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/).     

The superior olivary complex in C57BL/6 mice.

The cellular and cytoarchitectural features of the lateral superior olive, the medial superior olive, the superior paraolivary nucleus and the medial, lateral and ventral nuclei of the trapezoid body are described in C57BL/6 mice using Nissl, Bodian and Golgi techniques. Principal, spindle and marginal cells are present in a well-defined lateral superior olive. The dendrites of these cells run primarily within rostrocaudal sheets as in the cat. The principal cells of the medial nucleus of the trapezoid body are similar to the principal cells in the cat. Large multipolar cells characterize the lateral nucleus of the trapezoid body and bipolar cells with a medial-lateral orientation are found in the medial superior olive. The largest neurons are found in the superior paraolivary nucleus and the lateral superior olive, and the medial and ventral nuclei of the trapezoid body. While brain weight and neuronal packing density change with development, the characteristic location of cell groups and the shape and Nissl-staining pattern of neurons in the youngest brains examined were essentially unchanged in the adult mice, although dendritic maturation had occurred. The homologies of the C57BL/6 superior olivary complex nuclei with the same areas described in other mouse strains, rat and cat are discussed. This study expands our understanding of the organization of the superior olivary complex in an inbred strain of Mus musculus and relates it to other species. The data about changes occurring during postnatal maturation may aid in the interpretation of behavioral and physiological studies of neonatal plasticity of the auditory system.     

243,244Cm studies in C57BL/Do mice

Three groups of C57BL/Do mice were injected with different activities of 243,244Cm so that the long-term biological effects could be evaluated. The biological retention, R, of injected curium in the skeleton at t days after injection could be represented by the equations R = 0.245e-0.000379t and R = 0.208e-0.000494t for male and female mice, respectively. Effective skeletal retention equations were used to calculate the cumulative mean skeletal dose in rad at 140 days before death in each group of mice. The primary objective of this study was to determine the relative biological effectiveness (RBE) of 243,244Cm compared to 226Ra, using bone sarcoma induction as the end point. Combined data (bone sarcomas per 10(6) mouse-rad) for male and female mice permitted the RBE value +/- SD for 243,244Cm to be calculated as 4.4 +/- 1.8 compared to 1.0 for 226Ra. A comparison of RBE values form a previous study in this mouse strain and the value for 243,244Cm from this study suggests that the trivalent actinides 241Am, 243,244Cm, and 249Cf are about three times less effective for bone sarcoma induction than 239Pu.     

Hereditary hydronephrosis in C57BL/KsJ mice.

We sought to determine if the incidence of renal hydronephrosis in male C57BL/KsJ mice increased with age and if grossly normal kidneys would develop hydronephrosis over time. Spontaneous hydronephrosis was found incidentally in 32% of 234 male C57BL/KsJ mice killed as pancreas donors for islet transplantation experiments. The incidence of hydronephrosis increased with age; the incidence was 15% in 6- to 8-week-old mice, 52% in 8- to 10-week-old mice and 63% in 11- to 15-week-old mice (P less than 0.001). Additional mice received islet isografts beneath the renal capsule. Only mice with grossly normal kidneys received islet grafts. These same kidneys were then re-examined when the graft recipients were killed at the end of the experiment and the incidence of hydronephrosis was determined. The conversion of normal kidneys to hydronephrotic kidneys increased with the time since islet transplantation. Kidneys re-examined less than 4 weeks since transplantation had only 5.8% new hydronephrosis, while those re-examined later than 4 weeks after transplantation had a new hydronephrosis incidence rate of 40% (P less than 0.001). Our findings suggest that hydronephrosis is hereditary but not congenital, that it develops rapidly, and that it can complicate experiments using this strain. This may also represent a useful new animal model of progressive hydronephrosis.     

Pigmented spleens in C57BL mice

Approximately 15% of our highly inbred C57BL/6 mice show a spleen with a pigmented cranial part. Microscopically, abundant aggregates of pigment granules are observed primarily scattered between cells of the red pulp. They give a positive reaction with Perls' Prussian blue and stain heavily with silver-methenamine. Most of the granules are stored in macrophages. In some cases a number of granules are surrounded by a membrane. This abnormality may be designated haemosiderosis. In the present study we demonstrate that it is not related to the consumption of iron within the range normally found in laboratory animal diets. This suggests a genetic origin, although confirmation would require further research. If so, comparison with human idiopathic haemochromatosis is tempting. However, contrary to the human condition, organs other than the spleen are not affected, with exception of the liver. This organ contains minor deposits of pigment granules. Nor do affected animals appear to show any ill effects. Nevertheless, it seems worthwhile to investigate whether this phenomenon in C57BL mice could serve as a model for the human disease provided that a practical criterium becomes available for detection in the intact animal.     

Splenic eumelanin differs from hair eumelanin in C57BL/6 mice

The presence of melanin in spleens of black C57BL/6 mice has been known for long. Although its origin and biological functions are still obscure, the relation of splenic melanin to the hair follicle and skin pigmentation was suggested. Here, we demonstrated using for the first time electron paramagnetic resonance spectroscopy that black-spotted C57BL/6 spleens contain eumelanin. Its presence here is a "yes or no" phenomenon, as even in the groups which revealed the highest percentage of spots single organs completely devoid of the pigment were found. Percentage of the spotted spleens decreased, however, with the progress of telogen after spontaneously-induced hair growth. The paramagnetic properties of the spleen eumelanin differed from the hair shaft or anagen VI skin melanin. The splenic melanin revealed narrower signal, and its microwave power saturability betrayed more heterogenous population of paramagnetic centres than in the skin or hair shaft pigment. Interestingly, the pigment of dry hair shafts and of the wet tissue of depilated anagen VI skin revealed almost identical properties. The properties of splenic melanin better resembled the synthetic dopa melanin (water suspension, and to a lesser degree - powder sample) than the skin/hair melanin. All these findings may indicate a limited degradation of splenic melanin as compared to the skin/hair pigment. The splenic eumelanin may at least in part originate from the skin melanin phagocyted in catagen by the Langerhans cells or macrophages and transported to the organ.     

NK activity in C57BL/Ka mice during the development of radiation-induced lymphomas

Treatment of C57BL/Ka mice with a split dose wholebody irradiation (four weekly irradiations of 1,75 Gy) induces the development of thymic lymphomas. NK activity of spleen cells has been determined at several intervals after leukemogenic treatment. Two days after irradiations. NK activity is normal and decreases strongly after one week. This period of decline persists during about one month. Then, NK activity restores and reaches control values. Lymphomas appear in spite of NK activity restoration. The diminution of NK activity during the preleukemic period could favour preleukemic cells apparition.     

SAP suppresses the development of experimental autoimmune encephalomyelitis in C57BL/6 mice

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated disease of the central nervous system. Serum amyloid P component (SAP) is a highly conserved plasma protein named for its universal presence in amyloid deposits. Here we report that SAP-transgenic mice had unexpectedly attenuated EAE due to impaired encephalitogenic responses. Following induction with myelin oligodendroglial glycoprotein (MOG) peptide 35-55 in complete Freund's adjuvant, SAP-transgenic mice showed reduced spinal cord inflammation with lower severity of EAE attacks as compared with control C57BL/6 mice. However, in SAP-Knockout mice, the severity of EAE is enhanced. Adoptive transfer of Ag-restimulated T cells from wild type to SAP-transgenic mice, or transfer of SAP-transgenic Ag-restimulated T cells to control mice, induced milder EAE. T cells from MOG-primed SAP-transgenic mice showed weak proliferative responses. Furthermore, in SAP-transgenic mice, there is little infiltration of CD45-positive cells in the spinal cord. In vitro, SAP suppressed the secretion of interleukin-2 stimulated by P-selectin and blocked P-selectin binding to T cells. Moreover, SAP could change the affinity between α4-integrin and T cells. These data suggested that SAP could antagonize the development of the acute phase of inflammation accompanying EAE by modulating the function of P-selectin.     

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.     

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.     

Fertilizing ability of spermatozoa from aged C57BL/6NNia mice

Spermatozoa from C57BL/6NNia mice (7- and 25-month-old males that produced offspring and 25-month-old males incapable of producing offspring which either mated or did not mate after being paired for 1 month with proven-fertile females) were tested in in-vitro fertilization studies. The 7-month-old males fertilized the largest number of oocytes (80-86%) in vitro and 79% of them subsequently developed into blastocysts in culture. Aged males which failed to mate fertilized the lowest number of oocytes (11-19%) with 48% developing to blastocysts. This group of mice had the lowest number of spermatozoa in the cauda epididymidis (3.2 +/- 0.4 x 10(5)/mg tissue) with fewer motile spermatozoa (22.3 +/- 5.1%) than younger males. The percentage of spermatozoa retaining their acrosome after 3 h in culture was higher in aged males which had not mated when compared to younger males that had mated. After 4 h in culture, however, the number of spermatozoa that had lost their acrosome was almost identical in the two groups. Superovulated mice which were artificially inseminated with spermatozoa from 25-month-old mice that had not mated did not become pregnant. Testosterone concentrations were lowest in aged mice not mating. These concentrations may explain the poor behavioural response of these males, but whether they account for the inability of spermatozoa to fertilize ova in vitro or in vivo after artificial insemination is not known.     

Postnatal development of corticosteroid function of the adrenals in C57BL/6J-Ay mice

We studied postnatal development of corticosteroid function of the adrenals in mice during the period of elevated activity of the hypothalamic-pituitary-adrenal system and the influence of mutant gene Ay on this process. Normally, a corticosterone peak in blood and increased basal and stimulated steroidogenesis in vitro are observed in 3-week old mice. In 3-week old Ay/a mice (hyperexpression of protein agouti) a corticosterone peak in blood is lowered and genotypic differences in steroidogenesis in vitro are absent, as compared to a/A mice (absence of agouti), while at the ages of 10 and 15 weeks, there were no genotypic differences in the blood level of corticosterone and steroidogenesis in vitro was elevated. Thus, a high level of corticosterone during the period of elevated activity of the hypothalamic-pituitary-adrenal system in 3-week old mice is determined by enhanced steroidogenic function of the adrenals. Mutant gene Ay in male mice affected the postnatal development of the adrenal function: the peak of corticosterone in blood was lowered during the period of elevated activity of the system.     

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.     

Tumours of the bone in C57BL/10J mice

Tumours of the bone were found in 17 (12 females and 5 males) of 6768 (0.25%) C57BL/10J mice (3384 males and 3384 females) pooled from 14 oncogenic studies carried out from 1973 to 1987. Tumours of bone were found predominantly in female mice and were age related. The tumours were classified histologically as osteosarcoma (5 osteoblastic, 6 mixed type and 1 fibroblastic), chondroma (2), chondromyxoid fibroma (1), chondrosarcoma (1) and periosteal fibroma (1).     

Self-mutilation of the penis in C57BL/6N mice

A major cause of male reproductive failure in a C57BL/6N mouse production colony is self-inflicted mutilation of the penis. The extent of the damage ranged from loss of the distal end to loss of the entire penis. From January 1974 to August 1976, 645 adult male mice with mutilated penis were removed from this colony--where the monthly census was 9500 mice, mated 1 male to 4 females using a continuous mating system. On necropsy, it was observed that the substance blocking the urethra in the penile stump resulted in urine retention, grossly distending the urinary bladder. Proteus mirabilis and other bacteria isolated from the urethral plugs were considered secondary invaders.     

Age-related fibrillar deposits in brains of C57BL/6 mice

The present article reviews findings regarding the age-related occurrence of clusters of unusual granules in the brains of C57BL/6 (B6) mice and discusses the potential relevance of this phenomenon as a model of specific aspects of brain aging in humans. The granules occur predominatly in the hippocampus of B6 mice and represent aggregations of fibrillar material that are mostly associated with astrocytes. The deposits become evident at about 4 to 6 mo of age, and increase markedly in both number and size thereafter. Similar structures have been observed in adult senescence accelerated mice (SAM) and have been noted, although very rarely, in older mice from other strains. The deposits appear to manifest dominant genetic heritability. Heparan sulfate proteoglycan and laminin or related molecules have been identified as components of the granular material. Although the deposits do not represent senile plaques with β-amyloid deposition, they might mimic the deposition of extracellular matrix molecules that is thought to be an early event in amyloidogenesis in the aged brain and in Alzheimer's disease.     

Mast cells and macrophages in normal C57/BL/6 mice

Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.