Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Glycolipid stage-specific embryonic antigens (SSEA-1) in kidneys of male and female C57BL/6J and beige adult mice

SSEA-1 glycolipids from the kidneys of normal male and female as well as beige mutant mice were isolated and their structures were examined by component analysis, mass spectrometry, immunoblotting, and permethylation studies. These antigens were shown to be extended globoside derivatives as reported by Sekine et al. (1987. J. Biochem. 101:553-562). Quantitative high performance liquid chromatography analyses revealed that the concentration of SSEA-1 glycolipids were four-to fivefold greater in male than female mice. Essentially no SSEA-1 glycolipids were excreted in the urine of normal male mice and thus are not components of the multilamellar lysosomes normally excreted. Testosterone is known to induce the hypertrophy of proximal tubule cells that involves the formation of multilamellar lysosomes and results in the accumulation and excretion of these bodies and associated lysosomal enzymes and specific glycolipids. The present results indicate that in male mice there is also an increase in subcellular structures that contain SSEA-1 glycolipids. The amount of SSEA-1 glycolipids in male beige mice were greater than in normal mice on a per kidney basis. Thus, the increase is in proportion to the kidney hypertrophy seen in beige mouse kidneys. Beige mutant mice appear to have a primary defect in the excretion of multilamellar lysosomes which produces a secondary hypertrophy with an accompanying increase in SSEA-1 glycolipids.     

Teratogenicity of paraxanthine (1,7-dimethylxanthine) in C57BL/6J mice

The teratogenicity of caffeine, as well as two of its three dimethylated metabolites (theobromine and theophylline), has been established in animal studies. The third metabolite, paraxanthine, has not been reported as being tested for teratogenicity even though it is actually the major demethylated metabolite of caffeine metabolism in man. Pregnant C57BL/6J mice were treated i.p. with 175 or 300 mg/kg/day paraxanthine (1,7-dimethylxanthine) dissolved in deionized water at 4 p.m. on day 11 and 9 a.m. on day 12 of gestation. All dams were sacrificed on day 18, and fetuses were fixed for Wilson's razor blade sectioning or double-staining skeletal examination. A dose-related increase in total malformations, primarily cleft palate and limb malformations, was found. The pattern of malformations was similar to that reported for caffeine, theobromine, and theophylline, i.e., an asymmetric response with the left forelimb most often affected. A 21% resorption and a 46% malformation rate was observed at 300 mg/kg/day of paraxanthine, indicating that paraxanthine was slightly less toxic to the embryo than caffeine. Therefore, the parent compound, caffeine, as well as all three of its dimethylated metabolites--paraxanthine, theophylline, and theobromine--are teratogenic.     

Differences in anxiety-related behavior between apolipoprotein E-deficient C57BL/6 and wild type C57BL/6 mice

The influence of ApoE gene deletion on the anxiety state has not been previously investigated. The elevated plus maze was used in this study to determine differences in anxiety-related behavior between apoE-deficient and wild type C57BL/6 mice. The apoE-deficient mice demonstrated less anxiety on the elevated plus maze by spending more time in the open arms of the elevated plus maze compared to wild type mice (p<0.001). Additionally, female apoE-deficient mice visited the open arm of the maze more often than their apoE-deficient male counterpart (p<0.05). The anxiety state and/or sex are possible variables to be considered when designing physiological and/or behavioral studies involving mice that are apoE-deficient.     

Immune responses against allogeneic and syngeneic tumors in aged C57BL/6 mice

Aged C57BL/6 (B6) mice could reject allogeneic BALB/c RL male 1 tumor as efficiently as young B6 mice. However, in vitro analysis showed impaired generation of cytotoxic T cell response in aged B6 mice against allogeneic tumor. The reaction could be augmented by the addition of recombinant interleukin-2 (rIL-2). Enzyme-linked immunospots (ELISPOT) produced by CD8+ T cells purified from spleen cells showed no reduction in aged mice. The findings suggested that the number of CD8+ T cells capable of reacting against allogeneic H-2 antigens was similar in young and aged B6 mice. Low cytotoxic T lymphocyte (CTL) responsiveness in aged B6 mice appeared to have resulted from low responsiveness of CD4+ T cells producing IL-2. Although CTL generation was apparently impaired, strong multiple antigenicity of allogeneic tumor evoked a rejection response in aged B6 mice. On the other hand, no rejection response was observed against syngeneic EL4 tumor in aged B6 mice even after depletion of CD4+ CD25+ immunoregulatory cells. Depletion of CD4+ CD25+ cells caused rejection of EL4 tumor in young B6 mice. The findings suggested that aged B6 mice were incapable of inducing effector cells against weak tumor antigens. Only marginal CTL response and small number of ELISPOTs were generated in young but not aged B6 mice against EL4. Addition of rIL-2 to the culture augmented EL4 killing and ELISPOTs in spleen cells from young and aged B6 mice.     

Cardiovascular responses to caloric restriction and thermoneutrality in C57BL/6J mice

We utilized variations in caloric availability and ambient temperature (T(a)) to examine interrelationships between energy expenditure and cardiovascular function in mice. Male C57BL/6J mice (n = 6) were implanted with telemetry devices and housed in metabolic chambers for measurement of mean arterial pressure (MAP), heart rate (HR), O(2) consumption (VO(2)), and locomotor activity. Fasting (T(a) = 23 degrees C), initiated at the onset of the dark phase, resulted in large and transient depressions in MAP, HR, VO(2), and locomotor activity that occurred during hours 6-17, which suggests torporlike episodes. Food restriction (14 days, 60% of baseline intake) at T(a) = 23 degrees C resulted in progressive reductions in MAP and HR across days that were coupled with an increasing occurrence of episodic torporlike reductions in HR (<300 beats/min) and VO(2) (<1.0 ml/min). Exposure to thermoneutrality (T(a) = 30 degrees C, n = 6) reduced baseline light-period MAP (-14 +/- 2 mmHg) and HR (-184 +/- 12 beats/min). Caloric restriction at thermoneutrality produced further reductions in MAP and HR, but indications of torporlike episodes were absent. The results reveal that mice exhibit robust cardiovascular responses to both acute and chronic negative energy balance. Furthermore, we conclude that T(a) is a very important consideration when assessing cardiovascular function in mice.     

Substrains matter in phenotyping of C57BL/6 mice

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.     

The analgesic defect of C57BL/6J-bgJ/bgJ (beige-J: Chediak-Higashi syndrome) mice transmitted by adoptive transfer of spleen cells to normal littermates

We recently discovered and reported that C57BL/6J-bgJ/bgJ (beige-J) mice have a deficiency in their analgesic response to intracerebroventricularly-administered morphine in the tail-flick test. Postulating a link between these findings and the known immunological defect of beige-J mice (Chediak-Higashi syndrome), we examined the effect of splenectomy on beige-J mice and the adoptive transfer of their mononuclear spleen cells to normal littermate controls (2 x 10(7) cells via tail vein). Eight days after these interventions, the splenectomized beige-J mice responded nearly as well as normal mice to centrally administered morphine in the tail-flick test. The adoptive transfer recipients, in contrast, nearly completely lost their response to the analgesic action of morphine in this test. From the combined results, the spleen appears to be a significant factor in the analgesic defect of beige-J mice and, furthermore, mononuclear splenocytes appear to be the source of a substance that can transfer this defect to otherwise normal animals.     

寒冷对雌性C57BL/6小鼠动情周期的影响*

目的:研究寒冷对雌性C57BL/6小鼠动情周期的影响。方法:12只雌性小鼠随机分为对照组、低温组,每组6只;低温组每天4℃暴露4 h,每天阴道涂片法观察小鼠动情状况,对照组饲养于常温动物房;每2 d称量体重,2周后心脏取血、子宫和卵巢,检测小鼠血清E2、FSH、LH、Prl、P水平,进行子宫、卵巢的组织病理学检查。结果:与对照组比较,低温组小鼠体重无显著性差异(P＞0.05),小鼠子宫脏器系数明显较低、动情间期明显延长(P＜0.01),血清FSH显著升高、Prl显著降低(P＜0.01),小鼠子宫腺管扩张,卵巢卵泡数量明显减少。结论:寒冷可使雌性C57BL/6小鼠动情周期延长,进而可能影响生殖功能。     

Effects of baicalein and wogonin on drug-metabolizing enzymes in C57BL/6J mice

Effects of baicalein and wogonin, the major flavonoids of Scutellariae radix, on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. One-week treatment of mice with a liquid diet containing 5 mM baicalein resulted in 29%, 14%, 36%, 28%, and 46% decreases of hepatic benzo(a)pyrene hydroxylation (AHH), benzphetamine N-demethylation (BDM), N-nitrosodimethylamine N-demethylation (NDM), nifedipine oxidation (NFO), and erythromycin N-demethylation (EMDM) activities, respectively. Treatment with a liquid diet containing 5 mM wogonin resulted in 43%, 22%, 21%, 24%, and 35% decreases of hepatic AHH, BDM, NDM, NFO, and EMDM activities, respectively. However, hepatic 7-methoxyresorufin O-demethylation (MROD) activity was increased and decreased by baicalein- and wogonin-treatments, respectively. Similar modulation was observed with caffeine 3-demethylation (CDM) activity. Immunoblot analysis showed that the levels of hepatic CYP2E1 and CYP3A proteins were decreased by both baicalein- and wogonin-treatments. Hepatic CYP1A2 protein level was increased by baicalein but decreased by wogonin. In extrahepatic tissues, renal AHH activity was decreased by wogonin whereas pulmonary AHH, 7-ethoxyresorufin O-deethylation (EROD), and MROD activities were increased by both flavonoids. Both baicalein and wogonin strongly increased CYP1A protein level in mouse lung. Hepatic and renal UGT activities toward p-nitrophenol were suppressed by baicalein- and wogonin-treatments. However, cytosolic GST activity was not affected by flavonoids. These results suggest that ingestion of baicalein or wogonin can modulate drug-metabolizing enzymes and the modulation shows tissue specificity.     

Delayed matching-to-position performance in C57BL/6N mice

Delayed matching-to-sample is one of the most frequently employed behavioral tasks for assessing spatial working memory in animals. Although the advantages of the task have been widely acknowledged and it is used in the study of a variety of species, its application to mice has been rare. In the present study, we reported the efficacy of a delayed matching-to-position task in C57BL mice lever-pressing in an operant-conditioning chamber. Each trial started with the press of a back lever, followed by the presentation of either a left or right front lever. When the ratio requirement for presses to the front lever (sample) was met, a delay interval started. Delay interval continued until the mice made the first response after the elapse of the programmed delay interval. This was followed by the presentation of a choice of left or right front levers. The choice of the same front lever as the sample was reinforced, whereas the other was not. The proportion of correct choices showed a delay-dependent decrement. A higher ratio of response requirement to the sample resulted in increased accuracy, but the duration of the intertrial interval had no effect. Preceding trials also influenced response accuracy, indicating proactive interference. Overall, the results replicated the effects of parametric manipulations reported in other species, and thus, our findings validate the efficacy of the task for assessing spatial working memory in laboratory mice.     

Estrogen and progesterone affect responses to malaria infection in female C57BL/6 mice

Background: Previous data from our laboratory suggest that gonadally intact C57BL/6 male mice are more likely than their female counterparts to die from Plasmodium chabaudi infection, to recover more slowly from weight loss and hematocrit loss, and to have reduced interferon-γ (IFN-γ) and interleukin-10 (IL-10) responses. Removal of the ovaries, and hence, the primary production of sex steroids in females, reverses these differences.Objective: We hypothesized that sex differences in response to P chabaudi may be mediated by differential synthesis of IFN-γ and IL-10 that is influenced by estrogen, progesterone, or both.Methods: C57BL/6 female mice (n = 200; n = 10/time point/treatment/experiment) were ovariectomized and implanted with a 21-day controlled-release pellet containing either 0.1 mg of 17β-estradiol (E₂), 10 mg of progesterone (P₄), 0.1 mg of E₂ plus 10 mg of P₄, or cholesterol (placebo). Females were inoculated with 10⁶P chabaudi-infected erythrocytes. Body mass, body temperature, hematocrit, parasitemia, cytokine production, and antibody responses were monitored 0, 3, 5, 7, 10, 14, and 21 days postinoculation.Results: Administration of E₂, either alone or in combination with P₄, mitigated infection-induced weight loss, hematocrit loss, and hypothermia, compared with females receiving placebo pellets (P < 0.05 in each case). Hormone treatment did not affect levels of parasitemia. Females administered E₂ alone or in combination with P₄ produced 4 to 7 times higher IFN-γ and IL-10 during peak parasitemia than did females implanted with pellets containing either P₄ alone or placebo (P < 0.05 in each case). Exposure to E₂, either alone or in combination with P₄, increased anti-P chabaudi immunoglobulin G (IgG1) responses and the ratio of IgG1 to IgG2c (P < 0.05 in each case).Conclusion: This animal study suggests that physiological levels of estrogen, rather than progesterone, enhance immunity and, possibly, protect females from disease symptoms during malaria infection.     

Effects of pre-germinated brown rice treatment high-fat diet-induced metabolic syndrome in C57BL/6J mice

To investigate using pre-germinated brown rice (PGBR) to treat metabolic syndrome, we fed one group of mice standard-regular-diet (SRD) for 20 weeks and another group of mice high-fat-diet (HFD) for 16 weeks. We subdivided them into HFD group and HFD + PGBR group whose dietary carbohydrate was replaced with PGBR for 4 weeks. The HFD group gained more weight, had higher blood pressure, heart rate, blood glucose and lipids, liver levels of TG, feces TG and bile acid, lower adipose levels of adipocytokine, lower skeletal muscle IR, IRS-1, IRS-2, PI3 K, Akt/PKB, GLUT-1, GLUT-4, GCK and PPAR-γ; higher liver SREBP-1, SCD-1, FAS, HMGCR, LDLR, CYP7α1 and PPAR-α, and higher adipose SREBP-1, SCD-1, FAS, and lower adipose PPAR-α and adiponectin. The HFD + PGBR group had clearly improved blood pressure, biochemical parameters and above proteins expressions. PGBR successful treatment of metabolic syndrome was achieved through improvements in glucose and lipid synthesis and metabolism.     

Enhanced glucose tolerance in pancreatic-derived factor (PANDER) knockout C57BL/6 mice

Pancreatic-derived factor (PANDER; also known as FAM3B) is a uniquely structured protein strongly expressed within and secreted from the endocrine pancreas. PANDER has been hypothesized to regulate fasting and fed glucose homeostasis, hepatic lipogenesis and insulin signaling, and to serve a potential role in the onset or progression of type 2 diabetes (T2D). Despite having potentially pivotal pleiotropic roles in glycemic regulation and T2D, there has been limited generation of stable animal models for the investigation of PANDER function, and there are no models on well-established genetic murine backgrounds for T2D. Our aim was to generate an enhanced murine model to further elucidate the biological function of PANDER. Therefore, a pure-bred PANDER knockout C57BL/6 (PANKO-C57) model was created and phenotypically characterized with respect to glycemic regulation and hepatic insulin signaling. The PANKO-C57 model exhibited an enhanced metabolic phenotype, particularly with regard to enhanced glucose tolerance. Male PANKO-C57 mice displayed decreased fasting plasma insulin and C-peptide levels, whereas leptin levels were increased as compared with matched C57BL/6J wild-type mice. Despite similar peripheral insulin sensitivity between both groups, hepatic insulin signaling was significantly increased during fasting conditions, as demonstrated by increased phosphorylation of hepatic PKB/Akt and AMPK, along with mature SREBP-1 expression. Insulin stimulation of PANKO-C57 mice resulted in increased hepatic triglyceride and glycogen content as compared with wild-type C57BL/6 mice. In summary, the PANKO-C57 mouse represents a suitable model for the investigation of PANDER in multiple metabolic states and provides an additional tool to elucidate the biological function and potential role in T2D.KEY WORDS: Pancreatic-derived factor, FAM3B, Knockout model, Glycemic regulation, Hepatic insulin signaling, Glucose tolerance     

Substrain specific behavioral responses in male C57BL/6N and C57BL/6J mice to a shortened 21-hour day and high-fat diet

ABSTRACT

Altered circadian rhythms have negative consequences on health and behavior. Emerging evidence suggests genetics influences the physiological and behavioral responses to circadian disruption. We investigated the effects of a 21 h day (T = 21 cycle), with high-fat diet consumption, on locomotor activity, explorative behaviors, and health in male C57BL/6J and C57BL/6N mice. Mice were exposed to either a T = 24 or T = 21 cycle and given standard rodent chow (RC) or a 60% high-fat diet (HFD) followed by behavioral assays and physiological measures. We uncovered numerous strain differences within the behavioral and physiological assays, mainly that C57BL/6J mice exhibit reduced susceptibility to the obesogenic effects of (HFD) and anxiety-like behavior as well as increased circadian and novelty-induced locomotor activity compared to C57BL/6N mice. There were also substrain-specific differences in behavioral responses to the T = 21 cycle, including exploratory behaviors and circadian locomotor activity. Under the 21-h day, mice consuming RC displayed entrainment, while mice exposed to HFD exhibited a lengthening of activity rhythms. In the open-field and light-dark box, mice exposed to the T = 21 cycle had increased novelty-induced locomotor activity with no further effects of diet, suggesting daylength may affect mood-related behaviors. These results indicate that different circadian cycles impact metabolic and behavioral responses depending on genetic background, and despite circadian entrainment.     

In vivo IL-10 production reactivates chronic pulmonary tuberculosis in C57BL/6 mice

The production of immunosuppressive cytokines, such as IL-10 and TGF-beta, has been documented in individuals diagnosed with active tuberculosis. In addition, IL-10 production is increased within the lungs of mice that have chronic mycobacterial infection. Therefore, we hypothesized that the down-regulatory properties of IL-10 might contribute to the reactivation of chronic Mycobacterium tuberculosis infection in mice. To determine the influence of IL-10 on the course of infection, transgenic mice producing increased amounts of IL-10 under the control of the IL-2 promotor were infected with M. tuberculosis via the respiratory route. Mice that overexpressed IL-10 showed no increase in susceptibility during the early stages of infection, but during the chronic phase of the infection showed evidence of reactivation tuberculosis with a highly significant increase in bacterial numbers within the lungs. Reactivation was associated with the formation of macrophage-dominated lesions, decreased mRNA production for TNF and IL-12p40, and a decrease in Ag-specific IFN-gamma secretion. These data support the hypothesis that IL-10 plays a pivotal role during the chronic/latent stage of pulmonary tuberculosis, with increased production playing a potentially central role in promoting reactivation tuberculosis.