exposed to cold. Behavioral heat conservation mechanisms also displayed considerable amounts of genetic variability. However, due to strong evidence from numerous other studies that behavior serves an important adaptive role for temperature regulation in small mammals, we suggest that fluctuating selection pressures may have acted to maintain heritable variation in these traits.     

Propagation of MM Virus in Continuous Cell Lines

Baby hamster kidney (BHK), McCoy, and L cell lines were found to be suitable for isolation of MM virus from infected mouse brain tissue. The virus was recovered in high titer in the first passage in BHK and McCoy cells, with concomitant cytopathic effect (CPE). In L cells, virus yield was lower than in the other two cell lines and CPE was incomplete. Adaptation of the virus to BHK and McCoy cells by serial passages was evidenced by accelerated development of the CPE and increase in the virus titer. Plaques were obtained in all three cell lines when inoculated with infected mouse brain or with the tissue culture-propagated virus. In the BHK cells, the virus release preceded the appearance of CPE and maximal yield of virus was obtained after 1 to 3 days of incubation, depending on the size of inoculum. The BHK-propagated virus had the same lethality for mice as did the mouse brain-propagated stock, and there was no difference in the course of the disease caused by the two preparations.     

The ability to extrapolate the direction of movement in mice selected for large and small brain weight: the influence of environmental enrichment

Female mice from the strains selected for large and small brain weight (LB, SB) were kept either in "enriched" or in standard laboratory environment for 3 months. At the end of this period they were tested in the open field and for the ability to extrapolate the direction of the food stimulus movement. Scores of the LB mice kept in the "enriched" environment were significantly higher in the extrapolation test as compared to the LB mice kept in the standard environment as well as to the SB mice of both groups.     

Concurrent administration of coenzyme Q10 and alpha-tocopherol improves learning in aged mice

The main purpose of this study was to determine whether supplemental intake of coenzyme Q10 (CoQ) (ubiquinone-10) or alpha-tocopherol, either alone or together, could improve brain function of aged mice, as reflected in their cognitive or psychomotor performance. Separate groups of aged mice (24 months) were administered either CoQ (123 mg/kg/day), or alpha-tocopherol acetate (200 mg/kg/day), or both, or the vehicle (soybean oil) via gavage for a period of 14 weeks. Three weeks following the initiation of these treatments, mice were given a battery of age-sensitive behavioral tests for the assessment of learning, recent memory, and psychomotor function. In a test that required the mice to rapidly identify and remember the correct arm of a T-maze, and to respond preemptively in order to avoid an electric shock, the intake of alpha-tocopherol plus CoQ resulted in more rapid learning compared to the control group. Learning was not significantly improved in the mice receiving CoQ or alpha-tocopherol alone. None of the treatments resulted in a significant improvement of psychomotor performance in the old mice. In a separate study, treatment with higher doses of CoQ alone (250 or 500 mg/kg/day) for 14 weeks failed to produce effects comparable to those of the combination of alpha-tocopherol and CoQ. The apparent interaction of CoQ and alpha-tocopherol treatments is consistent with the previous suggestion, based on biochemical studies, that coenzyme Q and alpha-tocopherol act in concert. Overall, the findings suggest that concurrent supplementation of alpha-tocopherol with CoQ is more likely to be effective as a potential treatment for age-related learning deficits than supplementation with CoQ or alpha-tocopherol alone.     

The collaborative cross: a recombinant inbred mouse population for the systems genetic era

The mouse is the most extensively used mammalian model for biomedical and aging research, and an extensive catalogue of laboratory resources is available to support research using mice: classical inbred lines, genetically modified mice (knockouts, transgenics, and humanized mice), selectively bred lines, consomics, congenics, recombinant inbred panels, outbred and heterogeneous stocks, and an expanding set of wild-derived strains. However, these resources were not designed or intended to model the heterogeneous human population or for a systematic analysis of phenotypic effects due to random combinations of uniformly distributed natural variants. The Collaborative Cross (CC) is a large panel of recently established multiparental recombinant inbred mouse lines specifically designed to overcome the limitations of existing mouse genetic resources for analysis of phenotypes caused by combinatorial allele effects. The CC models the complexity of the human genome and supports analyses of common human diseases with complex etiologies originating through interactions between allele combinations and the environment. The CC is the only mammalian resource that has high and uniform genomewide genetic variation effectively randomized across a large, heterogeneous, and infinitely reproducible population. The CC supports data integration across environmental and biological perturbations and across space (different labs) and time.     

Growth and body composition of mice selected for high body weight

To elucidate the influence of high body weight selection on body compositional relationships, the accumulation of lipid, protein, and ash was investigated in two lines of mice selected for high 42-day body weight (H lines) and an unselected foundation population (FP). The two H lines differed in population size and were designated as the high-large (HL) and high-small (HS) lines. Logistic body growth curves revealed that HL mice exhibited an accelerated growth rate and reached a higher mature body weight than FP or HS mice. Over the range of body weights examined, HL mice had more lipid, less protein, and less ash than FP or HS mice of the same sex and body weight. However, HL lipid accumulation (relative to body weight increase) was not accelerated in comparison to that of FP mice. This study suggests that the existing model of selection-mediated compositional changes requires expansion to account for the ability of high-growth selection to direct an acceleration of body growth without a correlated enhancement of the relative rate of fat accumulation.     

Effects of stock differences in the acceleration of puberty in female mice

Newly born TO strain female mice were exposed daily to the urine from male albino mice of the same and CFLP strains, from feral mice carrying Robertsonian translocation chromosomes and to water as a control condition. At 21 days of age, when exposure was discontinued, there were differences in body weight between treatments which were not present when adult. Exposure to urine from mice with Robertsonian translocations did not accelerate puberty and the interval between vaginal opening and first oestrus was longer (4.2 days) than in mice exposed to the urine from the albino strains (1.8 days). Mice exposed to the urine from the Robertsonian stock were in dioestrus more often than those exposed to the urine from laboratory strains. The Robertsonian mice also differed in their behaviour in an open arena in that they passed fewer faecal pellets than those exposed to the urine from the albino mice. The water control mice defecated the least frequently. The mice exposed to the Robertsonian urine were less active than the laboratory strains but the differences did not reach an acceptable level (P less than 0.06) of significance.     

New in vivo and ex vivo models for the experimental study of sheep scrapie: development and perspectives

Sheep scrapie is a prototypical transmissible spongiform encephalopathy (TSE), and the most widespread of these diseases. Experimental study of TSE infectious agents from sheep and other species essentially depends on bioassays in rodents. Transmission of natural sheep scrapie to conventional mice commonly requires one or two years. In an effort to develop laboratory models in which investigations on the sheep TSE agent would be facilitated, we have established mice and cell lines that were genetically engineered to express ovine PrP protein and examined their susceptibility to the infection. A series of transgenic mice lines (tgOv) expressing the high susceptibility allele (VRQ) of the ovine PrP gene from different constructs was expanded. Following intracerebral inoculation with natural scrapie isolates, all animals developed typical TSE neurological signs and accumulated abnormal PrP in their brain. The survival time in the highest expressing tgOv lines ranged from 2 to 7 months, depending on the isolate. It was inversely related to the brain PrP content, and essentially unchanged on further passaging. Ovine PrP transgene expression thus enhanced scrapie disease transmission from sheep to mice. Such tgOv mice may bring new opportunities for analysing the natural variation of scrapie strains and measuring infectivity. As no relevant cell culture models for agents of naturally-occurring TSE exist, we have explored various strategies in order to obtain stable cell lines that would propagate the sheep agent ex vivo without prior adaptation to rodent. In one otherwise refractory rabbit epithelial cell line, a regulable expression of ovine PrP was achieved and found to enable an efficient replication of the scrapie agent in inoculated cultures. Cells derived from sheep embryos or from tgOv mice were also used in an attempt to establish permissive cell lines derived from the nervous system. Cells engineered to express PrP proteins of a specified sequence may thus represent a promising strategy to further explore, at the cellular level, various aspects of TSE diseases.     

Tissue-specific variation in repair activity for 3-methyladenine in DNA in two stocks of mice

Two stocks of mice, hybrid (C3H X 101)F1 and inbred SEC/R1, were compared for 3-methyladenine-DNA N-glycosylase activity which is involved in removal of 3-methyladenine, 7-methylguanine and some other N-methylpurines in DNA, in cell-free extracts of different tissues. Based on activity measured both per unit weight of tissue and per mass DNA, there is a significant organ-specific and stock-specific difference in N-glycosylase activity over a range of 0.5-8.7 fmoles of 3-methyladenine released per h at 37 degrees C per micrograms DNA of tissue extract. On a per cell basis, the repair activity for 3-methyladenine is the highest in stomach in both stocks. The tissue can be arranged in order of decreasing activity of glycolytic removal as stomach greater than kidney greater than lung greater than liver greater than spleen greater than brain greater than ovary for SEC/R1 mice and stomach greater than kidney greater than ovary greater than spleen, lung and brain greater than liver for the hybrid mice. For all tissues except ovary, SEC/R1 mice have 1.5-4-fold higher specific N-glycosylase activity than (C3H X 101)F1 mice. In contrast, the ovary of SEC/R1 stock has about half as much enzyme activity as that of the hybrid stock.     

Partial biopterin deficiency disturbs postnatal development of the dopaminergic system in the brain

Postnatal development of dopaminergic system is closely related to the development of psychomotor function. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine and requires tetrahydrobiopterin (BH4) as a cofactor. To clarify the effect of partial BH4 deficiency on postnatal development of the dopaminergic system, we examined two lines of mutant mice lacking a BH4-biosynthesizing enzyme, including sepiapterin reductase knock-out (Spr(-/-)) mice and genetically rescued 6-pyruvoyltetrahydropterin synthase knock-out (DPS-Pts(-/-)) mice. We found that biopterin contents in the brains of these knock-out mice were moderately decreased from postnatal day 0 (P0) and remained constant up to P21. In contrast, the effects of BH4 deficiency on dopamine and TH protein levels were more manifested during the postnatal development. Both of dopamine and TH protein levels were greatly increased from P0 to P21 in wild-type mice but not in those mutant mice. Serotonin levels in those mutant mice were also severely suppressed after P7. Moreover, striatal TH immunoreactivity in Spr(-/-) mice showed a drop in the late developmental stage, when those mice exhibited hind-limb clasping behavior, a type of motor dysfunction. Our results demonstrate a critical role of biopterin in the augmentation of TH protein in the postnatal period. The developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to high dependence of dopaminergic development on BH4 availability.     

Cortex weight: a genetic analysis in the mouse.

Differences in forebrain weight, forebrain weight/body weight ratios, and body weight were examined for the RI strains, their progenitor strains, and reciprocal F1 hybrids. The experiment was done in two widely separated laboratories with identical procedures for feeding, watering, and removal of brain tissue. The resulting strain distribution patterns suggested the influence of two or more loci in each laboratory. However, brain weights differed in the two laboratories. Some of the strains examined exhibited increased weights for the variables under consideration, while others exhibited decreased weights or no change at all. Implications for generalizations drawn from observations obtained in any single laboratory were discussed.     

The Heat Shock Response of Carrot : Protein Variations between Cultured Cell Lines

We have defined several parameters surrounding the heat shock response of cultured cells of carrot (Daucus carota L.) and have found that these cells exhibit a typical “higher plant” heat shock response. In particular, the resolution of the heat shock proteins (hsps) by two-dimensional polyacrylamide gel electrophoresis (PAGE) has revealed a pattern of proteins very similar to the hsps from soybean; specifically, the low molecular weight class is composed of approximately 15 to 20 different polypeptides which likely represent different members of a small gene family. In addition, we have compared the (2-D) PAGE profiles of hsps isolated from several different cultured cell lines currently maintained in our laboratory and have found notable differences in the low molecular weight hsps between cell lines. Some of the differences appear to be quantitative, while others may be qualitative. Each of the cell lines was derived from a different seedling of the same seed stock of the same cultivar; thus, genetic differences should be minimized. In addition, two of the cell lines, which show clear differences, were initially derived from a single parental line, and thus arose from a single genetic stock. Possible explanations for the cell line differences observed here are either partial aneuploidy or modified gene regulation resulting from molecular changes during the time in culture (i.e. somaclonal variation). These observations serve to highlight the potential for variation that exists in cells in culture even for such a highly conserved response and gene set as the heat shock genes.     

Trypanosoma cruzi: correlations of biological aspects of the life cycle in mice and triatomines.

The infection pattern in Swiss mice and Triatomine bugs (Rhodnius neglectus) of eleven clones and the original stock of a Trypanosoma cruzi isolate, derived from a naturally infected Didelphis marsupialis, were biochemically and biologically characterized. The clones and the original isolate were in the same zymodeme (Z1) except that two clones were found to be in zymodeme 2 when tested with G6PDH. Although infective, neither the original isolate nor the clones were highly virulent for the mice and lesions were only observed in mice infected with the original stock and one of the clones (F8). All clones and the original isolate infected bugs well while only the original isolate and clones E2 and F3 yielded high metacyclogenesis rates. An observed correlation between absence of lesions in the mammal host and high metacyclogenesis rates in the invertebrate host suggest a evolutionary trade off i.e. a fitness increase in one trait which is accompanied by a fitness reduction in a different one. Our results suggest that in a species as heterogeneous as T. cruzi, a cooperation effect among the subpopulations should be considered.     

Cryopreservation of transgenic mouse embryos — an eight years’ experience

A total of 70,629 predominantly transgenic mouse embryos prepared from 9,727 pregnant female donors were cryopreserved using a method leading to a high revitalisation rate. Against loss, 125 mutant mouse lines were protected. An average of 7.26 embryos (eight-cell embryos) per pregnant donor was received. To reduce the number of animals required as embryo donors, a special breeding exclusively for cryopreservation was omitted if possible and subsequently the number of animals used for freezing was reduced remarkably. The advantage of this strategy is that (mutant) mouse lines out of current use do not have to be kept in a breeding nucleus.In parallel, this procedure leads to rederivation and improves the export of mice to other facilities. The cryopreservation of these 125 mutant lines keeps the potential to save approximately 20,000 laboratory mice per year to be bred if they were kept in a breeding stock. This is a major contribution to the “3R” requirements developed by Russel and Burch to reduce the number of laboratory animals.     

Sleep-time variation for ethanol and the hypnotic drugs tribromoethanol, urethane, pentobarbital, and propofol within outbred ICR mice.

To evaluate the phenotypic variation within a commercial outbred mouse stock, we examined sleep-time (or duration of loss of righting reflex) of outbred ICR mice after i.p. injection of ethanol (4.0 g/kg of body weight), urethane (1.3 g), tribromoethanol (250 mg), and pentobarbital (60 mg), and after i.v. injection of propofol (30 mg). We observed high-grade individual differences in sleep-time that ranged from 0 to 179 min, 83.1 +/- 4.3 (mean and SEM of 100 mice) for ethanol; 0 to 169 min, 64.5 +/- 3.1 for pentobarbital; 0 to 160 min, 36.6 +/- 3.6 for urethane; 0 to 120 min, 21.5 +/- 2.2 for tribromoethanol; and 3 to 20.5 min, 7.1 +/- 0.3 for propofol. This extensive phenotypic variance within the outbred stock was as great as the variation reported among inbred strains or selected lines, and the varied susceptibility within the colony was inherited by Jcl:ICR-derived inbred strains IAI, ICT, IPI, and IQI. The range of sleep-time variance for ethanol, pentobarbital, urethane, tribromoethanol, and propofol within four-way cross hybrid Jcl:MCH(ICR) mice was 86.6%, 63.3%, 124%, 61.0%, and 53.1% that of outbred Jcl:ICR mice, respectively. The present study indicates that phenotypic variance within an outbred Jcl:ICR stock was at high risk for susceptibility to the drugs that depress the central nervous system and that Jcl:ICR-derived inbreds may be an excellent source of animal models for studying the anesthesia gene.     

Different human copper-zinc superoxide dismutase mutants, SOD1G93A and SOD1H46R, exert distinct harmful effects on gross phenotype in mice

Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of fatal neurodegenerative diseases characterized by a selective loss of motor neurons in the brain and spinal cord. Creation of transgenic mice expressing mutant Cu/Zn superoxide dismutase (SOD1), as ALS models, has made an enormous impact on progress of the ALS studies. Recently, it has been recognized that genetic background and gender affect many physiological and pathological phenotypes. However, no systematic studies focusing on such effects using ALS models other than SOD1(G93A) mice have been conducted. To clarify the effects of genetic background and gender on gross phenotypes among different ALS models, we here conducted a comparative analysis of growth curves and lifespans using congenic lines of SOD1(G93A) and SOD1(H46R) mice on two different genetic backgrounds; C57BL/6N (B6) and FVB/N (FVB). Copy number of the transgene and their expression between SOD1(G93A) and SOD1(H46R) lines were comparable. B6 congenic mutant SOD1 transgenic lines irrespective of their mutation and gender differences lived longer than corresponding FVB lines. Notably, the G93A mutation caused severer disease phenotypes than did the H46R mutation, where SOD1(G93A) mice, particularly on a FVB background, showed more extensive body weight loss and earlier death. Gender effect on survival also solely emerged in FVB congenic SOD1(G93A) mice. Conversely, consistent with our previous study using B6 lines, lack of Als2, a murine homolog for the recessive juvenile ALS causative gene, in FVB congenic SOD1(H46R), but not SOD1(G93A), mice resulted in an earlier death, implying a genetic background-independent but mutation-dependent phenotypic modification. These results indicate that SOD1(G93A)- and SOD1(H46R)-mediated toxicity and their associated pathogenic pathways are not identical. Further, distinctive injurious effects resulted from different SOD1 mutations, which are associated with genetic background and/or gender, suggests the presence of several genetic modifiers of disease expression in the mouse genome.     

Race specific resistance to lettuce downy mildew (Bremia lactucae) in the lettuce cultivar Bourguignonne Grosse Blonde d'Hiver

A stock of lettuce cv. Bourguignonne Grosse Blonde d'Hiver used in previous studies was shown to carry the race specific resistance factor R1 and not R9 as originally thought. Investigations into a commercial stock of cv. Bourguignonne which exhibited a heterogeneous reaction to some B. lactucae isolates showed that it was a mixture of two lines. Each line carried one of two R-factors (referred to as R9A and R9B) in a homozygous condition, although their identification was made difficult by the presence of R5/8. No plants were found which carried both R9A and R9B.