A locus for circadian period of locomotor activity on mouse proximal chromosome 3

Lengthened circadian period of locomotor activity is a characteristic of a congenic strain of mice carrying a nonsense mutation in exon 5 of the carbonic anhydrase II gene, car2. The null mutation in car2 is located on a DBA/2J inbred strain insert on proximal chromosome 3, on an otherwise C57BL/6J genomic background. Since reducing the size of the congenic region would narrow the possible candidate genes for period, two recombinant congenic strains (R1 and R2) were developed from the original congenic strain. These new congenic strains were assessed for period, genetic composition, and the presence of immunoreactive carbonic anhydrase II. R1 mice were homozygous DBA/2J for the distal portion of the original DBA/2J insert, while R2 mice were homozygous DBA/2J for the proximal portion. R1 mice had a significantly lengthened period compared to R2 mice and wild-type C57BL/6J mice, indicating that the gene(s) affecting period is likely found within the reduced DBA/2J insert (~1 cM) in the R1 mice. The R1 mice also possessed the null mutation in car2. This study confirmed the presence of a gene(s) affecting period on proximal chromosome 3 and significantly reduced the size of the congenic region and the number of candidate genes. Future studies will focus on identifying the gene influencing period.     

Intragastric self-infusion of ethanol in high- and low-drinking mouse genotypes after passive ethanol exposure

Two experiments examined the effect of 5 days of passive exposure to ethanol (or water) on later self-infusion of ethanol or water via surgically implanted intragastric (IG) catheters in mouse genotypes previously shown to drink high (C57BL/6J, HAP2) or low (DBA/2J, LAP2) amounts of ethanol in home-cage continuous-access two-bottle choice procedures. Intragastric ethanol self-infusion was affected by both genotype and a history of passive ethanol exposure, with greater intakes in the high-drinking genotypes and in groups that received passive exposure to ethanol. Passive ethanol exposure also increased preference for the flavor that signaled ethanol infusion (S+), eliminating genetic differences in this measure. The increases in ethanol intake and S+ preference induced by ethanol exposure might have been mediated jointly by development of tolerance to aversive post-absorptive ethanol effects and negative reinforcement because of alleviation of withdrawal. Bout analyses indicated that ethanol exposure increased ethanol self-infusion by increasing the total number of daily bouts rather than by increasing bout size. These analyses also showed that DBA/2J mice infused larger ethanol bouts and a greater percentage of their total intakes in large bouts than C57BL/6J mice. Overall, these studies suggest that the IG self-infusion procedure is a potentially useful new tool for studying genetic and environmental influences on excessive ethanol intake and preference in mice.     

Carcinogenicity test in B6C3F1 mice after parental and prenatal exposure to 50 Hz magnetic fields

Some epidemiological studies suggest association of childhood cancer with occupational exposure of the parents to magnetic fields. To test this relationship, 50 each of C57BL/6J female and C3H/HeJ male mice were exposed for 2 and 9 weeks, respectively, to 50 Hz sham (group A), 0.5 (group B), and 5 mT (group C) sinusoidal alternating magnetic fields. They were mated under the exposure for up to 2 weeks, and the exposure was continued until parturition. All the B6C3F1 offspring, without adjusting numbers of animals, were clinically observed without exposure to magnetic field for a nominal 78 weeks from 6-8 weeks of age after weaning and then euthanized for pathological examination according to a routine carcinogenicity test. 540 pups entered the test, and the survival rate was 96.7%. No F1 mouse died of tumoral diseases before a male in A group died of stomach cancer at 43 weeks of age. The first animal death in the exposed groups due to tumor occurred at 71 weeks of age. Eighteen animals died before necropsy at 84-86 weeks of age. No significant difference was detected in the final number of survivors and incidence of tumors between groups A and B, or A and C. Concerning reproduction total implants in group B were less than in group A and the difference was on the borderline of significance (P=.05). This difference was not reproduced in a later duplicate experiment.     

Establishment of a new embryonic stem cell line derived from C57BL/6 mouse expressing EGFP ubiquitously

Transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP) are useful as marker lines in chimera experiments. We established a new embryonic stem (ES) cell line (named B6G-2) from a C57BL/6 blastocyst showing ubiquitous EGFP expression. Undifferentiated B6G-2 cells showed strong green fluorescence and mRNAs of pluripotent marker genes. B6G-2 cells were transferred into a C57BL/6 blastocyst to generate a germline chimera, the progeny of which inherited ubiquitous EGFP expression. Mice derived completely from B6G-2 cells were also developed from the ES cells; these were tetraploid chimeras. The established B6G-2 cells were shown to be pluripotent and to be capable of differentiating into cells of all lineages. Thus, the new ES cell line expressing EGFP ubiquitously is useful for basic research in the field of regenerative medicine. The B6G-2 cell line is freely available from the BioResource Center, RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

MiR-34a通过抑制Pgc-1β和线粒体生成促进小鼠骨骼肌脂肪异位沉积

过氧化物酶体增殖物激活受体γ辅激活因子-1β（peroxisome proliferative activated receptor γ coactivator 1 β,Pgc-1β）与线粒体生成相关。已有研究证明,miR-34a在肝组织脂肪异位沉积中发挥重要作用,但是否与骨骼肌的脂肪异位沉积相关尚不清楚。本研究以C57Bl/6J小鼠为研究对象,通过尾静脉注射miR-34a模拟物,探讨miR-34a过表达对小鼠骨骼肌脂肪沉积的影响。组织切片进行油红O染色及甘油三酯含量测定揭示,miR-34a过表达的小鼠骨骼肌组织中脂滴积累及甘油三酯含量显著增加。实时荧光定量PCR（qRT-PCR）显示,与对照鼠比较,miR-34a处理的小鼠骨骼肌组织中的脂肪酸合成酶(Fas）表达显著上调,而脂肪酸氧化分解相关基因产物肉毒碱棕榈酰基转移酶1α（Cpt 1α）表达显著下调,提示miR-34a调控骨骼肌内脂肪的沉积机制可能是通过促进脂肪酸生成和抑制脂肪酸分解实现的。qRT-PCR和Western印迹证明,miR-34a可抑制Pgc-1β蛋白的表达。CoxⅡ/28S比例（线粒体定量指标）测定提示,注射miR-34a模拟物导致小鼠骨骼肌线粒体数目显著下调。生物信息分析显示,Pgc-1β mRNA的3′-UTR存在 miR-34a的潜在识别位点,因此miR-34a可能通过靶向识别Pgc-1β的3′-UTR抑制Pgc-1β表达,从而抑制线粒体生成。上述结果证明,miR-34a能通过靶向抑制PGC-1β表达,抑制线粒体生成,继而减少脂肪酸氧化分解,导致骨骼肌脂肪沉积增加。此外,上调脂肪酸合成酶也可能是miR-34a导致骨骼肌脂肪沉积增加的另一原因,其作用机制需进一步研究。     

Utility of a C57BL/6 ES line versus 129 ES lines for Targeted Mutations in Mice

Inbred ES lines, though useful for generating targeted mutations in mice, are used infrequently. To appreciate the relative efficiency of inbred ES lines, a C57BL/6 ES line was compared with 129 strain ES lines for effectiveness in chimera formation leading to the establishment of targeted mutations in mice. Data from a transgenic facility spanning 7 years were collected. C57BL/6 ES cells injected into Balb/c embryos results in lower coat color chimerism than do 129 ES cells injected into C57BL/6 embryos. Combined data indicate that five independent targeted C57BL/6 clones should be injected as compared to three independent 129 clones to generate enough chimeras to effectively test for germ-line transmission. Thus, although less efficient than 129 ES lines, the C57BL/6 ES line is a relatively competent line and useful for the routine generation of targeted mutations in mice on a defined genetic background.     

Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media

C57BL/6 is a well-characterized mouse strain that is used extensively for immunological and neurological research. The establishment of C57BL/6 ES cell lines has facilitated the study of gene-altered mice in a pure genetic background-however, relatively few such lines exist. Using a defined media supplement, knockout serum replacement (KSR) with knockout DMEM (KSR-KDMEM), we find that we can readily establish ES cell lines from blastocysts of C57BL/6J mice. Six lines were established, all of which were karyotypically normal and could be maintained in the undifferentiated state on mouse embryonic fibroblast (MEF) feeders. One line was further tested and found to be karyotypically stable and germline competent, both prior to manipulation and after gene targeting. For this cell line, efficiencies of cell cloning and chimera generation were greater when maintained in KSR-KDMEM. Our work suggests that the use of defined serum-free media may facilitate the generation of ES cells from inbred mouse strains.     

Effects of pre-germinated brown rice treatment high-fat diet-induced metabolic syndrome in C57BL/6J mice

To investigate using pre-germinated brown rice (PGBR) to treat metabolic syndrome, we fed one group of mice standard-regular-diet (SRD) for 20 weeks and another group of mice high-fat-diet (HFD) for 16 weeks. We subdivided them into HFD group and HFD + PGBR group whose dietary carbohydrate was replaced with PGBR for 4 weeks. The HFD group gained more weight, had higher blood pressure, heart rate, blood glucose and lipids, liver levels of TG, feces TG and bile acid, lower adipose levels of adipocytokine, lower skeletal muscle IR, IRS-1, IRS-2, PI3 K, Akt/PKB, GLUT-1, GLUT-4, GCK and PPAR-γ; higher liver SREBP-1, SCD-1, FAS, HMGCR, LDLR, CYP7α1 and PPAR-α, and higher adipose SREBP-1, SCD-1, FAS, and lower adipose PPAR-α and adiponectin. The HFD + PGBR group had clearly improved blood pressure, biochemical parameters and above proteins expressions. PGBR successful treatment of metabolic syndrome was achieved through improvements in glucose and lipid synthesis and metabolism.     

Presynaptic and postsynaptic organelles of synapses formed in cultures of previously dissociated mouse spinal cord

Summary This study describes some of the ultrastructural features of presynaptic and postsynaptic organelles at synapses developed in cultures of previously dissociated mouse spinal cord cells. Particular attention was paid to the agranular reticulum which is well developed at many presynaptic and postsynaptic sites, either in the form of simple tubules or cisternae, or more complex networks and often closely associated with mitochondria. In addition, the disposition of microtubules at and close to synaptic specializations is described. These and other features of synaptic zones, such as granular vesicles in presynaptic sites, are discussed in relation to cultures developed on feeder layers and synapses in vivo, and in relations to possible degenerative and regenerative events in the cell cultures.     

Differential expression of a subunit isoforms of the vacuolar-type proton pump ATPase in mouse endocrine tissues

Vacuolar-type proton ATPase (V-ATPase) is a multi-subunit enzyme that couples ATP hydrolysis to the translocation of protons across membranes. Mammalian cells express four isoforms of the a subunit of V-ATPase. Previously, we have shown that V-ATPase with the a3 isoform is highly expressed in pancreatic islets and is located in the membranes of insulin-containing granules in the β cells. The a3 isoform functions in the regulation of hormone secretion. In this study, we have examined the distribution of a subunit isoforms in endocrine tissues, including the adrenal, parathyroid, thyroid, and pituitary glands, with isoform-specific antibodies. We have found that the a3 isoform is strongly expressed in all these endocrine tissues. Our results suggest that functions of the a3 isoform are commonly involved in the process of exocytosis in regulated secretion. This research was supported in part by Grants-in-Aid from the Ministry of Education, Science, and Culture of Japan and by the Hayashi, Takeda, and Noda Foundations.     

Acute locomotor responses to cocaine in adolescents vs. adults from four divergent inbred mouse strains

Growing evidence suggests that adolescent mice display differential sensitivity to the acute locomotor activating effects of cocaine as compared to adults, but the direction of the difference varies across studies and the reasons are not clear. Few studies have directly examined genetic contributions to age differences in locomotor stimulation from cocaine. The goal of this study was to determine the extent to which reduced stimulation in C57BL/6J adolescents as compared to adults generalizes to other strains. Therefore, we examined male and female mice from four genetically divergent inbred stains (BALB/cByJ, C57BL/6J, DBA/2J and FVB/NJ) at two ages, postnatal day 30 and postnatal day 65. Mice received either saline or cocaine (15 or 30 mg/kg), and then immediately were placed back into their home cages. Locomotor activity was recorded continuously in the home cage by video tracking. Adolescents displayed reduced stimulation as compared to adults for C57BL/6J, BALB/cByJ and female FVB/NJ mice. No age differences were observed for DBA/2J or male FVB/NJ. No main effects of sex were observed. Strain differences in pharmacokinetics, neural development or physiology could contribute to the observed differences between ages across strains. Future comparative studies could discover biological differences between strains that explain age differences in cocaine sensitivity.     

Comparative gene expression analysis of the engulfment and cell motility (ELMO) protein family in the mouse brain

Engulfment and cell motility (ELMO) proteins bind to Dock180, a guanine nucleotide exchange factor (GEF) of the Rac family, and regulate GEF activity. The resultant ELMO/Dock180/Rac module regulates cytoskeletal reorganization responsible for the engulfment of apoptotic cells, cell migration, and neurite extension. The expression and function of Elmo family proteins in the nervous system, however, are not yet fully understood. Here, we characterize the comparative gene expression profiles of three Elmo family members (Elmo1, Elmo2, and Elmo3) in the brain of C57BL/6J mice, a widely used inbred strain, together with reeler mutant mice to understand gene expression in normal laminated brain areas compared with abnormal areas. Although all three Elmo genes showed widespread mRNA expression over various mouse tissues tested, Elmo1 and Elmo2 were the major types expressed in the brain, and three Elmo genes were up-regulated between the first postnatal week (infant stage) and the third postnatal week (juvenile, weaning stage). In addition, the mRNAs of Elmo genes showed distinct distribution patterns in various brain areas and cell-types; such as neurons including inhibitory interneurons as well as some non-neuronal cells. In the cerebral cortex, the three Elmo genes were widely expressed over many cortical regions, but the predominant areas of Elmo1 and Elmo2 expression tended to be distributed unevenly in the deep (a lower part of the VI) and superficial (II/III) layers, respectively, which also changed depending on the cortical areas and postnatal stages. In the dentate gyrus of the hippocampus, Elmo2 was expressed in dentate granule cells more in the mature stage rather than the immature-differentiating stage. In the thalamus, Elmo1 but not the other members was highly expressed in many nuclei. In the medial habenula, Elmo2 and Elmo3 were expressed at intermediate levels. In the cerebellar cortex, Elmo1 and Elmo2 were expressed in differentiating-mature granule cells and mature granule cells, respectively. In the Purkinje cell layer, Elmo1 and Elmo2 were expressed in Purkinje cells and Bergmann glia, respectively. Disturbed cellular distributions and laminar structures caused by the reeler mutation did not severely change expression in these cell types despite the disturbed cellular distributions and laminar structures, including those of the cerebrum, hippocampus, and cerebellum. Taken together, these results suggested that these three Elmo family members share their functional roles in various brain regions during prenatal-postnatal development.     

Hyperalgesic effect of emotional stress in mice of strains C57BL/6J and CBA/CaLac: Interaction with circadian rhythm-related effects

We studied emotional stress-induced modulations of the pain reaction evoked in mice of strains C57BL/6J and CBA/CaLac by subcutaneous injections of formalin; the measurements were performed at midtimes of a “dark” and a “light” phase of the pre-set fixed circadian rhythm. The magnitude of the pain reaction was estimated indirectly, according to characteristics of locomotion of the animal in a running wheel (the velocity of locomotion and the distance covered were considered values inversely correlating with the intensity of the pain response). We found that the intensity of the pain reaction within both phases of the circadian rhythm increased under the influence of stress, and that there were significant differences between the emotional stress-modulated intensities of the pain response observed in the examined genetic strains of mice. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 466–471, September–December, 2006.