C-fos expression is hypersensitive to serum-stimulation in cultured cystic kidney cells from the C57BL/6J-cpk mouse.

Cystic kidneys of the C57BL/6J-cpk murine model of polycystic kidney disease show a marked overexpression of the proto-oncogenes c-fos, c-myc, and c-Ki-ras, consistent with an increased rate of cell proliferation and an altered state of differentiation. To determine if cystic cells have increased responsiveness to stimulation with mitogenic agents, quiescent primary cultures from normal and cystic cpk kidneys were treated with fetal bovine serum (FBS), 8-bromo-cAMP (cAMP), or epidermal growth factor (EGF). The level of c-fos induction following stimulation by FBS was found to be dramatically higher in cystic cells than in normal cells; whereas induction by cAMP or EGF was essentially the same in both cell types and much less than that seen in FBS-stimulated cells. To determine if this serum hypersensitivity reflects an increased proliferative state in vivo, c-fos induction was examined in cultures derived from normal kidneys stimulated to regenerate by folic acid-induced acute renal injury. As with cystic kidneys, the folic acid-injured kidneys showed increased c-fos responsiveness to FBS in cell culture. These experiments suggest that cystic and regenerating kidneys have an altered phenotypic state in vivo that is manifested in cell culture by serum hypersensitivity. However, whereas the folic acid-injured kidneys ultimately reestablish normal kidney function, cystic kidneys further progress to renal failure, suggesting that cystic epithelial cells are locked in this altered state of differentiation.     

A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

Background

The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.

Results

We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.

Conclusions

Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.     

Characterization of the serotoninergic system in the C57BL/6 mouse skin.

We showed expression of the tryptophan hydroxylase gene and of tryptophan hydroxylase protein immunoreactivity in mouse skin and skin cells. Extracts from skin and melanocyte samples acetylated serotonin to N-acetylserotonin and tryptamine to N-acetyltryptamine. A different enzyme from arylalkylamine N-acetyltransferase mediated this reaction, as this gene was defective in the C57BL6 mouse, coding predominantly for a protein without enzymatic activity. Serotonin (but not tryptamine) acetylation varied according to hair cycle phase and anatomic location. Serotonin was also metabolized to 5-hydroxytryptophol and 5-hydroxyindole acetic acid, probably through stepwise transformation catalyzed by monoamine oxidase, aldehyde dehydrogenase and aldehyde reductase. Activity of the melatonin-forming enzyme hydroxyindole-O-methyltransferase was notably below detectable levels in all samples of mouse corporal skin, although it was detectable at low levels in the ears and in Cloudman melanoma (derived from the DBA/2 J mouse strain). In conclusion, mouse skin has the molecular and biochemical apparatus necessary to produce and metabolize serotonin and N-acetylserotonin, and its activity is determined by topography, physiological status of the skin, cell type and mouse strain.     

A model of sleep-disordered breathing in the C57BL/6J mouse.

To investigate the pathophysiological sequelae of sleep-disordered breathing (SDB), we have developed a mouse model in which hypoxia was induced during periods of sleep and was removed in response to arousal or wakefulness. An on-line sleep-wake detection system, based on the frequency and amplitude of electroencephalograph and electromyograph recordings, served to trigger intermittent hypoxia during periods of sleep. In adult male C57BL/6J mice (n = 5), the sleep-wake detection system accurately assessed wakefulness (97.2 +/- 1.1%), non-rapid eye movement (NREM) sleep (96.0 +/- 0.9%) and rapid eye movement (REM) sleep (85.6 +/- 5.0%). After 5 consecutive days of SDB, 554 +/- 29 (SE) hypoxic events were recorded over a 24-h period at a rate of 63.6 +/- 2.6 events/h of sleep and with a duration of 28.2 +/- 0.7 s. The mean nadir of fraction of inspired O(2) (FI(O(2))) on day 5 was 13.2 +/- 0.1%, and 137.1 +/- 13.2 of the events had a nadir FI(O(2)) <10% O(2). Arterial blood gases confirmed that hypoxia of this magnitude lead to a significant degree of hypoxemia. Furthermore, 5 days of SDB were associated with decreases in both NREM and REM sleep during the light phase compared with the 24-h postintervention period. We conclude that our murine model of SDB mimics the rate and magnitude of sleep-induced hypoxia, sleep fragmentation, and reduction in total sleep time found in patients with moderate to severe SDB in the clinical setting.     

Genetic polymorphisms among C57BL/6 mouse inbred strains

Mice from the inbred C57BL/6 strain have been commonly used for the generation and analysis of transgenic and knockout animal models. However, several C57BL/6 substrains exist, and these are genetically and phenotypically different. In addition, each of these substrains can be purchased from different animal providers and, in some cases, they have maintained their breeding stocks separated for a long time, allowing genetic differences to accumulate due to individual variability and genetic drift. With the aim of describing the differences in the genotype of several C57BL/6 substrains, we applied the Illumina^® Mouse Medium Density Linkage Mapping panel, with 1,449 single nucleotide polymorphisms (SNPs), to individuals from ten C57BL/6-related strains: C57BL/6JArc, C57BL/6J from The Jackson Lab, C57BL/6J from Crl, C57BL6/JRccHsd, C57BL/6JOlaHsd, C57BL/6JBomTac, B6(Cg)-Tyr ^c?2j /J, C57BL/6NCrl, C57BL/6NHsd and C57BL/6NTac. Twelve SNPs were found informative to discriminate among the mouse strains considered. Mice derived from the original C57BL/6J: C57BL/6JArc, C57BL/6J from The Jackson Lab and C57BL/6J from Crl, were indistinguishable. Similarly, all C57BL/6N substrains displayed the same genotype, whereas the additional substrains showed intermediate cases with substrain-specific polymorphisms. These results will be instrumental for the correct genetic monitoring and appropriate mouse colony handling of different transgenic and knockout mice produced in distinct C57BL/6 inbred substrains.     

Hippocampal lipids linked to spatial memory in the C57BL/6J mouse

Although the role of individual brain lipids for learning and memory has been reported, no systematic approach associating brain lipids with spatial memory has been carried out. It was therefore the aim of the study to determine brain lipids in hippocampus of mice forming and yoked controls that did not form spatial memory using the probe trial as the endpoint. 10 animals were trained in the Morris water maze (MWM) and 10 mice were serving as yoked controls i.e. no platform was used during the whole experiment. Hippocampal tissue lipids were extracted and data were acquired with Fourier transformation ion cyclotron resonance mass spectrometry (LTQ-FT) coupled to HPLC. Glycerophosphatidylethanolamines (18:0/22:6, 18:0/20:4 and 18:1/18:1), plasmalogens (16:0-10/22:6 and 18:0-10/22:6) and ceramides (18:0) showed higher levels in the trained group, while glycerolysophosphatidylcholines (16:0, 18:1, 18:0, 20:4), sphingomyelins (16:0, 24:1), ether linked glycerophosphatidylcholines (16:0-10/18:0), glycerophosphatidylcholines (16:0/18:1, 16:0/18:0, 18:0/18:1, 38:7, 18:1/20:1, 20:4/20:4, 22:1/18:1, 22:0/18:1, 20:4/22:6, 22:6/22:6), glucosylceramide (24:1) and plasmalogen (18:0-10/20:1) revealed lower levels in the trained group. Decreased levels of certain species of lysophosphatidylcholine, sphingomyelin, plasmenylphosphatidylcholine, phosphatidylcholine, glycosylceramide and plasmalogen at the probe trial for spatial memory may indicate catabolism in terms of consumption during this process. Increased hippocampal levels of long chain highly unsaturated phosphatidylethanolamines, plasmalogens and ceramides may reflect increased synthesis or decreased degradation at the endpoint of memory testing, probably representing interactions in the brain lipid pathways. The study shows pathways involved in spatial memory, may propose the use of individual brain lipids as probable cognitive enhancers and forms the basis for further studies on the role of brain lipids per se.     

Pavlovian biconditional discrimination learning in the C57BL/6J mouse

Four experiments using mice examined acquisition of Pavlovian biconditional discriminations in which two stimulus compounds were paired with food (AX+ and BY+) and two were not (AY- and BX-). Temporally asynchronous compounds were generated by using contextual stimuli (Experiment 1) and 15-s discrete visual cues (Experiments 2A, 2B and 3) to disambiguate when embedded noise or tone stimuli would be paired with food. When food pellets followed both reinforced compounds, successful acquisition was obtained in Experiment 1 but not in Experiments 2A and 2B even though the order of trials was modeled after that used in Experiment 1. However, when differential outcomes followed the reinforced compounds in Experiment 3, acquisition was obtained with discrete cue stimulus compounds. The implications of these results for modulatory models of conditional discrimination learning in animals are discussed.     

Quantitative trait loci analysis for the differences in susceptibility to atherosclerosis and diabetes between inbred mouse strains C57BL/6J and C57BLKS/J.

Mice from the inbred strain C57BLKS/J (BKS) exhibit increased susceptibility to both diabetes and atherosclerosis compared to C57BL/6J (B6) mice. To determine whether the differences in diabetes and atherosclerosis are related, we carried out a cross between B6-db/db and BKS. We selected 99 female F2-db/db progeny, tested the progeny for plasma lipids, plasma glucose, and fatty-streak lesions, and used quantitative trait loci (QTL) analysis to identify the chromosomal regions associated with these phenotypes. No major QTL were found for total cholesterol, VLDL-cholesterol, or triglycerides. Two suggestive QTL were found for HDL-cholesterol (LOD scores of 2. 7 and 2.8), and two suggestive loci were found for plasma glucose (LOD scores of 2.3 and 2.0). Lesion size was not correlated with plasma lipid levels or glucose. Lesion size was determined by a locus at D12Mit49 with a LOD score of 2.5 and a significant likelihood ratio statistic. The gene for apolipoprotein apoB lies within the region, but apoB levels were similar in strains B6 and BKS. The QTL on Chr 12 was confirmed by constructing a congenic strain with BKS alleles in the QTL region on a B6 genetic background. We conclude that susceptibilities to diabetes and atherosclerosis are not conferred by the same genes in these strains and that a major gene on Chr 12, which we name Ath6, determines the difference in atherosclerosis susceptibility.     

Morphometry and strain distribution of the C57BL/6 mouse aorta

The goal of the present study was to obtain a systematic set of data along the length of the mouse aorta to study variations of morphometry (diameter, wall thickness, and curvature), strain, and stress of the mouse aorta. Five mice were imaged with a 13-MHz ultrasound probe to determine the in vivo diameter along the aorta. A cast was made of these aortas to validate the ultrasonic diameter measurements. The root mean squared and systematic errors for these measurements were 12.6% and 6.4% of the mean ultrasound diameter, respectively. The longitudinal variations of geometry, stress, and strain from the aortic valve to the common iliac bifurcation were documented. Our results show that the residual circumferential strain leads to a uniformity of transmural strain of the aorta in the loaded state along the entire length of the aorta. Furthermore, we validated the incompressibility condition along the length of the aorta. These data of normal mice will serve as a reference state for the study of disease in future knockout models.     

Nuclear proteome profile of C57BL/6J mouse liver

The liver proteome can serve as a reference to better understand both disease mechanisms and possible therapeutics,since the liver is an important organ in the body that performs a large number of tasks.Here we identify the organelle proteome of C57BL/6J mouse liver nuclei as a promising strategy to enrich low abundance proteins,in the sense that analysis of whole liver cells is rather complex for current techniques and may not be suitable for proteins with low abundance.Evaluation of nucleus integrity and purity was performed to demonstrate the effectiveness of the optimized isolation procedure.The extracted nuclear proteins were identified by 2-DE MS analyses,and a total of 748 proteins were identified.Bioinformatic analyses were performed to demonstrate the physicochemical properties,cellular locations and functions of the proteins.     

Acetazolamide protects against posthypoxic unstable breathing in the C57BL/6J mouse.

Acetazolamide (Acz), a carbonic anhydrase inhibitor, is used to manage periodic breathing associated with altitude and with heart failure. We examined whether Acz would alter posthypoxic ventilatory behavior in the C57BL/6J (B6) mouse model of recurrent central apnea. Experiments were performed with unanesthetized, awake adult male B6 mice (n = 9), ventilatory behavior was measured using flow-through whole body plethysmography. Mice were given an intraperitoneal injection of either vehicle or Acz (40 mg/kg), and 1 h later they were exposed to 1 min of 8% O(2)-balance N(2) (poikilocapnic hypoxia) or 12% O(2)-3% CO(2)-balance N(2) (isocapnic hypoxia) followed by rapid reoxygenation (100% O(2)). Hypercapnic response (8% CO(2)-balance O(2)) was examined in six mice. With Acz, ventilation, including respiratory frequency, tidal volume, and minute ventilation, in room air was significantly higher and hyperoxic hypercapnic ventilatory responsiveness was generally lower compared with vehicle. Poikilocapnic and isocapnic hypoxic ventilatory responsiveness were similar among treatments. One minute after reoxygenation, animals given Acz exhibited posthypoxic frequency decline, a lower coefficient of variability for frequency, and no tendency toward periodic breathing, compared with vehicle treatment. We conclude that Acz improves unstable breathing in the B6 model, without altering hypoxic response or producing short-term potentiation, but with some blunting of hypercapnic responsiveness.     

Genetic differentiation within the inbred C57BL/6J mouse strain

The C57BL/6J (B6) inbred mouse strain is commonly used in biomedical researches. However, some unexpected inconsistency was reported compared with previous studies, and in most cases, it can be attributed to environmental, epigenetic or stochastic differences. The goal of this study was to investigate the genetic stability of the B6 strain maintained in different breeders. B6 mice purchased from five Chinese commercial breeders were examined, and mitochondrial D-loop sequence and 18 microsatellite loci were genotyped. There is no difference in the D-loop sequences, but variations exist in the nucleic microsatellite markers. Combining the data from MGI_4.01, a significant divergence is observed among those mice. The present study indicates that different B6 mice share the common maternal lineage and are still inbred in each breeder, but subline divergence occurs.     

Gender influences infectivity in C57BL/6 mice exposed to mouse minute virus

Two natural outbreaks of mouse minute virus (MMV) are described. Observations during management of the naturally infected colonies led to a study in which 4-wk-old C57BL/6NCr and C57BL/6Tac mice were inoculated oronasally with an immunosuppressive variant of MMV (MMVi), as were adult C57BL/6NCr lactating dams or their pups (age, 10 d). By day 28 postinoculation, 100% of the 4-wk-old male C57BL/6NCr and C57BL/6Tac mice, 56.2% of 4-wk-old C57BL/6NCr female and 62.5% of 4-wk-old C57BL/6Tac female mice, 100% of adult lactating C57BL/6NCr dams, and 100% of inoculated pups (10 d) had seroconverted. Serologically positive nursing dams did not infect their nursing pups. In contrast, when nursing pups were inoculated, 100% of their dams seroconverted by 28 d postinoculation. Only 1 of 4 facility sentinels (Tac:SW female mice) seroconverted to MMVi and none of the 4 research sentinels (Tac:SW female mice) seroconverted under a once-weekly bedding transfer program. Consequently, 4 new research Tac:SW sentinels of each gender (n = 8) were placed in known-positive cages at cage-change; 100% of the male mice but 0% of the females seroconverted by day 48. Study results suggest gender influences both infectivity and the ability to detect subclinical infections of MMVi. Other factors that may influence detection of MMV include mouse strain or stock, short shedding period, and prolonged time between cage changes. In light of the data from both the natural infections and the experimental cases, cessation of breeding likely will be beneficial when trying to eradicate this virus.     

Purification of the Ah receptor from C57BL/6J mouse liver

The photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, previously has been shown to selectively label two peptides in the cytosol fraction of C57BL/6J mouse liver: a 95-kDa peptide, the ligand binding moiety of the Ah receptor, and a 70-kDa proteolytic fragment formed from the larger peptide (Poland, A., Glover, E., Ebetino, F. H., and Kende, A.S. (1986) J. Biol. Chem. 261, 6352-6365). These two peptides were partially purified to an approximately 20,000-fold enrichment with a 15-20% yield by the following scheme: 1) photoaffinity labeling of the 35-55% ammonium sulfate fraction of liver cytosol; 2) chromatography on polyethyleneimine-Sepharose coupled at low charge density and heparin/Mn2+ precipitation of the dilute column eluate; 3) DEAE-Sepharose chromatography to remove heparin; 4) chromatography on heparin-Sepharose; 5) preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by electroelution of the protein and ion pair extraction to remove sodium dodecyl sulfate; and 6) high performance liquid chromatography on a reverse-phase C-4 column. Following initial chromatography on polyethyleneimine Sepharose, it was found that substantial subsequent purification could only be achieved under denaturing conditions.     

Proteins linked to extinction in contextual fear conditioning in the C57BL/6J mouse

Studying fear extinction is a major topic in neuroscience. No information on systematic studies on the linkage of contextual fear conditioning (cFC) with hippocampal protein levels is available and we were therefore interested in protein differences between animals with poor and good extinction. cFC was carried out in C57BL/6J mice, hippocampi were taken and proteins were run on two‐dimensional gel electrophoresis with subsequent quantification of protein spots. In‐gel digestion with trypsin and identification by ion trap MS/MS (high‐capacity ion trap) was used for the identification of significantly different hippocampal proteins between mice with good and poor performance of extinction. Signaling protein ras‐related protein rab‐7A and septin 8 levels were significantly higher in hippocampus of poor extinguishers, whereas ubiquitin carboxyterminal hydrolase isozyme L1 showed higher levels in animals with good extinction performance. A series of additional proteins showed significantly different levels between groups but the abovementioned were confirmed by immunoblotting. The abovementioned proteins have never been reported to be linked to extinction, memory, or learning and herein evidence for the involvement of several proteins in extinction mechanism as well as probably representing pharmaceutical targets is provided. Moreover, it is intriguing to demonstrate the differences between good and poor extinction performance at the protein level.     

1H NMR metabolomics study of metastatic melanoma in C57BL/6J mouse spleen

Melanoma is a malignant tumor of melanocytes. Although extensive investigations have been done to study metabolic changes in primary melanoma in vivo and in vitro, little effort has been devoted to metabolic profiling of metastatic tumors in organs other than lymph nodes. In this work, NMR-based metabolomics combined with multivariate data analysis is used to study metastatic B16-F10 melanoma in C57BL/6J mouse spleen. Principal component analysis, an unsupervised multivariate data analysis method, is used to detect possible outliers, while orthogonal projection to latent structure (OPLS), a supervised multivariate data analysis method, is employed to find important metabolites responsible for discriminating the control and the melanoma groups. Two different strategies, i.e. spectral binning and spectral deconvolution, are used to reduce the original spectral data before statistical analysis. Spectral deconvolution is found to be superior for identifying a set of discriminatory metabolites between the control and the melanoma groups, especially when the sample size is small. OPLS results show that the melanoma group can be well separated from its control group. It is found that taurine, glutamate, aspartate, O-phosphoethanolamine, niacinamide, ATP, lipids and glycerol derivatives are decreased statistically and significantly while alanine, malate, xanthine, histamine, dCTP, GTP, thymidine, 2′-deoxyguanosine are statistically and significantly elevated. These significantly changed metabolites are associated with multiple biological pathways and may be potential biomarkers for metastatic melanoma in spleen.