Organ-specific gene expressions in C57BL/6 mice after exposure to low-dose radiation

The possibility that radiation-induced alterations in gene expression are tissue specific and are related to apoptosis was examined using samples from brain, heart, lung, spleen and intestine from female C57BL6 mice after exposure to 0.2 Gy radiation. Apoptosis was the highest in spleen and intestine, moderate in lung, and absent in brain and heart. However, the mRNA expression of Trp53 and Cdkn1a (p21) after irradiation was not different among the organ types, and immunohistochemistry revealed that all the organs expressed these two proteins after irradiation. When expression patterns of 23 genes in the organs were examined by RT-PCR, neogenine, Apo1, nuclease sensitive element binding protein 1, syntaxin, cyclin G1, hNOP56, paraoxonase and glutathione peroxidase were overexpressed after irradiation in all the organs sampled, suggesting them as universal exposure markers for low-dose radiation. Sialyltransferase may be a candidate for radiation detection in spleen and intestine, which are radiosensitive organs. Because Sod1 (Cu/ZnSOD) and alphaB crystalline were expressed only in spleen, and protein tyrosine kinase and platelet membrane glycoprotein lib were expressed in both spleen and lung, these genes may also be potential markers for detection of radiation exposure, especially low-dose radiation, in these tissues. These data suggested possible tissue-specific markers of low-dose radiation exposure and suggested potential novel genetic modifiers of radiation response.     

Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm

Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%-12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%-88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%-63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.     

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.     

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.     

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.     

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/).     

Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells

Sperm cryopreservation provides an economical means of storing genetically engineered mouse strains in resource facilities. In general, relatively high fertilization rates are obtained for frozen/thawed sperm of the CBA/JN, DBA/2N, and C3H inbred strains and some F1 hybrid strains. However, the fertilization rate for frozen/thawed sperm of C57BL/6, which is the main strain of genetically engineered mice, remains very low. Therefore, it is necessary to establish an in vitro fertilization (IVF) method for cryopreserved C57BL/6 sperm that can obtain a high rate of fertilization after thawing. In the present study, we focused on the effects of methyl-beta-cyclodextrin (MBCD) on the fertilizing ability of frozen/thawed C57BL/6 sperm. Our results have shown that the highest fertilization rate for frozen/thawed sperm was obtained with MBCD at 1.0 mM for 30 min (63.7% +/- 11.0%), but the effects were attenuated by long-term incubation for 120 min at 1.0 or 2.0 mM. The embryos with frozen/thawed sperm showed good developmental potential, and the offspring had normal fertility. The efflux of cholesterol from frozen/thawed sperm was increased by MBCD in a dose-dependent manner and occurred much earlier and to a greater extent than bovine serum albumin. The localization of cholesterol labeled by filipin in the sperm plasma membrane was drastically decreased by MBCD. In summary, we suggest that MBCD is useful for developing an IVF method for frozen/thawed C57BL/6 mouse sperm achieving a high fertilization rate, being involved in the capacity to sequester cholesterol from sperm membrane.     

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.     

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.     

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.     

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.     

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.     

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.     

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.