Characterization and measurement of metallothionein messenger RNA of C57BL mouse liver

Liver poly(A)+RNA of Cd2+-treated C57BL mouse was characterized by cell-free translation, particularly intending to establish a procedure to measure the levels of messenger RNA coding for metallothioneins (MT-mRNA). Intact polysomes were obtained by Mg2+ precipitation from the liver cytoplasm of mice injected with 1 mg Cd2+/kg body wt. Poly(A)+RNA isolated from the polysomes was translated by a wheat germ cell-free system and the [35S]cysteine-labeled translation products were analyzed by sodium dodecyl sulfate (SDS)-15% polyacrylamide gel electrophoresis and fluorography. MTs were identified in the translation products directed by the RNA from the Cd2+-treated mice, but not in the translation products directed by the RNA from untreated mice. Relative incorporation of [35S]cysteine into MTs was determined by densitometrical quantification of the MT bands, and was found to be linear up to a RNA concentration of 150 micrograms/ml in the translation reaction mixture, showing that this system is suitable for the measurement of translatable MT-mRNA levels. Cd2+ stimulated the total levels of cell-free translation (1.4-fold at 20-60 micrograms/ml), not specifically to MT-mRNA. MT-mRNA sedimented at 9S in a sucrose gradient, and its size was comparable with rat and human MT-mRNAs.     

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.     

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.     

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.     

双歧杆菌C57BL小鼠抗辐射能力的研究

目的探讨双歧杆菌对电离辐射损伤的防护作用。方法选用SPF级C57BL小鼠30只,随机分成3组（每组10只）：A组：正常对照组;B组：放射损伤组;C组：双歧处理组,用双歧杆菌菌悬液灌肠28 d后,然后与放射损伤组一同进行^60Coγ射线一次性全身照射,形成急性放射损伤。24 h后按不同要求采集标本,对肝组织分别进行SOD和CAT活力及MDA的含量检测;用电镜观察脊神经组织细胞的超微结构;对脾脏组织用RT-PCR检测TLR2 mRNA水平的表达;并对肾脏组织用免疫组化检测bcl-2、NF-κBp65和Cytochrome C的蛋白表达情况。结果双歧杆菌处理组肝组织中SOD和CAT水平明显高于放射损伤组（P〈0.01）,同时MDA明显低于放射损伤组（P〈0.01）;电镜结果显示：放射损伤组与双歧处理组的细胞浆、细胞核和线粒体均明显肿胀,双歧处理组同时具有早期凋亡现象;RT-PCR结果显示：双歧处理组TLR2 mRNA的表达明显高于放射损伤组;免疫组化结果显示：双歧处理组Cytochrome C的表达明显优于放射损伤组,而NF-κBp65和bcl-2则相反。结论双歧杆菌可通过清除自由基、抗膜脂质氧化、提高免疫机能和诱导细胞凋亡等方式来降低辐射对小鼠的损伤。双歧杆菌具有一定的抗辐射作用。     

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.     

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.     

The influence of genetic background on expression of mutations at the diabetes locus in the mouse. I. C57BL/KsJ and C57BL/6J strains

Two new diabetic strains, C57BL/KsJ-db ^2J and C57BL/6J-db ^2J, have been developed. C57BL/KsJ-db ^2J/db^2J mice are indistinguishable from C57BL/KsJ-db/db mice, the original diabetes mutation. Both have severe diabetes characterized by hyperphagia, obesity, marked hyperglycemia, temporarily elevated plasma insulin concentrations, and typical degenerative changes in the islets of Langerhans. In contrast, C57BL/6J-db ^2J/db^2J mice, although also hyperphagic and obese, have mild diabetes characterized by transitory hyperglycemia and markedly elevated plasma insulin concentrations coupled with marked hypertrophy of the islets and increased proliferative capacity of beta cells. The mild diabetes-like syndrome produced by diabetes-2J on the C57BL/6J background is similar to that produced by the obese gene (ob) on the same background. The islet responses, whether atrophy or hypertrophy, appear to be due to the interaction of diabetes-2J (and possibly obese) with modifiers in the genetic background rather than being peculiar to the specific mutant. The markedly different disease patterns that result when the same gene is placed on different inbred backgrounds emphasize the importance of strict genetic control in biochemical and physiological studies with these and other obesity mutants.Supported in part by NIH Research Grants AM 14461 from the National Institute of Arthritis and Metabolic Diseases; CA 05873 from the National Cancer Institute; ACS E-162, a Janice M. Blood Memorial Grant for Cancer Research from the American Cancer Society; GB 27487 from the National Science Foundation; and an allocation from the Southwaite Foundation.     

Discriminating activation of CYP2B9 expression in male C57BL/6 mouse liver by beta-estradiol

The inducible expression of the cytochrome P450 2B subfamily was investigated in male C57BL/6 (B6) and DBA/2 (D2) mice, as well as their hybrids, B6D2F1, at the mRNA level. The expression of hepatic CYP2B mRNAs in B6 was lightly induced by beta-estradiol (ES), while that by phenobarbital (PB) or 1,1,1-trichloro-2, 2-bis(p-chlorophenyl) ethane (DDT) was prominent. Discriminating analysis showed a novelty that ES markedly induced CYP2B9 mRNA expression, whereas PB and DDT increased CYP2B10 more than CYP2B9 expression: albeit both mRNA species responded to all three inducers. Furthermore, the specific induction by ES of CYP2B9 mRNA in B6 male mice, but not D2 male mice, suggests strain dependency in the regulatory pathway of CYP2B9 expression.     

The adult β-globin genes of the “single” type mouse C57BL

A set of four clones containing the two adult β-globin genes of the “single” type mouse C57BL/10 (genotype Hbb^s/Hbb^s) were isolated from a library of cloned restriction fragments. The two genes, designated βs and βt, were physically mapped onto a 32 kb segment of the chromosome carried by the four clones. βs and βt form a stable heteroduplex 1850 bp long, indicating that they are intact and conserved at this level of resolution throughout their length, including their intervening sequences. The βs gene is allelic with the βdmaj gene of the BALB/ c mouse (genotype Hbb^d/Hbb^d). These two alleles, as well as their surrounding sequences, are highly conserved. In contrast, heteroduplexes of βt with its BALB/c allele, βdmin, revealed three extensive but localized rearrangements. One region of non-homology falls within the large intervening sequence, IVS2. To the 5′ side of the βt/βdmin gene position two unequal substitutions were observed; each results in the net insertion of about 1000 bp into the Hbb^d chromosome. The β/βdmin gene position is bracketed by a 1450 bp inverted repeat. One of the 1000 bp substitutions maps within this inverted repeat.     

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.     

Effects of atherogenic diet consumption on lipoproteins in mouse strains C57BL/6 and C3H

Inbred mouse strains C57BL/6J (B6) (susceptible) and C3H/HeJ (C3H) (resistant) differ in atherosclerosis susceptibility due to a single gene, Ath-1. Plasma lipoproteins from female mice fed chow or an atherogenic diet displayed strain differences in lipoprotein particle sizes and apolipoprotein (apo) composition. High density lipoprotein (HDL) particle sizes were 9.5 +/- 0.1 nm for B6 and 10.2 +/- 0.1 nm for C3H. No major HDL particle size subclasses were observed. Plasma HDL level in the B6 strain was reduced by the atherogenic diet consumption while the HDL level in the resistant C3H mice was unaffected. The reduction in HDL in the B6 strain was associated with decreases in HDL apolipoproteins A-I(-34%) and A-II(-60%). The HDL apoC content in mice fed chow was two-fold higher in C3H than B6. Lipoproteins containing apolipoprotein B (VLDL, IDL, LDL) shifted from a preponderance of the B-100 (chow diet) to a preponderance of the B-48 (atherogenic diet). The LDL-particle size distribution was strain-specific with the chow diet but not genetically associated with the Ath-1 gene. In both strains on each diet, apolipoprotein E was largely distributed in the VLDL, LDL, and HDL fractions. The B6 strain became sixfold elevated in total lipoprotein E content which in the C3H strain was not significantly affected by diet. However, the C3H LDL apoE content was reduced. On both diets, the C3H strain exhibited apolipoprotein E levels comparable to the atherogenic diet-induced levels of the B6 mice.     

Adrenal function and the effect of a high-fat diet on C57BL/6J and C57BL/6J-ob/ob mice.

In C57BL/6J mice and the ob/+ and ob/ob mutants total plasma corticosterone levels were found to be statistically different. In C57BL/6J mice the level was 1.9 +/- 0.2 mug/100 ml plasma, in ob/+ mice 8.6 +/- 1.6 mug/100 ml and in ob/ob mice 13.7 +/- 1.5 mug/100 ml. The percentage of protein-bound corticosterone as well as the free endogenous corticosterone levels were also different. Feeding a high-fat diet to young C57BL/6J and C57BL/6J-ob/ob mice for a period of 4 weeks had no effect upon blood glucose, plasma insulin and plasma corticosterone levels. The significantly higher increase in body weight of the high-fat diet groups of both lines of mice was mainly due to fat cell hypertrophy.     

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.