Mus Spretus Line-1 Sequences Detected in the Mus Musculus Inbred Strain C57bl/6j Using Line-1 DNA Probes

The inbred mouse strain, C57BL/6J, was derived from mice of the Mus musculus complex. C57BL/6J can be crossed in the laboratory with a closely related mouse species, M. spretus to produce fertile offspring; however there has been no previous evidence of gene flow between M. spretus and M. musculus in nature. Analysis of the repetitive sequence LINE-1, using both direct sequence analysis and genomic Southern blot hybridization to species-specific LINE-1 hybridization probes, demonstrates the presence of LINE-1 elements in C57BL/6J that were derived from the species M. spretus. These spretus-like LINE-1 elements in C57BL/6J reveal a cross to M. spretus somewhere in the history of C57BL/6J. It is unclear if the spretus-like LINE-1 elements are still embedded in flanking DNA derived from M. spretus or if they have transposed to new sites. The number of spretus-like elements detected suggests a maximum of 6.5% of the C57BL/6J genome may be derived from M. spretus.     

Genetic Analysis of Audiogenic Seizure Susceptibility in C57bl/6j x Dba/2j Recombinant Inbred Strains of Mice

The inheritance of susceptibility to audiogenic seizures (ASs) was studied in the C57BL/6J (B6) and DBA/2J (D2) progenitor strains, their reciprocal F₁ hybrids, backcross generations and in 21 B6 x D2 recombinant inbred (RI) strains of mice at 21 days of age. All of the D2 mice tested experienced ASs, whereas none of the B6 mice responded to the sound. Although 23% of the F₁ mice experienced wild running, they were generally as resistant to ASs as their B6 parents. Mice of the F₁ x B6 backcross generation were also resistant to ASs. In the F₁ x D2 backcross generation, however, a significant preponderance (72%) of AS-susceptible mice was found. No significant association was observed between any of the four coat-color phenotypes that were segregating in this generation and susceptibility to ASs. A continuous distribution of mean seizure severity scores and several new audiogenic response phenotypes, distinctly different from the phenotypes of either progenitor strain, were found among the 21 RI strains. These and the results from the F₁ x D2 backcross generation suggest that the difference in AS susceptibility between 21-day-old B6 and D2 mice cannot be under the control of a single locus. In addition, no association was found between AS susceptibility and the chromosome 4 markers Lyb-2, Mup-1 and b among the 21 RI strains. An association was observed, however, between AS susceptibility and the Ah locus. Several of the RI strains that were AS resistant at 21 days of age became AS susceptible as adults. One RI strain was susceptible to ASs at both young and adult ages. The B6, D2 and F₁ mice were completely resistant to ASs at adult ages. Genetic differences were found among the RI strains for the incidence, onset, duration, and type of severity of ASs. A remarkable amount of phenotypic variability in the audiogenic response, which can be attributed only to the influence of environmental factors, occurred within several of the RI strains. A multiple-factor mode of inheritance involving a physiological threshold can account for our observations.     

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.     

Genetic Differences among the a/J x C57bl/6j Recombinant Inbred Mouse Lines and Their Degree of Association with Glucocorticoid-Induced Cleft Palate

Hydrocortisone sodium phosphate was injected intramuscularly into A/J, C57BL/6J and recombinant inbred lines from these two parental lines to study the genetics of steroid-induced cleft palate in a situation of identical maternal and fetal genotypes. The strains were typed for H-2 (the major histocompatibility locus), beta-glucuronidase and beta 2-microglobulin, which served as markers on chromosomes 17, 5 and 2, respectively. Hepatic glucocorticoid binding capacity had been previously measured in Hepes buffer and Hepes buffer plus dithiothreitol (DTT). The level of glucocorticoid binding in Hepes buffer and in Hepes plus DTT, as well as their ratio, was compared to the incidence of steroid-induced cleft palate in the recombinant inbred lines. A correlation was found between the response of glucocorticoid binding to DTT (expressed as a ratio of binding in the presence of DTT to binding without DTT) and hydrocortisone-induced cleft palate. When analyzing the effect of the three chromosomal markers on hydrocortisone-induced cleft palate, the b alleles of beta 2-microglobulin and of beta-glucuronidase were associated with a higher incidence. Genetic analyses of the differences between these two inbred strains of mice in the incidence of steroid-induced cleft palate show it not to be monogenic.     

Diazinon-induced ulcerative keratitis in C57bl/6 mice

As a well-known organophosphate insecticide, diazinon (DZN) has been used for several decades in agriculture. The major signs of ophthalmic toxicity of DZN have been reported to be cholinergic overstimulation (lacrimation, myosis). Here, we report, for the first time, ulcerative keratitis in C57bl/6 mice secondary to sub-acute exposure to DZN. Four groups of female C57bl/6 mice were administered intraperitoneally either DZN (1, 5, 25 mg/kg/day) or vehicle for 14 consecutive days. Then, histopathological examinations on mice eyes were performed using light microscope and scored for corneal keratitis. Furthermore, blood cholinesterase activity, and hematologic examinations were performed. Data indicated a significant ulcerative keratitis with prompt vision loss in mice exposed to 25 and 5 mg/kg/day (P < 0.05) doses. These results suggest that diazinon might induce ulcerative keratitis secondary to its immunosuppresive effects at high doses in C57bl/6 mice.     

三个近交系C57BL/6J小鼠群体微卫星遗传变异分析(英文）

应用微卫星遗传标记对近交系C57BL/6J(B6)小鼠遗传稳定性进行分析。用FAM标记的引物PCR扩增了来自北京和上海三个实验动物生产单位提供的三个B6小鼠群体共15个微卫星位点并进行分型。结果显示,所有位点均处于纯合状态,其中7个位点为多态位点。研究表明各B6群体虽然为高度近交群体,但不同生产单位维持的B6群体之间存在遗传分化。     

Variation of mechanical properties along the length of the aorta in C57bl/6 mice

The objective of the present study is to obtain a systematic set of data on the mechanical properties along the entire length of the mouse aorta. The ascending aorta of seven mice was cannulated near the aortic valve, and the aorta was preconditioned with several cyclic changes in pressure. The perfusion pressure was then increased in 30-mmHg increments from 0 to 150 mmHg. Cab-O-Sil, colloidal silica, was mixed into the perfusate to prevent flow through the microvessels and hence attain zero-flow distensions. 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. This uniformity is attained in the range of 60-120 mmHg. At pressures <60 mmHg, the outer strain is greater than the inner strain, whereas at pressures >120 mmHg, the converse is true. Furthermore, we found that the circumferential and longitudinal stress-strain relationships are linear in the pressure range of 30-120 mmHg. Finally, the circumferential modulus is greatest (most rigid) near the diaphragm, and the majority of volume compliance (85%) is in the thoracic compared with the abdominal aorta. These findings are important for an understanding of the hemodynamics of the cardiovascular system of the normal mouse and will serve as a reference state for the study of various diseases in knock-in and knock-out models of this species.     

Genetic Difference of Hypothyroidism-Induced Cognitive Dysfunction in C57BL/6j and 129/Sv Mice

Adult-onset hypothyroidism induces cognitive impairments in learning and memory. Thyroxin (T4) replacement therapy appears to be effective in biochemically restoring euthyroidism, as evidenced by serum T4 and triiodothyronine concentrations within the normal range, although some the patients still exhibit cognitive dysfunctions. Here, we investigated the cognitive functions of propylthiouracil-induced hypothyroid mice in C57BL/6j and 129/Sv strains using the passive avoidance task and the novel object recognition test. Cognitive dysfunctions in hypothyroid mice were found only in the C57BL/6j strain, not in the 129/Sv strain. Further, we found that cholinergic neurons in the basal forebrain increased the membrane potential and input resistance with decreased capacitance, and that they decreased the amplitude and width of action potential in hypothyroid mice in the C57BL/6j strain but not in those in the 129/Sv strain, compared with the controls for each strain. Additionally, the excitability of cholinergic neurons in the basal forebrain was reduced in the hypothyroid mice in the C57BL/6j strain. These results indicated that transgenic mice with the C57BL/6j genetic background are more suitable for revealing the mechanism underlying hypothyroidism-induced cognitive dysfunction, and that the cholinergic basal forebrain may be the appropriate target for treating cognitive dysfunction in adult-onset hypothyroidism.

     

Generation of Live Offspring from Vitrified Mouse Oocytes of C57BL/6J Strain

In mammals, unfertilized oocytes are one of the most available stages for cryopreservation because the cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has generally been reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. C57BL/6J mice, an inbred strain, are used extensively for the production of transgenic and knockout mice. If the oocytes from C57BL/6J mice can be successfully cryopreserved, the cryopreservation protocol used will contribute to the high-speed production of not only gene-modified mice but also hybrid mice. Very recently, we succeeded in the vitrification of mouse oocytes derived from ICR (outbred) mice. However, our protocol can be applied to the vitrification of oocytes from an inbred strain. The aim of the present study was to establish the vitrification of oocytes from C57BL/6J mice. First, the effect of cumulus cells on the ability of C57BL/6J mouse oocytes to fertilize and develop in vitro was examined. The fertility and developmental ability of oocyte-removed cumulus cells (i.e., denuded oocytes, or DOs) after IVF were reduced compared to cumulus oocyte complexes (COCs) in both fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop into the 2-cell and blastocyst stages compared to the vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate, equivalent to the rate obtained with IVF using fresh COCs. Taken together, our results demonstrate that we succeeded for the first time in the vitrification of mouse oocytes from C57BL/6J mice. Our findings will also contribute to the improvement of oocyte vitrification not only in animals but also in clinical applications for human infertility.     

Structural and Mechanical Improvements to Bone Are Strain Dependent with Axial Compression of the Tibia in Female C57BL/6 Mice

Strain-induced adaption of bone has been well-studied in an axial loading model of the mouse tibia. However, most outcomes of these studies are restricted to changes in bone architecture and do not explore the mechanical implications of those changes. Herein, we studied both the mechanical and morphological adaptions of bone to three strain levels using a targeted tibial loading mouse model. We hypothesized that loading would increase bone architecture and improve cortical mechanical properties in a dose-dependent fashion. The right tibiae of female C57BL/6 mice (8 week old) were compressively loaded for 2 weeks to a maximum compressive force of 8.8N, 10.6N, or 12.4N (generating periosteal strains on the anteromedial region of the mid-diaphysis of 1700 με, 2050 με, or 2400 με as determined by a strain calibration), while the left limb served as an non-loaded control. Following loading, ex vivo analyses of bone architecture and cortical mechanical integrity were assessed by micro-computed tomography and 4-point bending. Results indicated that loading improved bone architecture in a dose-dependent manner and improved mechanical outcomes at 2050 με. Loading to 2050 με resulted in a strong and compelling formation response in both cortical and cancellous regions. In addition, both structural and tissue level strength and energy dissipation were positively impacted in the diaphysis. Loading to the highest strain level also resulted in rapid and robust formation of bone in both cortical and cancellous regions. However, these improvements came at the cost of a woven bone response in half of the animals. Loading to the lowest strain level had little effect on bone architecture and failed to impact structural- or tissue-level mechanical properties. Potential systemic effects were identified for trabecular bone volume fraction, and in the pre-yield region of the force-displacement and stress-strain curves. Future studies will focus on a moderate load level which was largely beneficial in terms of cortical/cancellous structure and cortical mechanical function.     

In situ analysis of centromere segregation in C57BL/6 x Mus spretus interspecific backcrosses

The analysis of major satellite sequence differences between Mus spretus and laboratory mice provides a robust method for analyzing the centromere location for the genetic maps of each mouse chromosome. Fluorescence in situ hybridization (FISH) of a genomic probe, pMR196, for the laboratory mouse major satellite sequences was used to identify C57BL/6Ros (B6) pericentromeric heterochromatin in progeny of reciprocal backcross matings. These included 80 (B6xM. spretus)F₁xM. spretus progeny (BSS) and 70 (B6xM. spretus)F₁xB6 (BSB) progeny. FISH analysis of pericentromeric heterochromatin was conducted on the same metaphase spreads that were karyotypically analyzed for chromosomespecific banding patterns. Analysis of chromosomal segregation suggested that there was not primary deviation from random assortment during meiosis in the interspecific hybrid female, because nearly all of the 190 pair-wise comparisons did not deviate from expected and because there was no consistent pattern of deviation of the same chromosomes in the reciprocal backcross progeny from similar (C57BL/6xM. spretus)F₁ hybrid females. These results affirm the value of using the major satellite to genetically mark pericentromeric heterochromatin in the analysis of the segregation and assortment of centromeres in Mus interspecific crosses.     

Cell-mediated immune response to high-passage Borrelia spirochetes in C57bl/6 mice is strictly dependent on antigen specificity

Inbred C57bl/6 mice were challenged with high-passage Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen specific T-cell response in vitro. Sonicated preparations of washed spirochetes were potent cell activators, capable of stimulating polyclonal proliferation after 72h of culture while increasing the incubation time up to 120h provoked specific cell-mediated response. Isolated murine spleocytes previously sensitized to B. burgdorferi sensu lato but not those from control mice could be induced for antigen-specific proliferation in vitro, as revealed by [3H]thymidine incorporation assay, Moreover, in mice presensitized to B. burgdorferi sensu lato, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies. The current study emphasises that the B. burgdorferi antigen-specific response may also be expected in different genospecies infections in men.     

DNA Sequence Analysis of Sry Alleles (Subgenus Mus) Implicates Misregulation as the Cause of C57bl/6j-Y(pos) Sex Reversal and Defines the Sry Functional Unit

The Sry (sex determining region, Y chromosome) open reading frame from mice representing four species of the genus Mus was sequenced in an effort to understand the conditional dysfunction of some M. domesticus Sry alleles when present on the C57BL/6J inbred strain genetic background and to delimit the functionally important protein regions. Twenty-two Sry alleles were sequenced, most from wild-derived Y chromosomes, including 11 M. domesticus alleles, seven M. musculus alleles and two alleles each from the related species M. spicilegus and M. spretus. We found that the HMG domain (high mobility group DNA binding domain) and the unique regions are well conserved, while the glutamine repeat cluster (GRC) region is quite variable. No correlation was found between the predicted protein isoforms and the ability of a Sry allele to allow differentiation of ovarian tissue when on the C57BL/6J genetic background, strongly suggesting that the cause of this sex reversal is not the Sry protein itself, but rather the regulation of SRY expression. Furthermore, our interspecies sequence analysis provides compelling evidence that the M. musculus and M. domesticus SRY functional domain is contained in the first 143 amino acids, which includes the HMG domain and adjacent unique region (UR-2).     

Strain differences in response to acute hypoxia: CD-1 versus C57BL/6J mice.

Some mammals respond to hypoxia by lowering metabolic demand for oxygen and others by maximizing efficiency of oxygen usage: the former strategy is generally held to be the more effective. We describe within the same species one outbred strain (CD-1) that lowers demand and another inbred strain (C57BL/6J) that maximizes oxygen efficiency to markedly extend hypoxic tolerance. Unanesthetized adult male mice (Mus musculus, CD-1 and C57BL/6J) between 20 and 35 g were used. Sham-conditioned (SC) C57BL/6J mice survived severe hypoxia (4.5% O(2), balance N(2)) roughly twice as long as SC CD-1 mice (median 211 and 93.5 s, respectively; P < 0.0001). Following acute hypoxic conditioning (HC), C57BL/6J mice survived subsequent hypoxia 10 times longer than HC CD-1 mice (median 2,198 and 238 s respectively; P < 0.0001). Therefore, C57BL/6J mice are both naturally more tolerant to hypoxia and show a greater increase in hypoxic tolerance in response to hypoxic conditioning. Indirect calorimetry indicates that CD-1 mice lower mass-specific oxygen consumption (V'o(2) in ml O(2).kg(-1).min(-1)) and carbon dioxide production (V'co(2) in ml CO(2).kg(-1).min(-1)) in response to HC (P = 0.002 and P < 0.0001, respectively), but C57BL/6J mice maintain V'o(2) and V'co(2) after HC. Respiratory exchange ratio and fluorometric assay of plasma ketones suggest that C57BL/6J mice rapidly switch to ketone metabolism, a more efficient substrate, while CD-1 mice reduce overall metabolic activity. We conclude that under severe hypoxia in mice, switching fuel, possibly to ketones, while maintaining V'o(2), may confer a greater survival advantage than simply lowering demand.