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Sample records for mouse metallothionein gene

  1. Expression of mouse metallothionein genes in tobacco

    SciTech Connect

    Maiti, I.B.; Yeargan, R.; Wagner, G.J.; Hunt, A.G. )

    1990-05-01

    We have expressed a mouse metallothionein (NT) gene in tobacco under control of the cauliflower mosaic virus (CaMV) 35S promoter and a pea ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) gene promoter. Seedlings in which MT gene expression is driven by the 35S promoter are resistant to toxic levels of cadmium. Mature plants carrying the 35S-MT gene accumulate less Cd in their leaves when exposed to low levels of Cd in laboratory growth conditions. Plants with the rbcS-MT construction express this gene in a light-regulated and tissue-specific manner, as expected. Moreover, the MT levels in leaves in these plants are about 20% of those seen in 35S-MT plants. These plants are currently being tested for Cd resistance. In addition, a small field evaluation of 35S-MT lines for Cd levels is being evaluated. These experiments will address the possibility of using MTs to alter Cd levels in crop species.

  2. Inheritance and expression of the mouse metallothionein gene in tobacco

    SciTech Connect

    Maiti, I.B.; Wagner, G.J.; Yeargan, R.; Hunt, A.G. )

    1989-11-01

    Genetically engineered seedlings obtained from self-fertilized transgenic tobacco (Nicotiana tabacum) contained and expressed the mouse metallothionein and kanamycin resistance marker genes and were more tolerant to cadmium stress than untransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration (0.02 micromolar) was about 20% lower than that in untransformed controls. Genetic analysis of R1 and R2 progeny showed inheritance of the marker gene to be as a dominant Mendelian trait. These results suggest the possibility of developing transgenic plants with modified tolerance to heavy metal stress and food crops having lower Cd content.

  3. Up-regulation of metallothionein gene expression in parkinsonian astrocytes.

    PubMed

    Michael, Gregory J; Esmailzadeh, Sharmin; Moran, Linda B; Christian, Lynne; Pearce, Ronald K B; Graeber, Manuel B

    2011-11-01

    The role of glial cells in Parkinson's disease (PD) is unclear. We have previously reported a striking up-regulation of DnaJB6 heat shock protein in PD substantia nigra astrocytes. Whole genome transcriptome analysis also indicated increased expression of metallothionein genes in substantia nigra and cortex of sporadic PD cases. Metallothioneins are metal-binding proteins in the CNS that are released by astrocytes and associated with neuroprotection. Metallothionein expression was investigated in 18 PD cases and 15 non-PD controls using quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridisation (ISH) and immunocytochemistry (ICC). We observed a strong increase in the expression of metallothioneins MT1E, MT1F, MT1G, MT1H, MT1M, MT1X and MT2A in both PD nigra and frontal cortex. Expression of LRP2 (megalin), the neuronal metallothionein receptor was also significantly increased. qRT-PCR confirmed metallothionein up-regulation. Astrocytes were found to be the main source of metallothioneins 1 and 2 based on ISH results, and this finding was confirmed by ICC. Our findings demonstrate metallothionein expression by reactive astrocytes in PD nigra and support a neuroprotective role for these cells. The traditional view that nigral astrocytes are non-reactive in PD is clearly incorrect. However, it is possible that astrocytes are themselves affected by the disease process which may explain their comparatively modest and previously overlooked response. PMID:21800131

  4. Molecular evolution of Drosophila metallothionein genes.

    PubMed

    Lange, B W; Langley, C H; Stephan, W

    1990-12-01

    The metallothionein genes of Drosophila melanogaster, Mtn and Mto, may play an important role in heavy metal detoxification. Several different tandem duplications of Mtn have been shown to increase cadmium and copper tolerance, as well as Mtn expression. In order to investigate the possibility of increased selection for duplications of these genes in natural populations exposed to high levels of heavy metals, we compared the frequencies of such duplications among flies collected from metal-contaminated and non-contaminated orchards in Pennsylvania, Tennessee and Georgia. Restriction enzyme analysis was used to screen 1666 wild third chromosomes for Mtn duplications and a subset (327) of these lines for Mto duplications. The frequency of pooled Mtn duplications found ranged from 0% to 20%, and was not significantly higher at the contaminated sites. No Mto duplications were identified. Estimates of sequence diversity at the Mtn locus among a subsample (92) of the duplication survey were obtained using four-cutter analysis. This analysis revealed a low level of polymorphism, consistent with both selection at the Mtn locus, and a fairly recent origin for the duplications. To further examine this hypothesis, we sequenced an Mtn allele of Drosophila simulans and measured the amount of nucleotide sequence divergence between D. simulans and its sibling species D. melanogaster. The levels of silent nucleotide polymorphism and divergence in the Mtn region were compared with those in the Adh region, using the neutrality test of R.R. Hudson, M. Kreitman and M. Aguad. PMID:1981765

  5. Molecular Evolution of Drosophila Metallothionein Genes

    PubMed Central

    Lange, B. W.; Langley, C. H.; Stephan, W.

    1990-01-01

    The metallothionein genes of Drosophila melanogaster, Mtn and Mto, may play an important role in heavy metal detoxification. Several different tandem duplications of Mtn have been shown to increase cadmium and copper tolerance, as well as Mtn expression. In order to investigate the possibility of increased selection for duplications of these genes in natural populations exposed to high levels of heavy metals, we compared the frequencies of such duplications among flies collected from metal-contaminated and non-contaminated orchards in Pennsylvania, Tennessee and Georgia. Restriction enzyme analysis was used to screen 1666 wild third chromosomes for Mtn duplications and a subset (327) of these lines for Mto duplications. The frequency of pooled Mtn duplications found ranged from 0% to 20%, and was not significantly higher at the contaminated sites. No Mto duplications were identified. Estimates of sequence diversity at the Mtn locus among a subsample (92) of the duplication survey were obtained using four-cutter analysis. This analysis revealed a low level of polymorphism, consistent with both selection at the Mtn locus, and a fairly recent origin for the duplications. To further examine this hypothesis, we sequenced an Mtn allele of Drosophila simulans and measured the amount of nucleotide sequence divergence between D. simulans and its sibling species D. melanogaster. The levels of silent nucleotide polymorphism and divergence in the Mtn region were compared with those in the Adh region, using the neutrality test of R. R. Hudson, M. Kreitman and M. Aguade. PMID:1981765

  6. Metallothionein gene expression in renal cell carcinoma

    PubMed Central

    Pal, Deeksha; Sharma, Ujjawal; Singh, Shrawan Kumar; Mandal, Arup Kumar; Prasad, Rajendra

    2014-01-01

    Introduction: Metallothioneins (MTs) are a group of low-molecular weight, cysteine-rich proteins. In general, MT is known to modulate three fundamental processes: (1) the release of gaseous mediators such as hydroxyl radical or nitric oxide, (2) apoptosis and (3) the binding and exchange of heavy metals such as zinc, cadmium or copper. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers. Most of the previous studies primarily used immunohistochemistry to analyze localization of MT in renal cell carcinoma (RCC). No information is available on the gene expression of MT2A isoform in different types and grades of RCC. Materials and Methods: In the present study, total RNA was isolated from 38 histopathologically confirmed cases of RCC of different types and grades. Corresponding adjacent normal renal parenchyma was taken as control. Real-time polymerase chain reaction (RT PCR) analysis was done for the MT2A gene expression using ?-actin as an internal control. All statistical calculations were performed using SPSS software. Results: The MT2A gene expression was found to be significantly increased (P < 0.01) in clear cell RCC in comparison with the adjacent normal renal parenchyma. The expression of MT2A was two to three-fold higher in sarcomatoid RCC, whereas there was no change in papillary and collecting duct RCC. MT2A gene expression was significantly higher in lower grade (grades I and II, P < 0.05), while no change was observed in high-grade tumor (grade III and IV) in comparison to adjacent normal renal tissue. Conclusion: The first report of the expression of MT2A in different types and grades of RCC and also these data further support the role of MT2A in tumorigenesis. PMID:25097305

  7. Tilapia metallothionein genes: PCR-cloning and gene expression studies.

    PubMed

    Cheung; Pok Lap, Andrew; Kwok Lim Lam, Vincent; Chan, King Ming

    2005-12-20

    Genomic PCR reactions were performed to isolate gene sequences of tilapia metallothionein (tiMT) from Oreochromis mossambicus and Oreochromis aureus. Two AP1 binding sites, four metal responsive elements, and a TATA box are the major cis-acting elements identified in the 800-bp 5' flanking region of the tiMTs obtained in this study. The tiMT gene promoter cloned from O. aureus was characterized in vitro using PLHC-1 cell-line, a hepatocellular carcinoma of a desert topminnow (Poecciliopsis lucida), following the administrations of Cd2+, Co2+, Cu2+, Ni2+, Pb2+ and Zn2+. Only Cd2+, Pb2+ and Zn2+ were able to induce the transcription of tiMT gene promoter in PLHC-1 cells in a dose-dependent manner. Zn2+ had the highest fold induction of tiMT gene promoter activity. Deletion mutants were tested for their abilities to drive the transcription of reporter gene following Cd2+ and Zn2+ administrations. However, Cu2+ and Ni2+ also induced the production of hepatic MT mRNA in vivo. Northern blot analysis showed that liver gave the highest fold induction of MT gene expression following the administration of heavy metal ions. These data indicated that hepatic MT mRNA level in tilapia is a potential sensitive biomarker of exposure to various metal ions including Cu2+, Cd2+, Ni2+, Pb2+, Hg2+ and Zn2+ ions. PMID:16309756

  8. Isolation and characterization of Mytilus edulis metallothionein genes.

    PubMed

    Leignel, Vincent; Laulier, Marc

    2006-01-01

    Metallothioneins (MTs) are crucial proteins in all organisms for the regulation of essential metals and the detoxification of heavy metals. Many studies have estimated MT levels in mussel tissues to detect marine metal pollution. In this study, we investigated the MT gene structures of the forms present in Mytilus edulis (blue mussel). One MT-10 (2413 bp) gene and one MT-20 (1906 bp) gene were obtained. These MT genes contain three exons and two long introns. The splicing signals for MT-10 and MT-20 were GTA(T/A)GT-(C/T)AG. The structural organization (length of intron, splicing signals, AT content) of MT-10 and MT-20 is compared with other MT genes. PMID:16326142

  9. Metallothionein gene activation in the earthworm (Lumbricus rubellus)

    PubMed Central

    Hckner, M.; Dallinger, R.; Strzenbaum, S.R.

    2015-01-01

    In order to cope with changing environmental conditions, organisms require highly responsive stress mechanisms. Heavy metal stress is handled by metallothioneins (MTs), the regulation of which is evolutionary conserved in insects and vertebrates and involves the binding of metal transcription factor 1 (MTF-1) to metal responsive elements (MREs) positioned in the promoter of MT genes. However, in most invertebrate phyla, the transcriptional activation of MTs is different and the exact mechanism is still unknown. Interestingly, although MREs are typically present also in invertebrate MT gene promoters, MTF-1 is notably absent. Here we use Lumbricus rubellus, the red earthworm, to study the elusive mechanism of wMT-2 activation in control and Cd-exposed conditions. EMSA and DNase I footprinting approaches were used to pinpoint functional binding sites within the wMT-2 promoter region, which revealed that the cAMP responsive element (CRE) is a promising candidate which may act as a transcriptional activator of invertebrate MTs. PMID:25797623

  10. Interleukin 6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures.

    PubMed

    Schroeder, J J; Cousins, R J

    1990-04-01

    Attention has focused on the cytokine interleukin 6 (IL-6) as a major mediator of acute-phase protein synthesis in hepatocytes in response to infection and tissue injury. We have evaluated the effects of IL-6 and IL-1 alpha as well as extracellular zinc and glucocorticoid hormone on metallothionein gene expression and cellular zinc accumulation in rat hepatocyte monolayer cultures. Further, we have evaluated the teleological basis for cytokine mediation by examining cytoprotection from CCl4-induced damage. Incubation of hepatocytes with IL-6 led to concentration-dependent and time-dependent increases in metallothionein-1 and -2 mRNA and metallothionein protein. The level of each was increased within 3 hr after the addition of IL-6 at 10 ng/ml (10 hepatocyte-stimulating factor units/ml). Maximal increases in metallothionein mRNA and metallothionein protein were achieved after 12 hr and 36 hr, respectively. In contrast, IL-1 alpha concentrations as high as 20 ng/ml (1000 lymphocyte-activating factor units/ml) had no effect. Concomitant with the up-regulation of metallothionein gene expression, IL-6 also increased cellular zinc. Responses to IL-6 required the synthetic glucocorticoid hormone dexamethasone and were optimized by increased extracellular zinc. In addition, IL-6 with dexamethasone, dexamethasone alone, and increased extracellular zinc each reversed, in decreasing potency, the deleterious effects of CCl4 on hepatocyte viability as measured by cell protein and lactate dehydrogenase activity of the medium. Thus, IL-6 is a major cytokine mediator of metallothionein gene expression and zinc metabolism in hepatocytes and provides cytoprotection from CCl4-induced hepatotoxicity via a mode consistent with dependence upon increased cellular metallothionein synthesis and zinc accumulation. PMID:2326272

  11. Metallothionein gene expression differs in earthworm populations with different exposure history.

    PubMed

    Mustonen, M; Haimi, J; Visnen, A; Knott, K E

    2014-11-01

    Metals are persistent pollutants in soils that can harm soil organisms and decrease species diversity. Animals can cope with metal contamination with the help of metallothioneins, small metal-binding proteins involved in homeostasis and detoxification of metals. We studied the expression of metallothionein with qPCR in a small, epigeic earthworm, Dendrobaena octaedra. We compared expression patterns and metal body content in earthworms collected from two sites with different metal contamination histories: Harjavalta, contaminated by a Cu-Ni smelter operational for over 50years, and Jyvskyl, an uncontaminated site. Earthworms from both sites were also experimentally exposed to different concentrations of Cu (control, 50, 100 or 200mg/kg) or Zn (control, 75, 150 or 300mg/kg) for 7, 14 or 28days to determine if there is a time related dose-response in gene expression. Population comparison showed that metallothionein expression was higher in earthworms from the contaminated site. In the exposure experiment, exposure time affected expression, but only in the earthworms from the uncontaminated site, suggesting that there is a delay in the metallothionein response of earthworms in this population. In contrast, earthworms from the contaminated site showed higher and constant levels of metallothionein expression at all exposure concentrations and durations. The constant metallothionein expression in earthworms from the contaminated site suggests that inducibility of metallothionein response could be lost in earthworms with metal exposure history. Adaptation of D. octaedra to metal exposure could explain the differences between the populations and explain the persistence of this species in contaminated forest soils. PMID:25179588

  12. Copper accumulation and compartmentalization in mouse fibroblast lacking metallothionein and copper chaperone, Atox1

    SciTech Connect

    Miyayama, Takamitsu; Suzuki, Kazuo T.; Ogra, Yasumitsu

    2009-06-01

    Copper (Cu) is the active center of some enzymes because of its redox-active property, although that property could have harmful effects. Because of this, cells have strict regulation/detoxification systems for this metal. In this study, multi-disciplinary approaches, such as speciation and elemental imaging of Cu, were applied to reveal the detoxification mechanisms for Cu in cells bearing a defect in Cu-regulating genes. Although Cu concentration in metallothionein (MT)-knockout cells was increased by the knockdown of the Cu chaperone, Atox1, the concentrations of the Cu influx pump, Ctr1, and another Cu chaperone, Ccs, were paradoxically increased; namely, the cells responded to the Cu deficiency despite the fact that cellular Cu concentration was actually increased. Cu imaging showed that the elevated Cu was compartmentalized in cytoplasmic vesicles. Together, the results point to the novel roles of MT and cytoplasmic vesicles in the detoxification of Cu in mammalian cells.

  13. Freezing of body fluids induces metallothionein gene expression in earthworms (Dendrobaena octaedra).

    PubMed

    Fisker, Karina Vincents; Holmstrup, Martin; Srensen, Jesper Givskov

    2016-01-01

    The molecular mechanisms activated by environmental contaminants and natural stressors such as freezing need to be investigated in order to better understand the mechanisms of interaction and potential effects that combined stressors may have on organisms. Using the freeze-tolerant earthworm Dendrobaena octaedra as model species, we exposed worms to freezing and exposure to sublethal copper in a factorial design and investigated the transcription of candidate genes for metal and cold stress. We hypothesised that both freezing and copper would induce transcription of genes coding for heat shock proteins (hsp10 and hsp70), metallothioneins (mt1 and mt2), and glutathione-S-transferase (gst), and that the combined effects of these two stressors would be additive. The gene transcripts hsp10, hsp70, and gst were significantly upregulated by freezing, but only hsp10 was upregulated by copper. We found that copper at the time of sampling had no effect on transcription of two metallothionein genes whereas transcription was strongly upregulated by freezing. Moreover, there was a significant interaction causing more than additive transcription rates of mt1 in the copper/freezing treatment suggesting that freeze-induced cellular dehydration increases the concentration of free copper ions in the cytosol. This metallothionein response to freezing is likely adaptive and possibly provides protection against freeze-induced elevated metal concentrations in the cytosol and excess ROS levels due to hypoxia during freezing. PMID:26325206

  14. Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosomes 3. Evidence for the role of chromosome rearrangement in gene amplification

    SciTech Connect

    Stallings, R.L.; Munk, A.C.; Longmire, J.L.; Hildebrand, C.E.; Crawford, B.D.

    1984-12-01

    Cadmium resistant (Cd/sup r/) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cd/sup r/ variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster x mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. The authors speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cd/sup r/ hamster cell lines. 34 references, 3 figures, 1 table.

  15. Structure, organization and expression of the metallothionein gene family in Arabidopsis.

    PubMed

    Zhou, J; Goldsbrough, P B

    1995-08-21

    Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Recent results indicate that plants also possess functional metallothionein genes. Here we report the cloning and characterization of five metallothionein genes from Arabidopsis thaliana. The position of the single intron in each gene is conserved. The proteins encoded by these genes can be divided into two groups (MT1 and MT2) based on the presence or absence of a central domain separating two cysteine-rich domains. Four of the MT genes (MT1a, MT1c, MT2a and MT2b) are transcribed in Arabidopsis. Several lines of evidence suggest that the fifth gene, MT1b, is inactive. There is differential regulation of the MT gene family. MT1 mRNA is expressed highly in roots, moderately in leaves and is barely detected in inflorescences and siliques. MT2a and MT2b mRNAs are more abundant in leaves, inflorescences and in roots from mature plants, but are also detected in roots of young plants, and in siliques. MT2a mRNA is strongly induced in seedlings by CuSO4, whereas MT2b mRNA is relatively abundant in this tissue and levels increase only slightly upon exposure to copper. MT1a and MT1c are located within 2 kb of each other and have been mapped to chromosome I. MT1b and MT2b map to separate loci on chromosome V, and MT2a is located on chromosome III. The locations of these MT genes are different from that of CAD1, a gene involved in cadmium tolerance in Arabidopsis. PMID:7565594

  16. Overexpression of Metallothionein-1 Modulates the Phenotype of the Tg2576 Mouse Model of Alzheimer's Disease.

    PubMed

    Manso, Yasmina; Comes, Gemma; Lpez-Ramos, Juan C; Belfiore, Mnica; Molinero, Amalia; Giralt, Mercedes; Carrasco, Javier; Adlard, Paul A; Bush, Ashley I; Delgado-Garca, Jos Mara; Hidalgo, Juan

    2016-01-19

    Alzheimer's disease (AD) is the most commonly diagnosed dementia, where signs of neuroinflammation and oxidative stress are prominent. In this study we intend to further characterize the roles of the antioxidant, anti-inflammatory, and heavy metal binding protein, metallothionein-1 (MT-1), by crossing Mt1 overexpressing mice with a well-known mouse model of AD, Tg2576 mice, which express the human amyloid-? protein precursor (hA?PP) with the Swedish K670N/M671L mutations. Mt1 overexpression increased overall perinatal survival, but did not affect significantly hA?PP-induced mortality and weight loss in adult mice. Amyloid plaque burden in ?14-month-old mice was increased by Mt1 overexpression in the hippocampus but not the cortex. Despite full length hA?PP levels and amyloid plaques being increased by Mt1 overexpression in the hippocampus of both sexes, oligomeric and monomeric forms of A?, which may contribute more to toxicity, were decreased in the hippocampus of females and increased in males. Several behavioral traits such as exploration, anxiety, and learning were altered in Tg2576 mice to various degrees depending on the age and the sex. Mt1 overexpression ameliorated the effects of hA?PP on exploration in young females, and potentiated those on anxiety in old males, and seemed to improve the rate of spatial learning (Morris water maze) and the learning elicited by a classical conditioning procedure (eye-blink test). These results clearly suggest that MT-1 may be involved in AD pathogenesis. PMID:26836194

  17. Expression response of duplicated metallothionein 3 gene to copper stress in Silene vulgaris ecotypes.

    PubMed

    Nevrtalova, Eva; Baloun, Jiri; Hudzieczek, Vojtech; Cegan, Radim; Vyskot, Boris; Dolezel, Jaroslav; Safar, Jan; Milde, David; Hobza, Roman

    2014-11-01

    Metallothioneins (MTs) were identified as important players in metal metabolism. MT3 gene presents a key metallothionein controlling copper homeostasis in plants. We have selected one cupricolous and one non-cupricolous ecotype to isolate and analyse the MT3 gene in Silene vulgaris. For expression data comparison, we have also included other metal-tolerant ecotypes. Based on a S. vulgaris BAC library screening, we have identified and sequenced a genomic clone containing MT3 gene (SvMT3). We found that SvMT3 gene has been locally duplicated in a tandem arrangement. Expression analysis and complementation studies using yeast mutants showed that both copies of the SvMT3 gene were functional. Moreover, we examined the expression of MT3 gene(s) in selected ecotypes under different copper treatments to show the tissue-specific expression response to copper stress. We demonstrated that higher copper concentrations specifically affected MT3 expression among ecotypes. Our analysis shows that MT3a has similar expression pattern in cupricolous ecotypes while MT3b has common expression features shared by all metallophyte S. vulgaris ecotypes. Our data indicate that down-regulation of MT3b root expression in higher copper concentrations is associated with copper stress. We propose that there might be a specific regulation of SvMT3s transcription depending on the type of heavy metal tolerance. PMID:24748066

  18. Metallothionein gene and protein expression as a biomarker for metal pollution in natural gudgeon populations.

    PubMed

    Knapen, Dries; Reynders, Hans; Bervoets, Lieven; Verheyen, Erik; Blust, Ronny

    2007-05-15

    Gudgeons (Gobio gobio) from historically Cd and Zn contaminated sites in Flanders (Belgium) were found to be resistant to elevated Cd levels. In previous work, this increased resistance was largely explained by increased metallothionein (MT) expression. Recently, environmental cleanup efforts resulted in a significant decrease in Cd concentrations in the surface water. In this study, we evaluated the use of hepatic metal and metallothionein (MT) concentrations as biomarkers of metal exposure before and after the cleanup. Hepatic MT mRNA levels were determined after the environmental metal levels decreased in order to assess the applicability of MT gene expression as an environmental biomarker in natural fish populations. Our data show that both metallothionein protein and gene expression have the potential to be sensitive biomarkers for metal exposure. Significant correlations were found (a) among accumulated metal concentrations and both MT protein and mRNA levels, and (b) between MT protein and mRNA levels. However, our data illustrated that while MT protein and gene expression give a quantitative picture of metal load at a single time point, quantitative information in natural populations cannot always be obtained when different time points (including different years) are compared, since MT gene and protein expression are affected by many other factors in addition to the metal load. Furthermore, the result of the environmental cleanup was reflected in a decrease of hepatic Cd concentrations. Zn remained the most important factor determining MT concentrations. Finally, two differently sized MT mRNAs were amplified to test the hypothesis that 3'-UTR length can offer a protective advantage in conditions of environmental stress. Our data provided no evidence to support this hypothesis. In contrast, the ratio of the long mRNA variant relative to total MT mRNA was surprisingly constant, and independent of exposure history. PMID:17379326

  19. CYTOKININ AND METALS REGULATE A TOBACCO METALLOTHIONEIN-LIKE GENE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To isolate cytokinin responsive genes, Nicotiana plumbaginifolia shoots/rosettes containing the heat shock inducible isopentenyl transferase (ipt) gene (HS-ipt) were heat shocked and used to prepare a cDNA library that was screened with a HS-induced subtractive probe. The cDNA clone pCkn16A1 (Access...

  20. Variation in metallothionein gene expression is associated with adaptation to copper in the earthworm Dendrobaena octaedra.

    PubMed

    Fisker, Karina Vincents; Holmstrup, Martin; Sørensen, Jesper Givskov

    2013-03-01

    Evolution of resistance to heavy metals has been reported for several populations of soil living organisms occurring at metal contaminated sites. Such genetically based and heritable resistance contribute to the persistence of populations in contaminated areas. Here we report on molecular responses to experimental copper in populations of the earthworm, Dendrobaena octaedra, originating from copper contaminated soil near Gusum (Sweden) where heavy metal pollution has been present for several decades. We studied gene expression of six genes potentially involved in resistance to copper toxicity using F2-generations of D. octaedra populations, originating from reference sites and contaminated (High, Medium and Low) sites around Gusum. The main result was different expression patterns of genes encoding for two different isoforms (mt1 and mt2) of metallothionein proteins during experimental exposure to copper contaminated soil. Expression of mt1 showed a fast and significant upregulation in the High population and a slower, albeit significant, upregulation in Medium and Low populations. However, in the three reference populations no upregulation were seen. In comparison, a fast upregulation was also seen for the High population in the isoform mt2, whereas, gene expression of all other populations, including reference populations, showed slower upregulation in response to experimental copper. The results indicate that copper resistance in D. octaedra from contaminated areas is related to an increased expression of metallothioneins. PMID:23237849

  1. Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: implications for mechanisms regulating metallothionein gene expression

    SciTech Connect

    Crawford, B.D.; Enger, M.D.; Griffith, B.B.; Griffith, J.K.; Hanners, J.L.; Longmire, J.L.; Munk, A.C.; Stallings, R.L.; Tesmer, J.G.; Walters, R.A.; Hildebrand, C.E.

    1985-02-01

    The authors describe here the derivation, characterization, and use of clonal cadmium-resistance (Cd/sup r) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylaminde gel electrophoresis, the authors showed that the stable Cd/sup r/ phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 M, coordinate amplifications of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which the authors have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cd/sup r/ variants expressing abundant MT and their corresponding Cd/sup s/ parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expressions of the isometallothioneins. 59 references, 8 figures.

  2. Regulation of the rat metallothionein-I gene by sodium butyrate.

    PubMed Central

    Birren, B W; Herschman, H R

    1986-01-01

    Sodium butyrate selectively induces accumulation of metallothionein-I (MT-I) RNA in H4IIE rat hepatoma cells. The induction is rapid; significant elevation in cytoplasmic MT-I RNA can be observed within three hours after exposure to 5 mM butyrate. Maximal levels of MT-I RNA are obtained after eight hours. Butyrate stimulates MT RNA accumulation in the absence of de novo protein synthesis, indicating that MT induction by butyrate is not a distal step in a cascade of gene activation events. Butyrate blocks the induction of tyrosine amino transferase by dexamethasone. In contrast, butyrate and dexamethasone induced MT RNA elevations are additive. Butyrate induced MT-I RNA transcripts initiate at the correct start site. Measurements of the transcriptional activity of the MT-I gene indicate that butyrate stimulates MT-I transcription. The rapid, direct nature of the induction of MT-I by butyrate, combined with the extensive characterization of the metallothionein gene, provide an excellent system in which to study the effects of butyrate on a small, well-defined, responsive region of chromatin. Images PMID:2868444

  3. Metallothionein genes: no association with Crohn's disease in a New Zealand population

    PubMed Central

    2012-01-01

    Metallothioneins (MTs) are excellent candidate genes for Inflammatory Bowel Disease (IBD) and have previously been shown to have altered expression in both animal and human studies of IBD. This is the first study to examine genetic variants within the MT genes and aims to determine whether such genetic variants have an important role in this disease. 28 tag SNPs in genes MT1 (subtypes A, B, E, F, G, H, M, X), MT2, MT3 and MT4 were selected for genotyping in a well-characterized New Zealand dataset consisting of 406 patients with Crohn's Disease and 638 controls. We did not find any evidence of association for MT genetic variation with CD. The lack of association indicates that genetic variants in the MT genes do not play a significant role in predisposing to CD in the New Zealand population. PMID:22284420

  4. The metallothionein gene, TaMT3, from Tamarix androssowii confers Cd2+ tolerance in tobacco.

    PubMed

    Zhou, Boru; Yao, Wenjing; Wang, Shengji; Wang, Xinwang; Jiang, Tingbo

    2014-01-01

    Cadmium (Cd) is a nonessential microelement and low concentration Cd2+ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys)-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS) with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum) through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd2+ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD) activity and chlorophyll concentration, but decreases of peroxidase (POD) activity and malondialdehyde (MDA) accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd2+ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd2+ stress. PMID:24918294

  5. The Metallothionein Gene, TaMT3, from Tamarix androssowii Confers Cd2+ Tolerance in Tobacco

    PubMed Central

    Zhou, Boru; Yao, Wenjing; Wang, Shengji; Wang, Xinwang; Jiang, Tingbo

    2014-01-01

    Cadmium (Cd) is a nonessential microelement and low concentration Cd2+ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys)-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS) with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum) through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd2+ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD) activity and chlorophyll concentration, but decreases of peroxidase (POD) activity and malondialdehyde (MDA) accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd2+ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd2+ stress. PMID:24918294

  6. Physiological, Diurnal and Stress-Related Variability of Cadmium-Metallothionein Gene Expression in Land Snails

    PubMed Central

    Pedrini-Martha, Veronika; Niederwanger, Michael; Kopp, Renate; Schnegg, Raimund; Dallinger, Reinhard

    2016-01-01

    The terrestrial Roman snail Helix pomatia has successfully adapted to strongly fluctuating conditions in its natural soil habitat. Part of the snail’s stress defense strategy is its ability to express Metallothioneins (MTs). These are multifunctional, cysteine-rich proteins that bind and inactivate transition metal ions (Cd2+, Zn2+, Cu+) with high affinity. In Helix pomatia a Cadmium (Cd)-selective, inducible Metallothionein Isoform (CdMT) is mainly involved in detoxification of this harmful metal. In addition, the snail CdMT has been shown to also respond to certain physiological stressors. The aim of the present study was to investigate the physiological and diurnal variability of CdMT gene expression in snails exposed to Cd and non-metallic stressors such as desiccation and oxygen depletion. CdMT gene expression was upregulated by Cd exposure and desiccation, whereas no significant impact on the expression of CdMT was measured due to oxygen depletion. Overall, Cd was clearly more effective as an inducer of the CdMT gene expression compared to the applied non-metallic stressors. In unexposed snails, diurnal rhythmicity of CdMT gene expression was observed with higher mRNA concentrations at night compared to daytime. This rhythmicity was severely disrupted in Cd-exposed snails which exhibited highest CdMT gene transcription rates in the morning. Apart from diurnal rhythmicity, feeding activity also had a strong impact on CdMT gene expression. Although underlying mechanisms are not completely understood, it is clear that factors increasing MT expression variability have to be considered when using MT mRNA quantification as a biomarker for environmental stressors. PMID:26935042

  7. Effect of metallothionein 2A gene polymorphism on allele-specific gene expression and metal content in prostate cancer

    SciTech Connect

    Krześlak, Anna; Forma, Ewa; Jóźwiak, Paweł; Szymczyk, Agnieszka; Bryś, Magdalena

    2013-05-01

    Metallothioneins (MTs) are highly conserved, small molecular weight, cysteine rich proteins. The major physiological functions of metallothioneins include homeostasis of essential metals Zn and Cu and protection against cytotoxicity of heavy metals. The aim of this study was to determine whether there is an association between the − 5 A/G single nucleotide polymorphism (SNP; rs28366003) in core promoter region and expression of metallothionein 2A (MT2A) gene and metal concentration in prostate cancer tissues. MT2A polymorphism was determined by the polymerase chain reaction–restriction fragment length polymorphism technique (PCR–RFLP) using 412 prostate cancer tissue samples. MT2A gene expression analysis was performed by real-time RT-PCR method. A significant association between rs28366003 genotype and MT2A expression level was found. The average mRNA level was found to be lower among minor allele carriers (the risk allele) than average expression among homozygotes for the major allele. Metal levels were analyzed by flamed atomic absorption spectrometer system. Highly statistically significant associations were detected between the SNP and Cd, Zn, Cu and Pb levels. The results of Spearman's rank correlation showed that the expressions of MT2A and Cu, Pb and Ni concentrations were negatively correlated. On the basis of the results obtained in this study, we suggest that SNP polymorphism may affect the MT2A gene expression in prostate and this is associated with some metal accumulation. - Highlights: • MT2A gene expression and metal content in prostate cancer tissues • Association between SNP (rs28366003) and expression of MT2A • Significant associations between the SNP and Cd, Zn, Cu and Pb levels • Negative correlation between MT2A gene expression and Cu, Pb and Ni levels.

  8. Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents.

    PubMed Central

    Kaina, B; Lohrer, H; Karin, M; Herrlich, P

    1990-01-01

    Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions. Images PMID:2320583

  9. Metallothionein gene expression in peripheral lymphocytes and renal dysfunction in a population environmentally exposed to cadmium

    SciTech Connect

    Lu Jian; Jin Taiyi . E-mail: tyjin@smhu.edu.cn; Nordberg, Gunnar; Nordberg, Monica . E-mail: monica.nordberg@imm.ki.se

    2005-08-07

    In order to study the validity of metallothionein (MT) gene expression in peripheral blood lymphocytes (PBLs) as a biomarker of cadmium exposure and susceptibility to renal dysfunction, MT mRNA levels were measured using reverse transcription polymerase chain reaction (RT-PCR) in PBLs from residents living in a cadmium-contaminated area. MT mRNA levels were found to increase with the increase of blood cadmium (BCd) and urinary cadmium (UCd) levels. Basal MT mRNA levels were significantly correlated with the logarithm of BCd levels and the logarithm of UCd levels confirming that MT expression in PBLs is a biomarker of cadmium exposure and internal dose. An inverse relationship was observed between in vitro induced MT-mRNA level in PBLs and urinary N-acetyl-{beta}-d-glucosaminidase (UNAG) suggesting that MT gene expression in PBLs may be used as a biomarker of susceptibility to renal toxicity of cadmium.

  10. Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins

    PubMed Central

    Shaw, Joseph R; Colbourne, John K; Davey, Jennifer C; Glaholt, Stephen P; Hampton, Thomas H; Chen, Celia Y; Folt, Carol L; Hamilton, Joshua W

    2007-01-01

    Background Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant. Results Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs. Conclusion The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences. PMID:18154678

  11. Differential sensitivity of snail embryos to cadmium: relation to age and metallothionein gene expression.

    PubMed

    Baurand, Pierre-Emmanuel; Dallinger, Reinhard; Niederwanger, Michael; Capelli, Nicolas; Pedrini-Martha, Veronika; de Vaufleury, Annette

    2016-02-01

    The aim of this study was to determine whether cadmium (Cd) sensitivity of Cantareus aspersus embryos is age-dependent and influenced by metallothionein (MT) gene expression. Hatching success and the expression of three MT isoform genes (Ca-CdMT, Ca-CuMT and Ca-Cd/CuMT) were measured in embryos exposed to increasing Cd concentrations for 24h starting on the sixth day of development. Isoform gene expression was quantified on days 7 and 12 after exposure. Results were compared to those of embryos exposed to the same conditions as above, but from the beginning of embryogenesis (day 0). Transcription of the Cd-specific MT gene (Ca-CdMT) was observed from the first day of development, whereas the two other genes did not respond to Cd at all. Overall, Cd sensitivity of embryos decreased with increasing age of development, as assessed by age-dependent increase of EC50 values for hatching rate, and increasing Cd threshold concentrations for Ca-CdMT expression. PMID:26341338

  12. Three-dimensional solution structure of mouse [Cd7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy.

    PubMed Central

    Zangger, K.; Oz, G.; Otvos, J. D.; Armitage, I. M.

    1999-01-01

    Sequential 1H-NMR assignments of mouse [Cd7]-metallothionein-1 (MT1) have been carried out by standard homonuclear NMR methods and the use of an accordion-heteronuclear multiple quantum correlation (HMQC) experiment for establishing the metal, 113Cd2+, to cysteine connectivities. The three-dimensional structure was then calculated using the distance constraints from two-dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys-Cd connectivities as input for a distance geometry-dynamical simulated annealing protocol in X-PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two separate domains, each containing one metal cluster. Because there were no interdomain constraints, the structure calculation for the N-terminal beta- and the C-terminal alpha-domain were carried out separately. The structures are based on 409 NMR constraints, consisting of 381 NOEs and 28 cysteine-metal connectivities. The only elements of regular secondary structure found were two short stretches of 3(10) helices along with some half-turns in the alpha-domain. Structural comparison with rat liver MT2 showed high similarity, with the beta-domain structure in mouse MT1 showing evidence of increased flexibility compared to the same domain in MT2. The latter was reflected by the presence of fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium-cadmium exchange rates found in the beta-domain of MT1. PMID:10631978

  13. METALLOTHIONEIN GENE TRANSCRIPTION AS AN INDICATOR OF METAL EXPOSURE IN FATHEAD MINNOWS

    EPA Science Inventory

    Metallothionein is a cysteine rich, low molecular weight, metal binding protein. Basal levels of endogenous metallothioneins (MT) have been reported in all eucaryotes. MT has been shown to play an essential role in regulating physiological requirements of essential metals such a...

  14. Cloning metallothionein gene in Zacco platypus and its potential as an exposure biomarker against cadmium.

    PubMed

    Lee, Sangwoo; Kim, Cheolmin; Kim, Jungkon; Kim, Woo-Keun; Shin, Hyun Suk; Lim, Eun-Suk; Lee, Jin Wuk; Kim, Sunmi; Kim, Ki-Tae; Lee, Sung-Kyu; Choi, Cheol Young; Choi, Kyungho

    2015-07-01

    Zacco platypus, pale chub, is an indigenous freshwater fish of East Asia including Korea and has many useful characteristics as indicator species for water pollution. While utility of Z. platypus as an experimental species has been recognized, genetic-level information is very limited and warrants extensive research. Metallothionein (MT) is widely used and well-known biomarker for heavy metal exposure in many experimental species. In the present study, we cloned MT in Z. platypus and evaluated its utility as a biomarker for metal exposure. For this purpose, we sequenced complete complementary DNA (cDNA) of MT in Z. platypus and carried out phylogenetic analysis with its sequences. The transcription-level responses of MT gene following the exposure to CdCl2 were also assessed to validate the utility of this gene as an exposure biomarker. Analysis of cDNA sequence of MT gene demonstrated high conformity with those of other fish. MT messenger RNA (mRNA) expression and enzymatic MT content significantly increased following CdCl2 exposure in a concentration-dependent manner. The level of CdCl2 that resulted in significant MT changes in Z. platypus was within the range that was reported from other fish. The MT gene of Z. platypus sequenced in the present study can be used as a useful biomarker for heavy metal exposure in the aquatic environment of Korea and other countries where this freshwater fish species represents the ecosystem. PMID:26092240

  15. Comparison of metallothionein gene expression and nonprotein thiols in ten Arabidopsis ecotypes. Correlation with copper tolerance.

    PubMed Central

    Murphy, A; Taiz, L

    1995-01-01

    Seedlings of 10 Arabidopsis ecotypes were compared with respect to copper tolerance, expression of two metallothionein genes (MT1 and MT2), and nonprotein thiol levels. MT1 was uniformly expressed in all treatments, and MT2 was copper inducible in all 10 ecotypes. MT1 and MT2 mRNA levels were compared with various growth parameters for the 10 ecotypes in the presence of 40 microM Cu2+. The best correlation (R = 0.99) was obtained between MT2 mRNA and the rate of root extension. MT2 mRNA levels also paralleled the recovery phase following inhibition by copper. Induction of MT2 mRNA was initiated at copper concentrations below the threshold for growth inhibition. In cross-induction experiments, Ag+, Cd2+, Zn2+, Ni2+, and heat shock all induced significant levels of MT2 gene expression, whereas Al3+ and salicylic acid did not. The correlation between copper tolerance and nonprotein thiol levels in the 10 ecotypes was not statistically significant. However, 2 ecotypes, Ws and Enkheim, previously shown to exhibit an acclimation response, had the highest levels of nonprotein thiols. We conclude that MT2 gene expression may be the primary determinant of ecotypic differences in the copper tolerance of nonpretreated Arabidopsis seedlings. PMID:8552721

  16. Transcription patterns of genes encoding four metallothionein homologs in Daphnia pulex exposed to copper and cadmium are time- and homolog-dependent.

    PubMed

    Asselman, Jana; Shaw, Joseph R; Glaholt, Stephen P; Colbourne, John K; De Schamphelaere, Karel A C

    2013-10-15

    Metallothioneins are proteins that play an essential role in metal homeostasis and detoxification in nearly all organisms studied to date. Yet discrepancies between outcomes of chronic and acute exposure experiments hamper the understanding of the regulatory mechanisms of their isoforms following metal exposure. Here, we investigated transcriptional differences among four identified homologs (mt1-mt4) in Daphnia pulex exposed across time to copper and cadmium relative to a control. Transcriptional upregulation of mt1 and mt3 was detected on day four following exposure to cadmium, whereas that of mt2 and mt4 was detected on day two and day eight following exposure to copper. These results confirm temporal and metal-specific differences in the transcriptional induction of genes encoding metallothionein homologs upon metal exposure which should be considered in ecotoxicological monitoring programs of metal-contaminated water bodies. Indeed, the mRNA expression patterns observed here illustrate the complex regulatory system associated with metallothioneins, as these patterns are not only dependent on the metal, but also on exposure time and the homolog studied. Further phylogenetic analysis and analysis of regulatory elements in upstream promoter regions revealed a high degree of similarity between metallothionein genes of Daphnia pulex and Daphnia magna, a species belonging to the same genus. These findings, combined with a limited amount of available expression data for D. magna metallothionein genes, tentatively suggest a potential generalization of the metallothionein response system between these Daphnia species. PMID:24113165

  17. Cloning, characterization, and expression of cadmium-induced metallothionein-2 gene from earthworm Pheretima aspergillum (E. Perrier).

    PubMed

    Gong, L; Li, W; Li, J; Li, W E; Wu, W R; Yu, L W

    2015-01-01

    Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich proteins, associated with metal accumulation and thus providing protection against toxic heavy metals such as cadmium (Cd). To investigate the mechanisms of enrichment of Cd in the earthworm Pheretima aspergillum, we isolated and cloned metallothionein-2 (MT-2) cDNA (538 bp) from P. aspergillum, analyzed its sequence, and examined MT-2 transcription levels by relative quantitative real-time PCR under different concentrations of Cd. The sequence of P. aspergillum MT-2 cDNA and its putative amino acid sequence were highly similar to sequences from other earthworms. The induction with Cd increased the MT-2 gene transcription level in a dose-dependent manner. In addition, earthworm recombinant MT-2 exhibited high Cd bioaccumulation ability in vitro. These results suggested that MT-2 plays an important role in tolerance and accumulation of Cd in P. aspergillum. PMID:26681024

  18. Pharmacokinetic evaluation of technetium-99-metallothionein-conjugated mouse monoclonal antibody B72. 3 in rhesus monkeys

    SciTech Connect

    Burchiel, S.W.; Hadjian, R.A.; Hladik, W.B.; Drozynski, C.A.; Tolman, G.L.; Haber, S.B.; Gallagher, B.M. )

    1989-08-01

    These studies were conducted to determine the biodistribution and pharmacokinetics of ({sup 99m}Tc)metallothionein-conjugated B72.3 ((Tc)MT-B72.3) in Rhesus monkeys (Macaca mulatta) that were performed as part of the preclinical evaluation of (Tc)MT-B72.3. The B72.3-MT conjugate was studied at three doses of B72.3 ranging from 0.03 mg/kg to 1 mg/kg to determine whether a relationship existed between the dose of total antibody administered intravenously and the biodistribution and clearance of the radiolabeled protein. Results indicated that (Tc)MT-B72.3 distributes rapidly to central body cavity organs and that there was no difference in the rate of blood elimination for the three doses of B72.3 studied. The terminal phase of blood elimination was found to be 26.2 +/- 6.1 hr for the combined groups of monkeys. Approximately one-half of injected {sup 99m}Tc activity was recovered in the urine within 24 hr. A second purpose of these studies was to evaluate the overall immunogenicity of the mouse monoclonal B72.3 IgG1 antibody in Rhesus monkeys. These results demonstrated that a single i.v. exposure to mouse monoclonal B72.3 at doses of 0.3 mg/kg or greater elicited antibody production to B72.3 in Rhesus monkeys within 3 wk. Analysis of (Tc)MT-B72.3 biodistribution and clearance in monkeys with circulating levels of antibodies to B72.3 (immunized monkeys) revealed that the liver was the primary site of clearance of the presumed immune complex and that blood elimination was greatly accelerated.

  19. Butyrate selectively activates the metallothionein gene in teratocarcinoma cells and induces hypersensitivity to metal induction.

    PubMed Central

    Andrews, G K; Adamson, E D

    1987-01-01

    The expression of metallothionein genes (MT-I and MT-II) was shown to be enhanced within 2 h of addition of 2.5-5 mM sodium butyrate to cultures of teratocarcinoma cells. Both undifferentiated stem cells (F9 and OC15) and differentiated cells (PSA5E and OC15 END) reacted similarly to butyrate by increased accumulation of MT mRNAs. As expected, all of the teratocarcinoma cells that were tested also responded to Zn2+ and Cd2+ by 5- to 10-fold increases in MT mRNA accumulation within 2-24 h of metal addition to the culture media. Surprisingly, MT genes in cells pretreated with butyrate were hypersensitive to metal induction, and this was demonstrated by accumulated transcript levels and by synthesis of MT protein. The maximal metal response was obtained by exposure of cells to butyrate for around 5-8 h together with 10 microM heavy metals. Metal additions to culture media over a range of concentrations and times only induced half the levels of MT mRNA that were achieved by butyrate plus metals. Butyrate enhanced the rate of accumulation of MT mRNA in response to metals, increased the sensitivity of the MT gene to metals, and protected cells from toxic effects of high concentrations of metals. The butyrate and metal ion responses were selective in that no accumulation of c-myc, c-fms, HSP-70, or AFP mRNA was detected. However, c-fos mRNA accumulated in cells exposed to toxic concentrations of metals (50 microM and higher) and this was also potentiated by butyrate treatment. These results suggest that butyrate alters the chromatin conformation of both the MT-I and MT-II genes leading to an accentuated transcriptional response to metals. Images PMID:3601676

  20. Characterization of three distinct metallothionein genes of the Ag-hyperaccumulating ectomycorrhizal fungus Amanita strobiliformis.

    PubMed

    Hlokov, Kate?ina; Mat?nov, Michaela; ?kov, Petra; Strnad, Hynek; Hrelov, Hana; Hroudov, Milue; Kotrba, Pavel

    2016-03-01

    Mechanisms evolved in eukaryotes to handle heavy metals involve cytosolic, metal-binding metallothioneins (MTs). We have previously documented that the sequestration of silver (Ag) in the Ag-hyperaccumulating Amanita strobiliformis is dominated by 34-amino-acid (AA) AsMT1a, 1b, and 1c isoforms. Here we show that in addition to AsMT1a, 1b, and 1c isogenes, the fungus has two other MT genes: AsMT2 encoding a 34-AA AsMT2 similar to MTs known from other species, but unrelated to AsMT1s; AsMT3 coding for a 62-AA AsMT3 that shares substantial identity with as-yet-uncharacterized conserved peptides predicted in agaricomycetes. Transcription of AsMT1s and AsMT3 in the A. strobiliformis mycelium was specifically inducible by treatments with Ag or copper (Cu) and zinc (Zn) or cadmium (Cd), respectively; AsMT2 showed a moderate upregulation in the presence of Cd. Expression of AsMTs in the metal-sensitive Saccharomyces cerevisiae revealed that all AsMTs confer increased Cd tolerance (AsMT3 proved the most effective) and that, unlike AsMT1 and AsMT2, AsMT3 can protect the yeasts against Zn toxicity. The highest level of Cu tolerance was observed with yeasts expressing AsMT1a. Our data indicate that A. strobiliformis can specifically employ different MT genes for functions in the cellular handling of Ag and Cu (AsMT1s) and Zn (AsMT3). PMID:26895864

  1. Cloning, characterization and gene expression of a metallothionein isoform in the edible cockle Cerastoderma edule after cadmium or mercury exposure.

    PubMed

    Paul-Pont, Ika; Gonzalez, Patrice; Montero, Natalia; de Montaudouin, Xavier; Baudrimont, Magalie

    2012-01-01

    Metallothionein (MT) genes encode crucial metal-binding proteins ubiquitously expressed in living organisms and which play important roles in homeostasis of essential metals and detoxification processes. Here, the molecular organization of the first metallothionein gene of the edible cockle Cerastoderma edule and its expression after cadmium (Cd) or mercury (Hg) exposures were determined. The resulting sequence (Cemt1) exhibits unusual features. The full length cDNA encodes a protein of 73 amino acids with nine classical Cys-X((1-3))-Cys motifs, but also one Cys-Cys not generally found in molluscan MT. Moreover, characterization of the molecular organization of the Cemt1 gene revealed two different alleles (A1 and A2) with length differences due to large deletion events in their intronic sequences involving direct Short Interspersed repeated Elements (SINE), while their exonic sequences were identical. To our knowledge, such large excision mechanisms have never before been reported in a bivalve gene sequence. After 10 days of Cd exposure at environmentally relevant doses, quantitative real-time PCR revealed a strong induction of Cemt1 in gills of C. edule. Surprisingly, neither induction of the Cemt1 gene nor of MT protein was shown after Hg exposure, despite the fact that this organism is able to bioaccumulate a high amount of this trace metal which is theoretically one of the most powerful inducers of MT biosynthesis. PMID:21963253

  2. The mouse cornichon gene family.

    PubMed

    Hwang, S Y; Oh, B; Zhang, Z; Miller, W; Solter, D; Knowles, B B

    1999-02-01

    As part of a large scale mouse Expressed Sequence Tag (EST) project to identify molecules involved in the initiation of mammalian development, a homolog of the Drosophila cornichon gene was detected as a mouse maternal transcript present in the two-cell embryo. Cornichon is a multigene family in the mouse: the new gene, Cnih, maps to mouse chromosome 10, another cornichon homolog, Cnil, maps to chromosome 14 and two additional cornichon-related loci, possibly pseudogenes, localize to chromosomes 3 and 10, respectively. Cnih encodes an open reading frame (ORF) of 144 amino acids that is 93% homologous (68% identical) to the Drosophila protein, whereas the ORF of Cnil contains two extra polypeptide regions not found in these other proteins. Transcripts of Cnih are highly abundant in the full grown oocyte and the ovulated unfertilized egg, while Cnil message is only detectable after activation of the embryonic genome at the eight-cell stage. In situ hybridization shows specific localization of Cnih transcripts to ovarian oocytes. The lack of cytoplasmic polyadenylation of the maternally inherited Cnih transcript suggests that Cnih mRNA is translated in the full grown oocyte before, but not after, ovulation. In Drosophila, cornichon is involved in the establishment of both anterior-posterior and dorso-ventral polarity via the epidermal growth factor (EGF)-receptor signaling pathway. Finding Cnih in the mammalian oocyte opens a new perspective on the investigation of EGF-signaling in the oocyte. PMID:10022955

  3. Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora

    SciTech Connect

    Kumar, Kalari Satish; Ravi Kumar, B.; Siddavattam, Dayananda; Subramanyam, Chivukula . E-mail: csubramanyam@hotmail.com

    2006-07-07

    In continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (CDRE) in the -3730 bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45-50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtained from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17 kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17 kDa protein matched with the regulatory {beta}-subunit of calcineurin (Ca{sup 2+}-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression.

  4. Two Metallothionein Genes in Oxya chinensis: Molecular Characteristics, Expression Patterns and Roles in Heavy Metal Stress

    PubMed Central

    Liu, Yaoming; Wu, Haihua; Kou, Lihua; Liu, Xiaojian; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

    2014-01-01

    Metallothioneins (MTs) are small, cysteine-rich, heavy metal-binding proteins involved in metal homeostasis and detoxification in living organisms. In the present study, we cloned two MT genes (OcMT1 and OcMT2) from Oxya chinensis, analyzed the expression patterns of the OcMT transcripts in different tissues and at varying developmental stages using real-time quantitative PCR (RT-qPCR), evaluated the functions of these two MTs using RNAi and recombinant proteins in an E. coli expression system. The full-length cDNAs of OcMT1 and OcMT2 encoded 40 and 64 amino acid residues, respectively. We found Cys-Cys, Cys-X-Cys and Cys-X-Y-Z-Cys motifs in OcMT1 and OcMT2. These motifs might serve as primary chelating sites, as in other organisms. These characteristics suggest that OcMT1 and OcMT2 may be involved in heavy metal detoxification by capturing the metals. Two OcMT were expressed at all developmental stages, and the highest levels were found in the eggs. Both transcripts were expressed in all eleven tissues examined, with the highest levels observed in the brain and optic lobes, followed by the fat body. The expression of OcMT2 was also relatively high in the ovaries. The functions of OcMT1 and OcMT2 were explored using RNA interference (RNAi) and different concentrations and treatment times for the three heavy metals. Our results indicated that mortality increased significantly from 8.5% to 16.7%, and this increase was both time- and dose-dependent. To evaluate the abilities of these two MT proteins to confer heavy metal tolerance to E. coli, the bacterial cells were transformed with pET-28a plasmids containing the OcMT genes. The optical densities of both the MT-expressing and control cells decreased with increasing concentrations of CdCl2. Nevertheless, the survival rates of the MT-overexpressing cells were higher than those of the controls. Our results suggest that these two genes play important roles in heavy metal detoxification in O. chinensis. PMID:25391131

  5. Differential expression and characterization of three metallothionein-like genes in Cavendish banana (Musa acuminata).

    PubMed

    Liu, Pei; Goh, Chong-Jin; Loh, Chiang-Shiong; Pua, Eng-Chong

    2002-02-01

    Metallothioneins (MTs) are cysteine-rich polypeptides that are involved in metal detoxification and homeostasis in both prokaryotes and eukaryotes. In this study, we report the isolation and characterization of three members (MT2A, MT2B and MT3) of the MT-like gene family from ripening banana fruit and their differential expression in various banana organs and during fruit development and ripening. All members of the MT-like gene encode small cysteine-rich polypeptides of 65-79 amino acid residues. MT2A shared a high sequence similarity (54-77%) with several type-2 MTs in plants, while MT3 was highly homologous (51-61%) with type-3 MTs. The three members expressed differentially in various organs but transcripts were generally more abundant in reproductive than vegetative organs. During fruit development, the MT2A transcript was barely detectable in ovary but increased to a high level in young fruit at 20 days after shooting (DAS) and declined gradually thereafter as fruit developed. In contrast, both MT2B and MT3 expressed poorly in young fruits (20-60 DAS) and transcripts were detected only in fruits at later stages of development. As ripening progressed, expression of MT2A decreased but that of MT3 increased. Expression of MT members during ripening appeared to be differentially regulated by ethylene, whose levels were low in FG and TY fruit but surged climacteristically in MG and declined sharply as ripening advanced further. Exogenous application of ethylene at 5 ppm or higher concentrations down-regulated MT2A expression and the inhibitory effect of ethylene could be partially suppressed by the presence of norbornadiene, an inhibitor of ethylene action. Ethylene had no effect on transcript accumulation of MT2B and MT3. However, MT3 expression was greatly enhanced in response to metals such as CdSO4, CuSO4 and ZnSO4. These results suggest that increased MT3 expression may be associated with excess metal ions present in ripening fruit tissues. This study also provided evidence, for the first time, that ethylene and metals play a regulatory role in expression of MT-like genes in banana. PMID:11903971

  6. The strong induction of metallothionein gene following cadmium exposure transiently affects the expression of many genes in Eisenia fetida: a trade-off mechanism?

    PubMed

    Brulle, F; Mitta, G; Leroux, R; Lemière, S; Leprêtre, A; Vandenbulcke, F

    2007-01-01

    Metal pollution causes disturbances at various levels of biological organization in most species. Important physiological functions could be affected in the exposed individuals and among the main physiological functions, immunity may provide one (or more) effector(s) whose expression can be directly affected by a metal exposure in various macroinvertebrates. Protein expressions were studied in order to test them as molecular biomarkers of metal exposure in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, transcriptionally controlled tumor protein, protein kinase C, ubiquitin and cyclophilin-A. The level of expression of each gene was analysed in whole organism following exposures to cadmium in soil using real-time PCR. Metallothionein, transcriptionally controlled tumor protein and cyclophilin-A expression were also measured following copper exposures in soil because these genes seemed to be sensitive to copper. This work enabled to distinguish metallothionein and cyclophilin-A among the 15 selected effectors. A strong decrease of the number of transcripts was also detected for most effectors soon after the exposure to cadmium suggesting that a trade-off mechanism occurs. PMID:17150412

  7. The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization

    PubMed Central

    Lin, Weiyu; Calatayud, Sara; Palacios, Òscar; Capdevila, Mercè; Atrian, Sílvia

    2016-01-01

    Fungal Cu-thioneins, and among them, the paradigmatic Neurospora crassa metallothionein (MT) (26 residues), were once considered as the shortest MTs -the ubiquitous, versatile metal-binding proteins- among all organisms, and thus representatives of their primeval forms. Nowadays, fungal MTs of diverse lengths and sequence features are known, following the huge heterogeneity of the Kingdom of Fungi. At the opposite end of N. crassa MT, the recently reported Cryptococcus neoformans CnMT1 and CnMT2 (122 and 186 aa) constitute the longest reported fungal MTs, having been identified as virulence factors of this pathogen. CnMTs are high-capacity Cu-thioneins that appear to be built by tandem amplification of a basic unit, a 7-Cys segment homologous to N. crassa MT. Here, we report the in silico, in vivo and in vitro study of a still longer fungal MT, belonging to Tremella mesenterica (TmMT), a saprophytic ascomycete. The TmMT gene has 10 exons, and it yields a 779-bp mature transcript that encodes a 257 residue-long protein. This MT is also built by repeated fragments, but of variable number of Cys: six units of the 7-Cys building blocks-CXCX3CSCPPGXCXCAXCP-, two fragments of six Cys, plus three Cys at the N-terminus. TmMT metal binding abilities have been analyzed through the spectrophotometric and spectrometric characterization of its recombinant Zn-, Cd- and Cu-complexes. Results allow it to be unambiguous classified as a Cu-thionein, also of extraordinary coordinating capacity. According to this feature, when the TmMT cDNA is expressed in MT-devoid yeast cells, it is capable of restoring a high Cu tolerance level. Since it is not obvious that T. mesenterica shares the same physiological needs for a high capacity Cu-binding protein with C. neoformans, the existence of this peculiar MT might be better explained on the basis of a possible role in Cu-handling for the Cu-enzymes responsible in lignin degradation pathways. PMID:26882011

  8. The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization.

    PubMed

    Iturbe-Espinoza, Paul; Gil-Moreno, Selene; Lin, Weiyu; Calatayud, Sara; Palacios, scar; Capdevila, Merc; Atrian, Slvia

    2016-01-01

    Fungal Cu-thioneins, and among them, the paradigmatic Neurospora crassa metallothionein (MT) (26 residues), were once considered as the shortest MTs -the ubiquitous, versatile metal-binding proteins- among all organisms, and thus representatives of their primeval forms. Nowadays, fungal MTs of diverse lengths and sequence features are known, following the huge heterogeneity of the Kingdom of Fungi. At the opposite end of N. crassa MT, the recently reported Cryptococcus neoformans CnMT1 and CnMT2 (122 and 186 aa) constitute the longest reported fungal MTs, having been identified as virulence factors of this pathogen. CnMTs are high-capacity Cu-thioneins that appear to be built by tandem amplification of a basic unit, a 7-Cys segment homologous to N. crassa MT. Here, we report the in silico, in vivo and in vitro study of a still longer fungal MT, belonging to Tremella mesenterica (TmMT), a saprophytic ascomycete. The TmMT gene has 10 exons, and it yields a 779-bp mature transcript that encodes a 257 residue-long protein. This MT is also built by repeated fragments, but of variable number of Cys: six units of the 7-Cys building blocks-CXCX3CSCPPGXCXCAXCP-, two fragments of six Cys, plus three Cys at the N-terminus. TmMT metal binding abilities have been analyzed through the spectrophotometric and spectrometric characterization of its recombinant Zn-, Cd- and Cu-complexes. Results allow it to be unambiguous classified as a Cu-thionein, also of extraordinary coordinating capacity. According to this feature, when the TmMT cDNA is expressed in MT-devoid yeast cells, it is capable of restoring a high Cu tolerance level. Since it is not obvious that T. mesenterica shares the same physiological needs for a high capacity Cu-binding protein with C. neoformans, the existence of this peculiar MT might be better explained on the basis of a possible role in Cu-handling for the Cu-enzymes responsible in lignin degradation pathways. PMID:26882011

  9. Changes in copper and zinc status and response to dietary copper deficiency in metallothionein-overexpressing transgenic mouse heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies have shown that metallothionein (MT) inhibits myocardial apoptosis induced by dietary copper restriction and that this inhibition is related to the antioxidant action of MT. However, the mechanism of action of MT in vivo is not known. Recent studies have suggested that zinc release ...

  10. Dual Opposing Roles of Metallothionein Overexpression in C57BL/6J Mouse Pancreatic β-Cells

    PubMed Central

    Chen, Suqin; Han, Junying; Liu, Yeqi

    2015-01-01

    Background Growing evidence indicates that oxidative stress (OS), a persistent state of excess amounts of reactive oxygen species (ROS) along with reactive nitrogen species (RNS), plays an important role in insulin resistance, diabetic complications, and dysfunction of pancreatic β-cells. Pancreatic β-cells contain exceptionally low levels of antioxidant enzymes, rendering them susceptible to ROS-induced damage. Induction of antioxidants has been proposed to be a way for protecting β-cells against oxidative stress. Compared to other antioxidants that act against particular β-cell damages, metallothionein (MT) is the most effective in protecting β-cells from several oxidative stressors including nitric oxide, peroxynitrite, hydrogen peroxide, superoxide and streptozotocin (STZ). We hypothesized that MT overexpression in pancreatic β-cells would preserve β-cell function in C57BL/6J mice, an animal model susceptible to high fat diet-induced obesity and type 2 diabetes. Research Design and Methods The pancreatic β-cell specific MT overexpression was transferred to C57BL/6J background by backcrossing. We studied transgenic MT (MT-tg) mice and wild-type (WT) littermates at 8 weeks and 18 weeks of age. Several tests were performed to evaluate the function of islets, including STZ in vivo treatment, intraperitoneal glucose tolerance tests (IPGTT) and plasma insulin levels during IPGTT, pancreatic and islet insulin content measurement, insulin secretion, and islet morphology assessment. Gene expression in islets was performed by quantitative real-time PCR and PCR array analysis. Protein levels in pancreatic sections were evaluated by using immunohistochemistry. Results The transgenic MT protein was highly expressed in pancreatic islets. MT-tg overexpression significantly protected mice from acute STZ-induced ROS at 8 weeks of age; unexpectedly, however, MT-tg impaired glucose stimulated insulin secretion (GSIS) and promoted the development of diabetes. Pancreatic β-cell function was significantly impaired, and islet morphology was also abnormal in MT-tg mice, and more severe damage was detected in males. The unique gene expression pattern and abnormal protein levels were observed in MT-tg islets. Conclusions MT overexpression protected β-cells from acute STZ-induced ROS damages at young age, whereas it impaired GSIS and promoted the development of diabetes in adult C57BL/6J mice, and more severe damage was found in males. PMID:26335571

  11. Transgenic Brassica napus and tobacco plants harboring human metallothionein gene are resistant to toxic levels of heavy metals

    SciTech Connect

    Misra, S. )

    1989-04-01

    A chimeric gene containing a cloned human metallothionein-II (MT-II) processed gene was introduced into Brassica napus and tobacco cells on a disarmed Ti plasmid of Agrobacterium tumefaciens. Transformants expressed MT protein as a nuclear trait, and in a constitutive manner. Seeds from self-fertilized transgenic plants were germinated on media containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal. The growth of root and shoot of transformed seedlings was unaffected by up to 100{mu}M CdCl{sub 2}, whereas, control seedlings showed severe inhibition of root and shoot growth and chlorosis of leaves. The results of these experiments indicate that agriculturally important plants such a B. napus can be genetically engineered for heavy metals tolerance/sequestration and eventually for partitioning of heavy metals in non-consumed plant tissues.

  12. Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxity in cultured rat liver cells

    SciTech Connect

    Coogan, T.P.; Bare, R.M.; Bjornson, E.J.; Waalkes, M.P. )

    1994-01-01

    Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that appear to play an important role in the cellular defense system against cadmium toxicity. Although substantial evidence exists demonstrating a reduction in cadmium toxicity concomitant with MT induction, little is known about the possible effects of stimulation of MT synthesis on cadmium-induced genotoxicity. Thus, the alkaline elution technique was used to assess single-strand DNA damage (SSD) in TRL-1215 cells, a liver-derived cell line shown to have inducible MT Gene expression. The SSD accumulated over a 2-h time period in a time-dependent manner following exposure to 500 [mu]M CdCl[sub 2]. Low concentration cadmium pretreatment (10 [mu]M CdCl[sub 2], 24 h) provided protection against the genotoxicity of high-concentration cadmium (500 [mu]M CdCl[sub 2], 2 h). A 2-h exposure to 500 [mu]M CdCl[sub 2], had no effect on viability, as assessed using a tetrazolium-dye based assay, in cells from either the pretreated or nonpretreated group. Metallothionein was induced in a time-dependent manner by low-concentration cadmium pretreatment: Exposure for 24 and 48 h resulted in 3.3- and 6.4-fold increases, respectively. In addition, a 24-h exposure to low-concentration cadmium resulted in an increase in MT-I gene expression. Cadmium accumulation was 2.6-fold greater in low-concentration cadmium-pretreated cells as compared to non-pretreated cells. These data demonstrate that low-concentration cadmium pretreatment provides protection against cadmium-induced single-strand DNA damage and support the hypothesis that this protection is due to stimulation of MT gene expression. 38 refs., 6 figs.

  13. Metallothionein gene expression in embryos of the terrestrial snail (Cantareus aspersus) exposed to cadmium and copper in the Bordeaux mixture.

    PubMed

    Baurand, Pierre-Emmanuel; Dallinger, Reinhard; Capelli, Nicolas; de Vaufleury, Annette

    2016-02-01

    The response specificity of three metallothionein (MT) genes (CdMT, CuMT and Cd/CuMT) was assessed after long-term exposure (20days) of Cantareus aspersus eggs to cadmium (Cd) (2 to 6mg/L) or to the fungicide Bordeaux mixture (BM) (2.5 and 7.5g/L). MT gene expression measured by quantitative real-time PCR (qRT-PCR) revealed that in the unexposed embryos, the transcript levels of the three MT genes decreased significantly through embryonic development. However, the CdMT gene was strongly upregulated with increasing Cd exposure concentration, whereas the transcript levels of the other two genes increased less pronouncedly, but significantly above an exposure concentration of 4mg Cd/L. Upon exposure to BM, all three MT genes were significantly upregulated above a BM concentration of 2.5g/L. It is concluded that long-term Cd exposure in hatched snails induced patterns of MT gene expression that differed from those obtained after short-term exposure (24h). PMID:26514570

  14. A cadmium metallothionein gene of ridgetail white prawn Exopalaemon carinicauda (Holthuis, 1950) and its expression

    NASA Astrophysics Data System (ADS)

    Zhang, Jiquan; Wang, Jing; Xiang, Jianhai

    2013-11-01

    Metallothioneins (MTs) are a group of low molecular weight cysteine-rich proteins capable of binding heavy metal ions. A cadmium metallothionein ( EcMT — Cd) cDNA with a 189 bp open reading frame (ORF) that encoded a 62 amino acid protein was obtained from Exopalaemon carinicauda. Seventeen cysteines were in the deduced amino acid sequence, and the cysteine (Cys)-rich characteristic was revealed in different metallothioneins in other species. In addition, the deduced amino acid sequence did not contain any aromatic amino acid residues, such as tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe). EcMT—Cd mRNA was expressed in all tested tissues (the ovary, muscle, stomach, and hepatopancreas), and its expression profiles in the hepatopancreas were very different when shrimps were exposed to seawater containing either 50 μmol/L CuSO4 or 2.5 μmol/L CdCl 2. The expression of EcMT-Cd was significantly up-regulated in shrimp exposed to CuSO4 for 12 h and down-regulated in shrimps exposed to CdCl2 for 12 h. After 24 h exposure to both metals, its expression was down-regulated. By contrast, at 48 h the EcMT-Cd was up-regulated in test shrimps exposed to CdCl2. The transcript of EcMT-Cd was very low or even absent before the zoea stage, and the expression of EcMT-Cd was detected from mysis larvae-I, then its expression began to rise. In conclusion, a cadmium MT exists in E. carinicauda that is expressed in different tissues and during different developmental stages, and responds to the challenge with heavy metal ions, which provides a clue to understanding the function of cadmium MT.

  15. Cancer gene discovery in mouse and man

    PubMed Central

    Mattison, Jenny; van der Weyden, Louise; Hubbard, Tim; Adams, David J.

    2009-01-01

    The elucidation of the human and mouse genome sequence and developments in high-throughput genome analysis, and in computational tools, have made it possible to profile entire cancer genomes. In parallel with these advances mouse models of cancer have evolved into a powerful tool for cancer gene discovery. Here we discuss the approaches that may be used for cancer gene identification in both human and mouse and discuss how a cross-species oncogenomics approach to cancer gene discovery represents a powerful strategy for finding genes that drive tumourigenesis. PMID:19285540

  16. cDNA sequence encoding metallothionein protein from Aegiceras corniculatum and its gene expression induced by Pb? and Cd? stresses.

    PubMed

    Yuhong, Li; Atagana, Harrison I; Jingchun, Liu; Wenlin, Wu; Shijun, Wu

    2013-12-01

    Constructing various green wetland examples for mangrove wetland systems is a useful way to use natural power to remediate the polluted wetlands at intertidal zones. Metallothioneins (MT) are involved in heavy metal tolerance, homeostasis, and detoxification of intracellular metal ions in plants. In order to understand the mechanism of heavy metal uptake in Aegiceras corniculatum, we isolated its metallothionein gene and studied the MT gene expression in response to heavy metals contamination. Here, we report the isolation and characterization of MT2 genes from young stem tissues of A. corniculatum growing in the cadmium (Cd) and lead (Pb) polluted wetlands of Quanzhou Bay, southeast of China. The obtained cDNA sequence of MT is 512 bp in length, and it has an open reading frame encoding 79 amino acid residues with a molecular weight of 7.92 kDa and the theoretical isoelectric point of 4.55. The amino acids include 14 cysteine residues and 14 glycine residues. It is a non-transmembrane hydrophilic protein. Sequence and homology analysis showed the MT protein sequence shared more than 60% homology with other plant type 2 MT-like protein genes. The results suggested that the expression level of MT gene of A. corniculatum young stems induced by a certain range concentration of Cd(2+) and Pb(2+) stresses (0.2 mmol L(-1) Pb(2+), 1 mmol L(-1) Pb(2+), 0.2 mmol L(-1) Pb(2+), and 40 ?mmol L(-1) Cd(2+); 1 mmol L(-1) Pb(2+) and 40 ?mol L(-1) Cd(2+)) compared with control might show an adaptive protection. The expression levels of MT gene at 20 h stress treatment were higher than those at 480 h stress treatment. The expression levels of MT gene with 0.2 mmol L(-1) Pb(2+) stress treatment were higher than those with 0.2 mmol L(-1) Pb(2+) and 40 ?mol L(-1) Cd(2+) stress treatment, and the MT gene expression levels with 1 mmol L(-1) Pb(2+) treatment were higher than those with 1 mmol L(-1) Pb(2+) and 40 ?mol L(-1) Cd(2+) treatment. There exists an antagonistic action between Pb(2+) and Cd(2+) in the MT metabolization of A. corniculatum. PMID:23856811

  17. Characterization of a Type 1 Metallothionein Gene from the Stresses-Tolerant Plant Ziziphus jujuba.

    PubMed

    Yang, Mingxia; Zhang, Fan; Wang, Fan; Dong, Zhigang; Cao, Qiufen; Chen, Mingchang

    2015-01-01

    Plant metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, and metal-binding proteins, which play an important role in the detoxification of heavy metal ions, osmotic stresses, and hormone treatment. Sequence analysis revealed that the open-reading frame (ORF) of ZjMT was 225 bp, which encodes a protein composed of 75 amino acid residues with a calculated molecular mass of 7.376 kDa and a predicated isoelectric point (pI) of 4.83. ZjMT belongs to the type I MT, which consists of two highly conserved cysteine-rich terminal domains linked by a cysteine free region. Our studies showed that ZjMT was primarily localized in the cytoplasm and the nucleus of cells and ZjMT expression was up-regulated by NaCl, CdCl2 and polyethylene glycol (PEG) treatments. Constitutive expression of ZjMT in wild type Arabidopsis plants enhanced their tolerance to NaCl stress during the germination stage. Compared with the wild type, transgenic plants accumulate more Cd2+ in root, but less in leaf, suggesting that ZjMT may have a function in Cd2+ retension in roots and, therefore, decrease the toxicity of Cd2+. PMID:26213917

  18. Arbuscular mycorrhiza affects nickel translocation and expression of ABC transporter and metallothionein genes in Festuca arundinacea.

    PubMed

    Shabani, Leila; Sabzalian, Mohammad R; Mostafavi Pour, Sodabeh

    2016-01-01

    Mycorrhizal fungi are key microorganisms for enhancing phytoremediation of soils contaminated with heavy metals. In this study, the effects of the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae (=Glomus mosseae) on physiological and molecular mechanisms involved in the nickel (Ni) tolerance of tall fescue (Festuca arundinacea?=?Schedonorus arundinaceus) were investigated. Nickel addition had a pronounced negative effect on tall fescue growth and photosynthetic pigment contents, as well as on AMF colonization. Phosphorus content increased markedly in mycorrhizal plants (M) compared to non-inoculated (NM) ones. However, no significant difference was observed in root carbohydrate content between AMF-inoculated and non-inoculated plants. For both M and NM plants, Ni concentrations in shoots and roots increased according to the addition of the metal into soil, but inoculation with F. mosseae led to significantly lower Ni translocation from roots to the aboveground parts compared to non-inoculated plants. ABC transporter and metallothionein transcripts accumulated to considerably higher levels in tall fescue plants colonized by F. mosseae than in the corresponding non-mycorrhizal plants. These results highlight the importance of mycorrhizal colonization in alleviating Ni-induced stress by reducing Ni transport from roots to shoots of tall fescue plants. PMID:26041568

  19. Characterization of a Type 1 Metallothionein Gene from the Stresses-Tolerant Plant Ziziphus jujuba

    PubMed Central

    Yang, Mingxia; Zhang, Fan; Wang, Fan; Dong, Zhigang; Cao, Qiufen; Chen, Mingchang

    2015-01-01

    Plant metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, and metal-binding proteins, which play an important role in the detoxification of heavy metal ions, osmotic stresses, and hormone treatment. Sequence analysis revealed that the open-reading frame (ORF) of ZjMT was 225 bp, which encodes a protein composed of 75 amino acid residues with a calculated molecular mass of 7.376 kDa and a predicated isoelectric point (pI) of 4.83. ZjMT belongs to the type I MT, which consists of two highly conserved cysteine-rich terminal domains linked by a cysteine free region. Our studies showed that ZjMT was primarily localized in the cytoplasm and the nucleus of cells and ZjMT expression was up-regulated by NaCl, CdCl2 and polyethylene glycol (PEG) treatments. Constitutive expression of ZjMT in wild type Arabidopsis plants enhanced their tolerance to NaCl stress during the germination stage. Compared with the wild type, transgenic plants accumulate more Cd2+ in root, but less in leaf, suggesting that ZjMT may have a function in Cd2+ retension in roots and, therefore, decrease the toxicity of Cd2+. PMID:26213917

  20. Effect of cadmium on glutathione S-transferase and metallothionein gene expression in coho salmon liver, gill and olfactory tissues

    PubMed Central

    Espinoza, Herbert M.; Williams, Chase R.; Gallagher, Evan P.

    2012-01-01

    The glutathione S-transferases (GSTs) are a multifunctional family of phase II enzymes that detoxify a variety of environmental chemicals, reactive intermediates, and secondary products of oxidative damage. GST mRNA expression and catalytic activity have been used as biomarkers of exposure to environmental chemicals. However, factors such as species differences in induction, partial analyses of multiple GST isoforms, and lack of understanding of fish GST gene regulation, have confounded the use of GST as markers of pollutant exposure. In the present study, we examined the effect of exposure to cadmium (Cd), a prototypical environmental contaminant and inducer of mammalian GST, on GST mRNA expression in coho salmon (Oncorhynchus kisutch) liver, gill, and olfactory tissues. GST expression data were compared to those for metallothionein (MT), a prototypical biomarker of metal exposure. Data mining of genomic databases led to the development of quantitative real-time PCR (qPCR) assays for salmon GST isoforms encompassing 9 subfamilies, including alpha, mu, pi, theta, omega, kappa, rho, zeta and microsomal GST. In vivo acute (8-48 hr) exposures to low (3.7 ppb) and high (347 ppb) levels of Cd relevant to environmental scenarios elicited a variety of transient, albeit minor changes (<2.5-fold) in tissue GST profiles, including some reductions in GST mRNA expression. In general, olfactory GSTs were the earliest to respond to cadmium, whereas, more pronounced effects in olfactory and gill GST expression were observed at 48 hr relative to earlier time points. Although evaluation of GSTs reflected a cadmium-associated oxidative stress response, there was no clear GST isoform in any tissue that could serve as a reliable biomarker of acute cadmium exposure. By contrast, metallothionein (MT) mRNA was consistently and markedly induced in all three tissues by cadmium, and among the tissues examined, olfactory MT was the most sensitive marker of cadmium exposures. In summary, coho salmon exhibit a complex GST tissue profile consisting of at least 9 isoforms, all of which are present in the peripheral olfactory system. Short-term exposure to environmental levels of Cd causes transient changes in salmon GST consistent with oxidative stress, and in some cases, includes a loss of GST. In a biomarker context, however, monitoring of tissue MT mRNA expression, especially in the peripheral olfactory system, may be of greater utility for assessing short-term environmental exposures to cadmium. PMID:22257444

  1. Partial structure of the mouse glucokinase gene

    SciTech Connect

    Ishimura-Oka, Kazumi; Chu, Mei-Jin; Sullivan, M.; Oka, Kazuhiro

    1995-10-10

    A complementary DNA for glucokinase (GK) was cloned from mouse liver total RNA by a combination of the polymerase chain reaction (PCR) and mouse liver cDNA library screening. Liver- and {beta}-cell-specific exons 1 were isolated by PCR using mouse and rat genomic DNAs. These clones were then used to screen a mouse genomic library; three genomic clones were isolated and characterized. The mouse GK gene spans over 20 kb, containing 11 exons including a liver- or {beta}-cell-specific exon 1, which encodes a tissue-specific 15-aa peptide at the N-terminus of the protein. Both types of GK contain 465 amino acid residues. The predicted amino acid sequence of mouse {beta}-cell-specific GK showed 98 and 96% identity to the rat and human enzymes, respectively; the corresponding values are 98 and 95% respectively, for the liver-specific GK. Several transcription factor-binding consensus sequences are identified in the 5{prime} flanking region of the mouse GK gene. 21 refs., 1 fig.

  2. Identification of two metallothionein genes and their roles in stress responses of Musca domestica toward hyperthermy and cadmium tolerance.

    PubMed

    Tang, Ting; Huang, Da-wei; Zhang, Di; Wu, Yin-jian; Murphy, Robert W; Liu, Feng-song

    2011-10-01

    Stress proteins such as metallothioneins (MTs) play a key role in cellular protection against environmental stressors. In nature, insects such as houseflies (Musca domestica) are commonly exposed to multiple stressors including heavy metals (e.g. Cadmium, Cd) and high temperatures. In this paper, we identify two novel MT genes from the cDNAs of M. domestica, MdMT1 and MdMT2, which putatively encode 40 and 42 amino acid residues respectively. Expression of the two MTs' mRNAs, which are examined in the fat body, gut, hemocyte, and the epidermis. From our study, we saw that the expression of MdMT1 and MdMT2 are enhanced by Cd and thermal stress. Levels of expression are highest at 10 mM Cd(2+) within a 24-h period, and expressions increase significantly with exposure to 10 mM Cd for 12h. Levels of the mRNAs are up-regulated after heat shock and that of MdMT2 reaches its maximum peak faster than MdMT1. Both of the MT genes might be involved in a transient systemic tolerance response to stressors and they may play important roles in heavy metal and high temperature tolerance in the housefly. To detect whether or not the MTs bind heavy metals, the target genes are cloned into the prokaryotic expression vector pET-DsbA to obtain fusion protein expressed in Escherichia coli BL21 (DE3). Recombinant DsbA-MdMT1 significantly increases tolerance of the host bacteria to Cd(2+), but DsbA-MdMT2 is absent. These differential characteristics will facilitate future investigations into the physiological functions of MTs. PMID:21762786

  3. Molecular cloning of a metallothionein-like gene from Nicotiana glutinosa L. and its induction by wounding and tobacco mosaic virus infection.

    PubMed Central

    Choi, D; Kim, H M; Yun, H K; Park, J A; Kim, W T; Bok, S H

    1996-01-01

    The cloning and characterization of genes expressed in plant disease resistance could be an initial step toward understanding the molecular mechanisms of disease resistance. A metallothionein-like gene that is inducible by tobacco mosaic virus and by wounding was cloned in the process of subtractive cloning of disease resistance-response genes in Nicotiana glutinosa. One 530-bp cDNA clone (KC9-10) containing an open reading frame of 81 amino acids was characterized. Genomic Southern blot hybridization with the cDNA probe revealed that tobacco metallothionein-like genes are present in few or in one copy per diploid genome. Northern blot hybridization detected strong induction of a 0.5-kb mRNA by wounding and tobacco mosaic virus infection, but only mild induction was detected when copper was tested as an inducer. Methyl jasmonate, salicylic acid, and ethylene were also tested as possible inducers of this gene, but they had no effect on its expression. The possible role of this gene in wounded and pathogen-stressed plants is discussed. PMID:8819331

  4. Metallothionein gene expression under different time in testicular Sertoli and spermatogenic cells of rats treated with cadmium.

    PubMed

    Ren, Xu Yi; Zhou, Yong; Zhang, Jian Peng; Feng, Wei Hua; Jiao, Bing Hua

    2003-01-01

    The rodent testes are generally more susceptible to cadmium (Cd)-induced toxicity than the liver. To clarify the molecular mechanism underlying tissue and cell differences in Cd sensitivity, we compared metallothionein (MT) gene expression, MT protein accumulation, and Cd retention under different times in freshly isolated testicular Sertoli and spermatogenic cells and liver of rats treated with Cd. Adult male Sprague-Dawley rats received a s.c. injection of 4.0 micromol Cd/kg and 1, 3, 6, or 24h later and untreated animals (0h) tissue were sampled and testicular Sertoli and spermatogenic cells isolated. MT1 and MT2 mRNA levels were determined by semi-quantitative RT-PCR analysis followed by densitometry scanning, and MT was estimated by the enzyme-linked immunosorbent assay (ELISA) method. Cadmium content was determined by atomic absorption spectrophotometry. Testicular lesions were not grossly or histologically observed in rats treated with 4.0 micromol Cd/kg. In the present study, we demonstrated that the rat testis indeed expressed MT1 and MT2, the major isoforms. We also found that untreated animals contained relatively high basal levels of both isoform mRNA, which were increased after Cd treatment in liver and peaked at 3h, followed by a decline, in contrast, the mRNA levels in Sertoli cells peaked at 6h. Interestingly, the induction of MT1 mRNA was lower than MT2 mRNA in Sertoli cells and liver of rats treated with Cd. However, the MT1 mRNA levels of spermatogenic cells decreased 0-3h after Cd treatment, followed by an increase; in contrast, MT2 mRNA levels increased 0-3h after Cd treatment, followed by a reduction, but induced extents of them are lower than those of Sertoli cells and liver. Cd exposure substantially increased hepatic MT, but did not increase MT translation in Sertoli and spermatogenic cells. These results indicate: (1) that Cd-induced MT mRNA expression is cell- and time-dependent; (2) that the inability to induce the metal-detoxicating MT protein in response to Cd, might account for higher susceptibility of testes to Cd toxicity and carcinogenesis relative to liver. PMID:12642155

  5. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    PubMed

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and fluid obtained from metal implant sites. PMID:25594566

  6. Promoter region of mouse Tcrg genes

    SciTech Connect

    Ishimi, Y.; Huang, Y.Y.; Ohta, S.

    1996-06-01

    The mouse T-cell receptor (Tcr){gamma} chain is characterized by a specific expression of V gene segments in the thymus corresponding to consecutive developmental stages; i.e., the Vg5 in fetal, Vg6 in neonatal, and Vg4 and Vg7 in adult. The order of the Vg gene usage correlates with the localization of the Vg gene segment on the chromosome; i.e., the Vg5 gene, being most proximal to the Jg1, is used first, followed by the Vg segments away from the Jg1 in a sequential manner. Since they all rearrange to the same Jg1 gene segment, the sequences in the coding region and/or in the 5{prime} upstream region are responsible for the stage-specific transcription. Also, Goldman and co-workers reported the germline transcription of Vg genes preceding their rearrangement. Therefore, the stage-specific transcription may be involved in the regulation of the stage-specific rearrangement; we sequenced and analyzed the 5{prime} flanking regions of the Vg5, Vg6, Vg4, and Vg7 genes to study the transcriptional relation. 18 refs., 2 figs., 1 tab.

  7. The structure of the mouse parvalbumin gene.

    PubMed

    Schleef, M; Zhlke, C; Jockusch, H; Schffl, F

    1992-01-01

    Parvalbumin (PV) is a calcium-binding protein of the EF-hand family, expressed mainly in fast contracting/relaxing muscles of vertebrates. We have isolated five overlapping genomic PV clones which overall span 28 kilobase pairs (kb) around the Pva locus on mouse Chromosome (Chr) 15. The positions of four introns were determined by DNA sequencing. They interrupt the coding sequences at positions corresponding to those in rat and human PV genes. The transcription start site, 25 bp downstream from the TATA-box, was mapped by oligonucleotide primer extension on poly(A)(+)-RNA. The analysis of 0.4 kb promoter sequence of the mouse PV gene revealed CCAAT- and TATA-box sequences and a 59 bp GC-rich stretch between positions -59 and -118. Similar motifs have been found in the parvalbumin genes of rat and human. A perfect 11-bp repeat upstream to positions -149 and -163 respectively is homologous only to the rat promoter. These results will be related to tissue and species differences in PV expression. PMID:1611216

  8. The biology of novel animal genes: Mouse APEX gene knockout

    SciTech Connect

    MacInnes, M.; Altherr, M.R.; Ludwig, D.; Pedersen, R.; Mold, C.

    1997-07-01

    This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The controlled breeding of novel genes into mice, including the gene knockout (KO), or conversely by adding back transgenes provide powerful genetic technologies that together suffice to determine in large part the biological role(s) of novel genes. Inbred mouse remains the best understood and most useful mammalian experimental system available for tackling the biology of novel genes. The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE), is involved in a key step in the repair of spontaneous and induced AP sites in DNA. Efficient repair of these lesions is imperative to prevent the stable incorporation of mutations into the cellular genome which may lead to cell death or transformation. Loss or modulation of base excison repair activity in vivo may elevate the spontaneous mutation rate in cells, and may lead to a substantial increase in the incidence of cancer. Despite extensive biochemical analysis, however, the significance of these individual APE functions in vivo has not been elucidated. Mouse embryonic stem (ES) cells heterozygous for a deletion mutation in APE have been generated and whole animals containing the APE mutation have been derived from these ES cells. Animals homozygous for the APE null mutation die early in gestation, underscoring the biological significance of this DNA repair gene.

  9. In silico and experimental analyses predict the therapeutic value of an EZH2 inhibitor GSK343 against hepatocellular carcinoma through the induction of metallothionein genes

    PubMed Central

    Liu, Tsang-Pai; Hong, Yi-Han; Tung, Kwang-Yi; Yang, Pei-Ming

    2016-01-01

    There are currently no effective molecular targeted therapies for hepatocellular carcinoma (HCC), the third leading cause of cancer-related death worldwide. Enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 (H3K27)-specific methyltransferase, has been emerged as novel anticancer target. Our previous study has demonstrated that GSK343, an S-adenosyl-L-methionine (SAM)-competitive inhibitor of EZH2, induces autophagy and enhances drug sensitivity in cancer cells including HCC. In this study, an in silico study was performed and found that EZH2 was overexpressed in cancerous tissues of HCC patients at both gene and protein levels. Microarray analysis and in vitro experiments indicated that the anti-HCC activity of GSK343 was associated with the induction of metallothionein (MT) genes. In addition, the negative association of EZH2 and MT1/MT2A genes in cancer cell lines and tissues was found in public gene expression database. Taken together, our findings suggest that EZH2 inhibitors could be a good therapeutic option for HCC, and induction of MT genes was associated with the anti-HCC activity of EZH2 inhibitors.

  10. ScMT2-1-3, a Metallothionein Gene of Sugarcane, Plays an Important Role in the Regulation of Heavy Metal Tolerance/Accumulation

    PubMed Central

    Guo, Jinlong; Xu, Liping; Su, Yachun; Wang, Hengbo; Gao, Shiwu; Xu, Jingsheng; Que, Youxiong

    2013-01-01

    Plant metallothioneins (MTs), which are cysteine-rich, low-molecular-weight, and metal-binding proteins, play important roles in detoxification, metal ion homeostasis, and metal transport adjustment. In this study, a novel metallothionein gene, designated as ScMT2-1-3 (GenBank Accession number JQ627644), was identified from sugarcane. ScMT2-1-3 was 700 bp long, including a 240 bp open reading frame (ORF) encoding 79 amino acid residues. A His-tagged ScMT2-1-3 protein was successfully expressed in Escherichia coli system which had increased the host cell's tolerance to Cd2+, Cu2+, PEG, and NaCl. The expression of ScMT2-1-3 was upregulated under Cu2+ stress but downregulated under Cd2+ stress. Real-time qPCR demonstrated that the expression levels of ScMT2-1-3 in bud and root were over 14 times higher than those in stem and leaf, respectively. Thus, both the E. coli assay and sugarcane plantlets assay suggested that ScMT2-1-3 is significantly involved in the copper detoxification and storage in the cell, but its functional mechanism in cadmium detoxification and storage in sugarcane cells needs more testification though its expressed protein could obviously increase the host E. coli cell's tolerance to Cd2+. ScMT2-1-3 constitutes thus a new interesting candidate for elucidating the molecular mechanisms of MTs-implied plant heavy metal tolerance/accumulation and for developing sugarcane phytoremediator varieties. PMID:23781509

  11. Expression of a metallothionein A1 gene of Pisum sativum in white poplar enhances tolerance and accumulation of zinc and copper.

    PubMed

    Turchi, Adelaide; Tamantini, Ivano; Camussi, Alessandro M; Racchi, Milvia Luisa

    2012-02-01

    Metallothioneins (MT) play an important role in heavy metal detoxification and homeostasis of intracellular metal ions in plant. In this study, two transgenic lines expressing MT type 2 gene (PsMT(A1)) from Pisum sativum, a regenerated non transformed line NT and clone AL22, selected as heavy metal tolerant, were characterized in presence of the heavy metals for the ability to accumulate zinc and copper and to activate antioxidative enzyme defences: superoxide dismutase, catalase, ascorbate peroxidase. The levels of expression of MT type 2 gene assessed by RT-qPCR confirmed the gene over-expression in transgenic lines and evidenced in NT and AL22 the up-regulation of gene transcription by zinc and copper. Transgenic poplar lines during heavy metal stress displayed increased ability to translocate and accumulate zinc and copper compared with NT and AL22. The antioxidant enzyme defence was differently activated in response to metals in the transgenic lines without a significant increase of ROS. These results suggested that PsMT(A1) could play a role in ROS scavenging leading to enhanced metal tolerance and increased zinc and copper sequestration in root and leaf. PMID:22195577

  12. Role of Oxidative Stress in the Induction of Metallothionein-2A and Heme Oxygenase-1 Gene Expression by the Antineoplastic Agent Gallium Nitrate in Human Lymphoma Cells

    PubMed Central

    Yang, Meiying; Chitambar, Christopher R.

    2008-01-01

    The mechanisms of action of gallium nitrate, an antineoplastic drug, are only partly understood. Using a DNA microarray to examine genes induced by gallium nitrate in CCRF-CEM cells, we found that gallium increased metallothionein-2A (MT2A) and heme oxygenase-1 (HO-1) gene expression and altered the levels of other stress-related genes. MT2A and HO-1 were increased after 6 and 16 h of incubation with gallium nitrate. An increase in oxidative stress, evidenced by a decrease in cellular GSH and GSH/GSSG ratio, and an increase in dichlorodihydrofluoroscein (DCF) fluorescence, was seen after 1 – 4 h incubation of cells with gallium nitrate. DCF fluorescence was blocked by the mitochondria-targeted antioxidant mitoquinone. N-acetyl-L-cysteine blocked gallium-induced MT2A and HO-1 expression and increased gallium’s cytotoxicity. Studies with a zinc-specific fluoroprobe suggested that gallium produced an expansion of an intracellular labile zinc pool, suggesting an action of gallium on zinc homeostasis. Gallium nitrate increased the phosphorylation of p38 mitogen-activated protein kinase and activated Nrf-2, a regulator of HO-1 gene transcription. Gallium-induced Nrf-2 activation and HO-1 expression were diminished by a p38 MAP kinase inhibitor. We conclude that gallium nitrate induces cellular oxidative stress as an early event which then triggers the expression of HO-1 and MT2A through different pathways. PMID:18586083

  13. Expression of the rgMT gene, encoding for a rice metallothionein-like protein in Saccharomyces cerevisiae and Arabidopsis thaliana.

    PubMed

    Jin, Shumei; Sun, Dan; Wang, Ji; Li, Ying; Wang, Xinwang; Liu, Shenkui

    2014-12-01

    Metallothioneins (MTs) are cysteine-rich proteins of low molecular weight with many attributed functions, such as providing protection against metal toxicity, being involved in regulation of metal ions uptake that can impact plant physiology and providing protection against oxidative stress. However, the precise function of the metallothionein-like proteins such as the one coded for rgMT gene isolated from rice (Oryza sativa L.) is not completely understood. The whole genome analysis of rice (O. sativa) showed that the rgMT gene is homologue to the Os11g47809 on chromosome 11 of O. sativa sp. japonica genome. This study used the rgMT coding sequence to create transgenic lines to investigate the subcellular localization of the protein, as well as the impact of gene expression in yeast (Saccharomyces cerevisiae) and Arabidopsis thaliana under heavy metal ion, salt and oxidative stresses. The results indicate that the rgMT gene was expressed in the cytoplasm of transgenic cells. Yeast cells transgenic for rgMT showed vigorous growth compared to the nontransgenic controls when exposed to 7 mM CuCl2, 10 mM FeCl2, 1 M NaCl, 24 mM NaHCO3 and 3.2 mM H2O2, but there was no significant difference for other stresses tested. Similarly, Arabidopsis transgenic for rgMT displayed significantly improved seed germination rates over that of the control when the seeds were stressed with 100 ?M CuCl2 or 1 mM H2O2. Increased biomass was observed in the presence of 100 ?M CuCl2, 220 ?M FeCl2, 3 mM Na2CO3, 5 mM NaHCO3 or 1 mM H2O2. These results indicate that the expression of the rice rgMT gene in transgenic yeast and Arabidopsis is implicated in improving their tolerance for certain salt and peroxide stressors. PMID:25572229

  14. Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium.

    PubMed

    Chaturvedi, Ruchi; Archana, G

    2014-06-01

    Due to its exemplary resistance to ionising radiation, oxidative stress, desiccation and several DNA damaging agents, Deinococcus radiodurans R1 (DR1) is considered as one of the most appropriate candidates for the bioremediation of the nuclear waste sites. However, the high sensitivity of this bacterium to heavy metals, which are usually preponderant at nuclear waste dump sites, precludes its application for bioremediation. This study deals with the expression two metal binding peptides in DR1 as an attractive strategy for developing metal tolerance in this bacterium. A synthetic gene (EC20) encoding a phytochelatin analogue with twenty repeating units of glutamate and cysteine was constructed by overlap extension and expressed in DR1. The cyanobacterial metallothionein (MT) gene, smtA was cloned for intracellular expression in DR1. Both the genes were expressed under the native groESL promoter. DR1 strain carrying the recombinant EC20 demonstrated 2.5-fold higher tolerance to Cd(2+) and accumulated 1.21-fold greater Cd(2+) as opposed to the control while the heterologous expression of MT SmtA in DR1 imparted the transformant superior tolerance to Cd(2+) amassing 2.5-fold greater Cd(2+) than DR1 expressing EC20. PMID:24578153

  15. Metallothionein and the Biology of Aging

    PubMed Central

    Swindell, William R.

    2010-01-01

    Metallothionein (MT) is a low molecular weight protein with anti-apoptotic properties that has been demonstrated to scavenge free radicals in vitro. MT has not been extensively investigated within the context of aging biology. The purpose of this review, therefore, is to discuss findings on MT that are relevant to basic aging mechanisms and to draw attention to the possible role of MT in pro-longevity interventions. MT is one of just a handful of proteins that, when overexpressed, has been demonstrated to increase mouse lifespan. MT also protects against development of obesity in mice provided a high fat diet as well as diet-induced oxidative stress damage. Abundance of MT is responsive to caloric restriction (CR) and inhibition of the insulin / insulin-like signaling (IIS) pathway, and elevated MT gene expression has been observed in tissues from fasted and CR-fed mice, long-lived dwarf mice, worms maintained under CR conditions, and long-lived daf-2 mutant worms. The dysregulation of MT in these systems is likely to have tissue-specific effects on aging outcomes. Further investigation will therefore be needed to understand how MT contributes to the response of invertebrates and mice to CR and the endocrine mutations studied by aging researchers. PMID:20933613

  16. Structure and chromosomal localization of the mouse SNAP-23 gene.

    PubMed

    Vaidyanathan, V V; Roche, P A

    2000-04-18

    SNAP-23 plays an important role in the regulation of vesicle trafficking in mammalian cells. In this report, we have determined the exon/intron organization of the mouse SNAP-23 gene. The SNAP-23 gene spans 31kb of the mouse genome and consists of eight exons interrupted by seven introns. The exon organization of the mouse SNAP-23 gene is identical to that of the related SNAP-25 gene in both chicken and Drosophila, suggesting that SNAP-23 arose by duplication of the SNAP-25 gene. Primer extension analysis revealed a major transcription start site approximately 112bp upstream of the translation start site. Like many ubiquitously expressed housekeeping genes, the proximal promoter region for the mouse SNAP-23 gene lacks consensus TATA and CAAT boxes. The SNAP-23 gene was localized to mouse chromosome 2 at band 2E5 using both fluorescence in-situ hybridization and radiation hybrid panel mapping studies. The identification of the structure of the mouse SNAP-23 gene reveals that the overall exon organization of SNAP-25 family members is conserved throughout evolution. PMID:10773458

  17. Cloning and characterization of a tilapia (Oreochromis aureus) metallothionein gene promoter in Hepa-T1 cells following the administration of various heavy metal ions.

    PubMed

    Chan, William Wai Lun; Chan, King Ming

    2008-01-20

    Metallothioneins (MTs) are highly conserved intracellular metal-binding proteins that contribute to the homeostasis of essential metals and the detoxification of non-essential heavy metals. MT gene expression is induced by various heavy metal ions, and Zn(2+) is able to bind and activate a transcription factor associated with the MT gene that is known as the metal responsive element (MRE) binding transcription factor-1 (MTF-1). Heavy metals other than Zn(2+), such as Cd(2+) and Cu(2+), fail to activate the binding of MTF-1 to MREs despite their ability to induce the transcription of the MT gene. To study how different metal ions regulate MT gene expression, a tilapia (ti)-MT gene promoter was cloned and its responses to activation by various metal ions measured using a Hepa T1 cell culture model. The tiMT gene promoter contains six functional MREs within 2118bp 5' of the translational start site. A transient gene expression study showed the tiMT gene promoter fragment to be responsive to Cd(2+), Cu(2+), Hg(2+), Pb(2+), and Zn(2+). Deletions from the 5' end and the site-directed mutagenesis of individual MREs in the tiMT gene promoter confirmed that both proximal and distal clusters of MREs were required for the maximal metal induction of the tiMT gene. The distal cluster of MREs greatly enhanced the induction of tiMT gene expression by several of the heavy metal ions, and especially the non-Zn(2+) ions. Individual MREs showed a different responsiveness to metal ions, with MREe being the most potent, MREb being responsive to Zn(2+) but not to other metal ions, and MREa being mainly for the basal expression of the tiMT gene. Electrophoretic mobility shift assay (EMSA) identified a transcription factor that was able to bind most of the MREs, with the exception of MREd, but the binding was only activated by the in vivo administration of Zn(2+), not the administration of Cd(2+) or Cu(2+). In conclusion, the results of this study on a Hepa T1 cell model suggest that the mechanism of MT gene activation by non-Zn(2+) metal ions is different from that of activation by Zn(2+), and that different MREs may be involved in the activation of the tiMT gene by different metal ions without enhancing the binding of MTF-1 to MREs. PMID:18023887

  18. Mapping of the mouse homologue of the Wilson disease gene to mouse chromosome 8

    SciTech Connect

    Reed, V.; Williamson, P.; Boyd, Y.

    1995-08-10

    ATP7B, the gene altered in Wilson disease (WD) patients, lies in a block of homology shared between human chromosome 13q14 and the central region of mouse chromosome 14. However, we have mapped the murine homologue of ATP7B (Atp7b) to mouse chromosome 8 by somatic cell hybrid analysis. Analysis of 80 interspecific backcross offspring was used to position Atp7b close to D8Mit3 and another ATPase locus, Atp4b, on mouse chromosome 8. ATP4B lies in 13q34 and is separated from ATP7B by several loci whose mouse homologues map to mouse chromosome 14. The assignment of Atp7b to mouse chromosome 8 identifies a previously unrecognized region of homology between this chromosome and human chromosome 13. This assignment suggests a possible location for the toxic milk mutation in the mouse, which has been proposed as a homologue of WD. 17 refs., 2 figs.

  19. Oxidative stress, neurotoxicity, and metallothionein (MT) gene expression in juvenile rock fish Sebastes schlegelii under the different levels of dietary chromium (Cr(6+)) exposure.

    PubMed

    Kim, Jun-Hwan; Kang, Ju-Chan

    2016-03-01

    Juvenile Sebastes schlegelii were exposed for 4 weeks with the different levels of dietary chromium (Cr(6+)) concentration (0, 30, 60, 120 and 200mg/kg). The superoxide dismutase (SOD) activity, glutathione S-transferase (GST) activity, and glutathione (GSH) level of liver and gill were evaluated after 4 weeks exposure. The SOD and GST activity of liver and gill was significantly increased in the concentration of 240mg/kg after 2 weeks and over 120mg/kg after 4 weeks, whereas a considerable decrease in the concentration of 240mg/kg after 2 weeks and over 120mg/kg after 4 weeks was observed in the GSH levels of liver and gill. In neurotoxicity, AChE activity was significatly inhibited in brain in the concentration of 240mg/kg after 2 weeks and over 60mg/kg after 4 weeks and muscle in the concentration of 240mg/kg after 2 weeks and over 120mg/kg after 4 weeks. Metallothionein (MT) gene in liver was considerably increased over 120mg/kg after 2 weeks and at 30, 120, and 240mg/kg after 4 weeks by dietary chromium exposure. The results indicate that dietary Cr exposure over 120mg/kg can induce substantial alterations in antioxidant responses, AChE activity and MT gene expression. PMID:26680530

  20. Effect of duplicate genes on mouse genetic robustness: an update.

    PubMed

    Su, Zhixi; Wang, Junqiang; Gu, Xun

    2014-01-01

    In contrast to S. cerevisiae and C. elegans, analyses based on the current knockout (KO) mouse phenotypes led to the conclusion that duplicate genes had almost no role in mouse genetic robustness. It has been suggested that the bias of mouse KO database toward ancient duplicates may possibly cause this knockout duplicate puzzle, that is, a very similar proportion of essential genes (PE) between duplicate genes and singletons. In this paper, we conducted an extensive and careful analysis for the mouse KO phenotype data and corroborated a strong effect of duplicate genes on mouse genetics robustness. Moreover, the effect of duplicate genes on mouse genetic robustness is duplication-age dependent, which holds after ruling out the potential confounding effect from coding-sequence conservation, protein-protein connectivity, functional bias, or the bias of duplicates generated by whole genome duplication (WGD). Our findings suggest that two factors, the sampling bias toward ancient duplicates and very ancient duplicates with a proportion of essential genes higher than that of singletons, have caused the mouse knockout duplicate puzzle; meanwhile, the effect of genetic buffering may be correlated with sequence conservation as well as protein-protein interactivity. PMID:25110693

  1. Protection against zinc toxicity by metallothionein and zinc transporter 1.

    PubMed

    Palmiter, Richard D

    2004-04-01

    Cells protect themselves from zinc toxicity by inducing proteins such as metallothionein (MT) that bind it tightly, by sequestering it in organelles, or by exporting it. In this study, the interplay between zinc binding by MT and its efflux by zinc transporter 1 (ZnT1) was examined genetically. Inactivation of the Znt1 gene in baby hamster kidney (BHK) cells that do not express their Mt genes results in a zinc-sensitive phenotype and a high level of "free" zinc. Restoration of Mt gene expression increases resistance to zinc toxicity approximately 4-fold, but only slightly reduces free zinc levels. Expression of ZnT1 provides greater protection (approximately 7-fold) and lowers free zinc substantially. Selection for zinc resistance in BHK cells that cannot synthesize either MT or ZnT1 is ineffective. However, parental BHK cells that grow in high concentrations (>500 microM) of zinc can be selected; these cells have amplified their endogenous Znt1 genes. The Znt1 gene is also amplified in zinc-resistant mouse cells that cannot induce their Mt genes. However, if Mt genes can be expressed, then they are preferentially amplified. Thus, both ZnT1 and MT genes contribute to zinc resistance in BHK cells, whereas ZnT1 plays a larger role in regulating free zinc levels. PMID:15041749

  2. Cloning and characterization of TsMT3, a type 3 metallothionein gene from salt cress (Thellungiella salsuginea).

    PubMed

    Quan, Xian Q; Wang, Zeng L; Zhang, Hui; Bi, Yu P

    2008-06-01

    A full-length type 3 plant metallothionein cDNA was isolated from 200 mM NaCl stressed shoots of the salt cress (Thellungiella salsuginea). The 447 bp TsMT3 cDNA sequence has a 207 bp open reading frame (ORF) and encodes a deduced 69 residue peptide of molecular weight 7.52 kDa. Southern blot analysis indicates that, there is only one copy of TsMT3 in the T. salsuginea genome. The accumulation of TsMT3 mRNA is enhanced by the stress imposed by PEG6000, 200 mM NaCl, 50 microM ABA, 4 degrees C, 40 microM CuSO(4) or 25 microM CdCl2. The expression vector pET28-TsMT3 was heterologously expressed in Escherichia coli to define the contribution of TsMT3 to heavy metal tolerance. In the presence of 2 mM CuSO4, 0.3 mM Pb(NO3)2 or 0.4 mM CdCl2, TsMT3 expressing cells exhibited enhanced metal tolerance and accumulated more metal than the controls. We believe that TsMT3 is probably involved in the processes of metal homeostasis, tolerance, and reactive oxygen species (ROS) scavenging. PMID:17852348

  3. Myocardial Overexpression of Mecr, a Gene of Mitochondrial FAS II Leads to Cardiac Dysfunction in Mouse

    PubMed Central

    Chen, Zhijun; Leskinen, Hanna; Liimatta, Erkki; Sormunen, Raija T.; Miinalainen, Ilkka J.; Hassinen, Ilmo E.; Hiltunen, J. Kalervo

    2009-01-01

    It has been recently recognized that mammalian mitochondria contain most, if not all, of the components of fatty acid synthesis type II (FAS II). Among the components identified is 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (Etr1/Mecr), which catalyzes the NADPH-dependent reduction of trans-2-enoyl thioesters, generating saturated acyl-groups. Although the FAS type II pathway is highly conserved, its physiological role in fatty acid synthesis, which apparently occurs simultaneously with breakdown of fatty acids in the same subcellular compartment in mammals, has remained an enigma. To study the in vivo function of the mitochondrial FAS in mammals, with special reference to Mecr, we generated mice overexpressing Mecr under control of the mouse metallothionein-1 promoter. These Mecr transgenic mice developed cardiac abnormalities as demonstrated by echocardiography in vivo, heart perfusion ex vivo, and electron microscopy in situ. Moreover, the Mecr transgenic mice showed decreased performance in endurance exercise testing. Our results showed a ventricular dilatation behind impaired heart function upon Mecr overexpression, concurrent with appearance of dysmorphic mitochondria. Furthermore, the data suggested that inappropriate expression of genes of FAS II can result in the development of hereditary cardiomyopathy. PMID:19440339

  4. Cloning and transcript analysis of type 2 metallothionein gene (SbMT-2) from extreme halophyte Salicornia brachiata and its heterologous expression in E. coli.

    PubMed

    Chaturvedi, Amit Kumar; Mishra, Avinash; Tiwari, Vivekanand; Jha, Bhavanath

    2012-05-15

    Salicornia brachiata is an extreme halophyte growing luxuriantly in the coastal marshes and frequently exposed to various abiotic stresses including heavy metals. A full length type 2 metallothionein (SbMT-2) gene was isolated using RACE and its copy number was confirmed by southern blot analysis. Transcript expression of SbMT-2 gene was analyzed by semi-quantitative Rt-PCR and real time quantitative (qRT) PCR. Expression of SbMT-2 gene was up-regulated concurrently with zinc, copper, salt, heat and drought stress, down regulated by cold stress while unaffected under cadmium stress. Heterologous expression of SbMT-2 gene enhances metal accumulation and tolerance in E. coli. Metal-binding characteristics of SbMT-2 protein show its possible role in homeostasis and/or detoxification of heavy metals. Significant tolerance was observed by E. coli cells expressing recombinant SbMT-2 for Zn(++), Cu(++) and Cd(++) compared to cells expressing GST only. Sequestration of zinc was 4-fold higher compared to copper and in contrast SbMT-2 inhibits the relative accumulation of cadmium by 1.23-fold compared to GST protein. Fusion protein SbMT-2 showed utmost affinity to zinc (approx. 2.5 fold to Cu(++) and Cd(++)) followed by copper and cadmium ions with same affinity. Halophyte S. brachiata has inherent resilience of varying abiotic tolerance therefore SbMT-2 gene could be a potential candidate to be used for enhanced metal tolerance and heavy metal phytoremediation. PMID:22441126

  5. Comprehensive comparative homeobox gene annotation in human and mouse

    PubMed Central

    Wilming, Laurens G.; Boychenko, Veronika; Harrow, Jennifer L.

    2015-01-01

    Homeobox genes are a group of genes coding for transcription factors with a DNA-binding helix-turn-helix structure called a homeodomain and which play a crucial role in pattern formation during embryogenesis. Many homeobox genes are located in clusters and some of these, most notably the HOX genes, are known to have antisense or opposite strand long non-coding RNA (lncRNA) genes that play a regulatory role. Because automated annotation of both gene clusters and non-coding genes is fraught with difficulty (over-prediction, under-prediction, inaccurate transcript structures), we set out to manually annotate all homeobox genes in the mouse and human genomes. This includes all supported splice variants, pseudogenes and both antisense and flanking lncRNAs. One of the areas where manual annotation has a significant advantage is the annotation of duplicated gene clusters. After comprehensive annotation of all homeobox genes and their antisense genes in human and in mouse, we found some discrepancies with the current gene set in RefSeq regarding exact gene structures and coding versus pseudogene locus biotype. We also identified previously un-annotated pseudogenes in the DUX, Rhox and Obox gene clusters, which helped us re-evaluate and update the gene nomenclature in these regions. We found that human homeobox genes are enriched in antisense lncRNA loci, some of which are known to play a role in gene or gene cluster regulation, compared to their mouse orthologues. Of the annotated set of 241 human protein-coding homeobox genes, 98 have an antisense locus (41%) while of the 277 orthologous mouse genes, only 62 protein coding gene have an antisense locus (22%), based on publicly available transcriptional evidence. PMID:26412852

  6. Determination of metallothionein in biological fluids using enzyme-linked immunoassay with commercial antibody.

    PubMed

    Milnerowicz, Halina; Bizoń, Anna

    2010-01-01

    Metallothionein (MT) is a low molecular weight cysteine-rich protein with a number of roles in the pro/antioxidant balance and homeostasis of essential metals, such as zinc and copper, and in the detoxification of heavy metals, such as cadmium and mercury. Until now, detection of metallothionein in biological fluids remained difficult because of a lack of a broadly reactive commercial test. Meaningful comparison of the values of metallothionein concentrations reported by different authors using their specific isolation procedures and different conditions of enzyme-linked immunoassay is difficult due to the absence of a reference material for metallothionein. Therefore in the present study, we describe a quantitative assay for metallothionein in biological fluids such as plasma and urine performed by a direct enzyme-linked immunoassay using a commercially available monoclonal mouse anti-metallothionein clone E9 antibody and commercial standards of metallothionein from rabbit liver and a custom preparation of metallothionein from human liver. The sensitivity of the assay for the standard containing two isoforms MT-I and MT-II from human liver was 140 pg/well. The reactivity of the commercial standards and standards containing two isoforms MT-I and MT-II isolated from human liver in our laboratory with a commercial monoclonal mouse anti-metallothionein clone E9 antibody were similar. This suggests that the described ELISA test can be useful for determination of metallothionein concentration in biological fluids. The concentrations of metallothionein in human plasma, erythrocyte lysate and in urine of smoking and non-smoking healthy volunteers are reported. Tobacco smoking increases the extracellular metallothionein concentration (plasma and urine) but does not affect the intracellular concentration (erythrocyte lysate). PMID:20349027

  7. Inducible and combinatorial gene manipulation in mouse brain

    PubMed Central

    Dogbevia, Godwin K.; Marticorena-Alvarez, Ricardo; Bausen, Melanie; Sprengel, Rolf; Hasan, Mazahir T.

    2015-01-01

    We have deployed recombinant adeno-associated viruses equipped with tetracycline-controlled genetic switches to manipulate gene expression in mouse brain. Here, we show a combinatorial genetic approach for inducible, cell type-specific gene expression and Cre/loxP mediated gene recombination in different brain regions. Our chemical-genetic approach will help to investigate when, where, and how gene(s) control neuronal circuit dynamics, and organize, for example, sensory signal processing, learning and memory, and behavior. PMID:25954155

  8. Inducible and combinatorial gene manipulation in mouse brain.

    PubMed

    Dogbevia, Godwin K; Marticorena-Alvarez, Ricardo; Bausen, Melanie; Sprengel, Rolf; Hasan, Mazahir T

    2015-01-01

    We have deployed recombinant adeno-associated viruses equipped with tetracycline-controlled genetic switches to manipulate gene expression in mouse brain. Here, we show a combinatorial genetic approach for inducible, cell type-specific gene expression and Cre/loxP mediated gene recombination in different brain regions. Our chemical-genetic approach will help to investigate 'when', 'where', and 'how' gene(s) control neuronal circuit dynamics, and organize, for example, sensory signal processing, learning and memory, and behavior. PMID:25954155

  9. A reanalysis of mouse ENCODE comparative gene expression data

    PubMed Central

    Gilad, Yoav; Mizrahi-Man, Orna

    2015-01-01

    Recently, the Mouse ENCODE Consortium reported that comparative gene expression data from human and mouse tend to cluster more by species rather than by tissue. This observation was surprising, as it contradicted much of the comparative gene regulatory data collected previously, as well as the common notion that major developmental pathways are highly conserved across a wide range of species, in particular across mammals. Here we show that the Mouse ENCODE gene expression data were collected using a flawed study design, which confounded sequencing batch (namely, the assignment of samples to sequencing flowcells and lanes) with species. When we account for the batch effect, the corrected comparative gene expression data from human and mouse tend to cluster by tissue, not by species. PMID:26236466

  10. Metallothionein-protein interactions.

    PubMed

    Atrian, Slvia; Capdevila, Merc

    2013-04-01

    Metallothioneins (MTs) are a family of universal, small proteins, sharing a high cysteine content and an optimal capacity for metal ion coordination. They take part in a plethora of metal ion-related events (from detoxification to homeostasis, storage, and delivery), in a wide range of stress responses, and in different pathological processes (tumorigenesis, neurodegeneration, and inflammation). The information on both intracellular and extracellular interactions of MTs with other proteins is here comprehensively reviewed. In mammalian kidney, MT1/MT2 interact with megalin and related receptors, and with the transporter transthyretin. Most of the mammalian MT partners identified concern interactions with central nervous system (mainly brain) proteins, both through physical contact or metal exchange reactions. Physical interactions mainly involve neuronal secretion multimers. Regarding metal swap events, brain MT3 appears to control the metal ion load in peptides whose aggregation leads to neurodegenerative disorders, such as A? peptide, ?-synuclein, and prion proteins (Alzheimer's and Parkinson's diseases, and spongiform encephalopathies, respectively). Interaction with ferritin and bovine serum albumin are also documented. The intercourse of MTs with zinc-dependent enzymes and transcription factors is capable to activate/deactivate them, thus conferring MTs the role of metabolic and gene expression regulators. As some of these proteins are involved in cell cycle and proliferation control (p53, nuclear factor ?B, and PKC?), they are considered in the context of oncogenesis and tumor progression. Only one non-mammalian MT interaction, involving Drosophila MtnA and MtnB major isoforms and peroxiredoxins, has been reported. The prospective use for biomedical applications of the MT-interaction information is finally discussed. PMID:25436572

  11. Transcriptional response of two metallothionein genes (OcMT1 and OcMT2) and histological changes in Oxya chinensis (Orthoptera: Acridoidea) exposed to three trace metals.

    PubMed

    Liu, Yaoming; Wu, Haihua; Yu, Zhitao; Guo, Yaping; Zhang, Jianzhen; Zhu, Kun Yan; Ma, Enbo

    2015-11-01

    This study evaluated the transcriptional responses of two metallothionein (MT) genes (OcMT1 and OcMT2) in various tissues (brain, optic lobe, Malpighian tubules, fat bodies, foregut, gastric caeca, midgut and hindgut) of Oxya chinensis (Thunberg) (Orthoptera: Acridoidea) after exposed to the trace metals cadmium (Cd), copper (Cu) and zinc (Zn) for 48h. The study revealed that the exposure of O. chinensis to each of the three metals at the median lethal concentration (LC50) or lower concentration(s) up-regulated the transcriptions of both OcMT1 and OCMT2 in the eight tissues except for OcMT1 and OcMT2 with Cd in brain and gastric caeca, respectively, and OcMT2 with Cu in gastric caeca. These results suggested that the exposure of O. chinensis to the metals may enhance MT biosynthesis that protects tissues by binding these metals in various tissues. To examine possible histopathological effect of the metals, we examined the histological changes in the fat bodies after O. chinensis was exposed to each of these metals at LC50. The exposure of Cd significantly reduced the size and number of adipocytes as compared with the control. However, such an effect was not observed in O. chinensis exposed to either Cu or Zn. These results suggested that fat bodies might be either significantly affected by Cd or play a crucial role in detoxification of excessive trace metals. PMID:26159299

  12. The heterologous expression of the Iris lactea var. chinensis type 2 metallothionein IlMT2b gene enhances copper tolerance in Arabidopsis thaliana.

    PubMed

    Gu, Chun-Sun; Liu, Liang-Qin; Deng, Yan-Ming; Zhu, Xu-Dong; Huang, Su-Zhen; Lu, Xiao-Qing

    2015-02-01

    Iris lactea var. chinensis (I. lactea var. chinensis) is a widely adapted perennial species with a high level of copper tolerance. To evaluate the role of metallothioneins (MTs) in copper tolerance in I. lactea var. chinensis, a full-length cDNA homologue of MT2, designated IlMT2b (GenBank accession No. AB907788), was cloned using the RACE-PCR method. The expression level of IlMT2b in the leaves and roots of I. lactea var. chinensis was induced in response to copper (Cu) treatment. Ectopic expression of IlMT2b in Arabidopsis thaliana increased the Cu concentration and reduced H2O2 production in the transgenic plants. After treatment with 50 and 100 μM Cu, the root length of two transgenic seedlings was respectively about 1.5- and 3-fold longer than that of the wild-type. Together, these results suggested that IlMT2b may represent a useful target gene for the phytoremediation of Cu-polluted soil. PMID:25533567

  13. Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins

    PubMed Central

    Holmes, Roger S.; Wright, Matthew W.; Laulederkind, Stanley J. F.; Cox, Laura A.; Hosokawa, Masakiyo; Imai, Teruko; Ishibashi, Shun; Lehner, Richard; Miyazaki, Masao; Perkins, Everett J.; Potter, Phillip M.; Redinbo, Matthew R.; Robert, Jacques; Satoh, Tetsuo; Yamashita, Tetsuro; Yan, Bingfan; Yokoi, Tsuyoshi; Zechner, Rudolf; Maltais, Lois J.

    2011-01-01

    Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and “CES” (human) and “Ces” (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding “P” and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species. PMID:20931200

  14. Activation of Pattern Recognition Receptors Upregulates Metallothioneins, Thereby Increasing Intracellular Accumulation of Zinc, Autophagy, and Bacterial Clearance by Macrophages

    PubMed Central

    Lahiri, Amit; Abraham, Clara

    2014-01-01

    Background & Aims Continuous stimulation of pattern recognition receptors (PRRs), including nucleotide-binding oligomerization domain-2 (NOD2) (variants in NOD2 have been associated with Crohn's disease), alters the phenotype of myeloid-derived cells, reducing production of inflammatory cytokines and increasing clearance of microbes. We investigated the mechanisms by which microbial clearance increases in macrophages under these conditions. METHODS Monocytes were purified from human peripheral blood mononuclear cells and differentiated to monocyte-derived macrophages (MDMs). We also isolated human intestinal macrophages. Bacterial clearance by MDMs was assessed in gentamicin protection assays. Effects of intracellular zinc and autophagy were measured by flow cytometry, immunoblot, reverse transcription PCR, and microscopy experiments. Small interfering RNAs were used to knock down specific proteins in MDMs. NOD2/ and C57BL/6J mice, maintained in a specific pathogen-free facility, were given antibiotics, muramyl dipeptide (to stimulate NOD2), or dextran sodium sulfate; intestinal lamina propria cells were collected and analyzed. RESULTS Chronic stimulation of human MDMs through NOD2 upregulated the expression of multiple genes encoding metallothioneins, which bind and regulate levels of intracellular zinc. Intestinal myeloid-derived cells are continually stimulated through PRRs; metallothionein expression was upregulated in human and mouse intestinal myeloid-derived cells. Continuous stimulation of NOD2 increased levels of intracellular zinc, thereby increasing autophagy and bacterial clearance. The metal-regulatory transcription factor-1 (MTF-1) was required for regulation of metallothionein genes in human MDMs. Knockdown of MTF-1 did not affect baseline clearance of bacteria by MDMs. However, the increase in intracellular zinc, autophagy, and bacterial clearance observed with continuous NOD2 stimulation was impaired in MDMs upon MTF-1 knockdown. Addition of zinc or induction of autophagy restored bacterial clearance to MDMs following metallothionein knockdown. NOD2 synergized with the PRRs TLR5 and TLR9 to increase the effects of metallothioneins in MDMs. In mice, the intestinal microbiota contributed to the regulation in expression of metallothioneins, levels of zinc, autophagy, and bacterial clearance by intestinal macrophages. CONCLUSIONS In studies of human MDMs and in mice, continuous stimulation of PRRs induces expression of metallothioneins. This leads to increased levels of intracellular zinc and enhanced clearance of bacteria via autophagy in macrophages. PMID:24960189

  15. Differential gene expression in mouse retina related to regional differences in vulnerability to hyperoxia

    PubMed Central

    Natoli, Riccardo; Valter, Krisztina; Stone, Jonathan

    2010-01-01

    Purpose In the C57BL/6J mouse retina, hyperoxia-induced degeneration of photoreceptors shows strong regional variation, beginning at a locus ~0.5 mm inferior to the optic disc. To identify gene expression differences that might underlie this variability in vulnerability, we have used microarray techniques to describe regional (superior-inferior) variations in gene expression in the retina. Methods Young adult C57BL/6J mice raised in dim cyclic illumination (12 h at 5 lx and 12 h in darkness) were exposed to hyperoxia (75% oxygen for two weeks). Retinas were collected from hyperoxia-exposed and control animals without fixation and divided into superior and inferior halves. RNA was extracted from each sample, purified, and hybridized to Mouse Gene 1.0 ST arrays (Affymetrix). The consistency of the microarray results was assessed using quantitative PCR for selected genes. Expression data were analyzed to identify genes and ncRNAs whose differential expression between the superior and inferior retina could be associated with relative vulnerability to hyperoxia. Results In control retinas, only two genes showed a fold difference in expression >2 between the superior and inferior retina; another 25 showed a fold difference of 1.5–2.0. Of these 27, the functions of six genes, including ventral anterior homeobox containing gene 2 (Vax2) and T-box 5 (Tbox5), are related to parameters of anatomic development and the functions of five are related to sensory perception. Among the latter, short-wave-sensitive cone opsin (Opn1sw) was more strongly expressed in the inferior retina and medium-wave-sensitive cone opsin (Opn1mw) in the superior retina. This is consistent with known differences in S- and M-cone distribution, confirming our separation of retinal regions. The highest fold difference was reported for membrane metalloendopeptidase (Mme), a member from the metallothionein group of cytoprotective proteins. To identify genes whose regulation by hyperoxia was significantly different between the inferior and superior retina, we calculated the “fold margin” (FM, the difference between hyperoxia-induced regulation in the inferior and superior retina) for each gene, and identified genes for which abs(FM) > 0.5. Genes thus identified numbered 112, and included many immune-, cell defense-, and inflammation- related genes. Conclusions Gene expression analysis revealed relatively subtle differences between inferior and superior regions of control C57BL/6J retinas, with only 27 genes showing an expression difference >1.5 fold. Among these, genes related to cytoprotection and apoptosis were included, along with genes related to central projections and cone-type differences. After hyperoxia-induced photoreceptor degeneration had begun, the number of genes that showed significant expression differences between the inferior and superior retina more than quadrupled, with genes related to immune processes, defense processes, and inflammation being numerically dominant. PMID:20454693

  16. Immunologic Applications of Conditional Gene Modification Technology in the Mouse

    PubMed Central

    Sharma, Suveena; Zhu, Jinfang

    2014-01-01

    Since the success of homologous recombination in altering mouse genome and the discovery of Cre-loxP system, the combination of these two breakthroughs has created important applications for studying the immune system in the mouse. Here, we briefly summarize the general principles of this technology and its applications in studying immune cell development and responses; such implications include conditional gene knockout and inducible and/or tissue-specific gene over-expression, as well as lineage fate mapping. We then discuss the pros and cons of a few commonly used Cre-expressing mouse lines for studying lymphocyte development and functions. We also raise several general issues, such as efficiency of gene deletion, leaky activity of Cre, and Cre toxicity, all of which may have profound impacts on data interpretation. Finally, we selectively list some useful links to the Web sites as valuable mouse resources. PMID:24700321

  17. Characteristics of the mouse genomic histamine H1 receptor gene

    SciTech Connect

    Inoue, Isao; Taniuchi, Ichiro; Kitamura, Daisuke

    1996-08-15

    We report here the molecular cloning of a mouse histamine H1 receptor gene. The protein deduced from the nucleotide sequence is composed of 488 amino acid residues with characteristic properties of GTP binding protein-coupled receptors. Our results suggest that the mouse histamine H1 receptor gene is a single locus, and no related sequences were detected. Interspecific backcross analysis indicated that the mouse histamine H1 receptor gene (Hrh1) is located in the central region of mouse Chromosome 6 linked to microphthalmia (Mitfmi), ras-related fibrosarcoma oncogene 1 (Raf1), and ret proto-oncogene (Ret) in a region of homology with human chromosome 3p. 12 refs., 3 figs.

  18. RADIOIMMUNOASSAY OF METALLOTHIONEIN

    EPA Science Inventory

    The goal of this project was to develop a radioimmunoassay for metallothionein. Since this protein is involved with the transport of cadmium in biological systems and may in fact protect against cadmium poisoning, the ability to monitor the levels in the human population is of th...

  19. Cloning of the mouse steroid sulfatase (Sts) gene

    SciTech Connect

    Salido, E.C. |; Li, X.M.; Shapiro, L.J.

    1994-09-01

    In humans, the STS gene is located on the distal short arm of the X chromosome, proximal to the pseudoautosomal region (PAR). STS activity can be detected in mouse tissues using the same substrates as for the human STS assay, and quantitative differences in STS levels among various mouse strains allowed the mapping of Sts to the PAR. However, several attempts to clone the mouse Sts gene using human reagents have failed, which has been taken as evidence of substantial divergence between these genes. We report the cloning of the mouse Sts gene by using the Sts cDNA from an intermediate species, the rat. The coding region of the rat Sts cDNA was used as a probe to screen mouse fibroblast and liver cDNA libraries, and 5 clones were isolated. DNA sequence of the 2.5 kb cDNA revealed 75% similarity with rat Sts, while it was only 63% and 60% similar to the human STS at the DNA and protein levels, respectively. Interestingly, the mouse Sts cDNA revealed a high GC content, including 225 CpG dinucleotides in the coding region, compared to 88 and 44 CpGs in the same regions of the rat and human STS genes, respectively. Despite the low degree of conservation between these genes, all the point mutations described so far in human STS-deficient patients occur at amino acid residues that are conserved between these three species. Using a panel of mouse-hamster somatic cell hybrids, the mouse Sts cDNA sequences were mapped to the mouse X and Y chromosomes, with restriction fragments of the same size for both chromosomes, consistent with localization of Sts in the PAR. The pseudoautosomal pattern of inheritance was ascertained in back-crosses between C3H/An and SW mice, making use of STS activity assays and RFLPs. RT-PCR experiments using cDNA from a panel of hamster-mouse somatic cell hybrids containing either the inactive or the active X chromosome indicated that the mouse Sts gene escapes X-inactivation, as expected for a pseudoautosomal gene.

  20. Cloning, characterization and targeting of the mouse HEXA gene

    SciTech Connect

    Wakamatsu, N.; Trasler, J.M.; Gravel, R.A.

    1994-09-01

    The HEXA gene, encoding the {alpha} subunit of {beta}-hexosaminidase A, is essential for the metabolism of ganglioside G{sub M2}, and defects in this gene cause Tay-Sachs disease in humans. To elucidate the role of the gene in the nervous system of the mouse and to establish a mouse model of Tay-Sachs disease, we have cloned and characterized the HEXA gene and targeted a disruption of the gene in mouse ES cells. The mouse HEXA gene spans {approximately}26 kb and consists of 14 exons, similar to the human gene. A heterogeneous transcription initiation site was identified 21-42 bp 5{prime} of the initiator ATG, with two of the sites fitting the consensus CTCA (A = start) as seen for some weak initiator systems. Promoter analysis showed that the first 150 bp 5{prime} of the ATG contained 85% of promoter activity observed in constructs containing up to 1050 bp of 5{prime} sequence. The active region contained a sequence matching that of the adenovirus major late promoter upstream element factor. A survey of mouse tissues showed that the highest mRNA levels were in (max to min): testis (5.5 x brain cortex), adrenal, epididymis, heart, brain, lung, kidney, and liver (0.3 x brain cortex). A 12 kb BstI/SalI fragment containing nine exons was disrupted with the insertion of the bacterial neo{sup r} gene in exon 11 and was targeted into 129/Sv ES cells by homologous recombination. Nine of 153 G418 resistant clones were correctly targeted as confirmed by Southern blotting. The heterozygous ES cells were microinjected into mouse blastocysts and implanted into pseudo-pregnant mice. Nine male chimeric mice, showing that 40-95% chimerism for the 129/Sv agouti coat color marker, are being bred in an effort to generate germline transmission of the disrupted HEXA gene.

  1. Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR

    SciTech Connect

    Zorita, I.; Bilbao, E.; Schad, A.; Cancio, I.; Soto, M.; Cajaraville, M.P. . E-mail: mirenp.cajaraville@ehu.es

    2007-04-15

    Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles and oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv{sub BAS}) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv{sub BAS} increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals.

  2. Structure of a cluster of mouse histone genes.

    PubMed Central

    Sittman, D B; Graves, R A; Marzluff, W F

    1983-01-01

    The four mouse histone genes (2 H3 genes, an H2b gene and an H2a gene) present in a cloned 12.9 kilobase fragment of DNA have been completely sequenced including both 5' and 3' flanking regions. These genes are expressed in cultured mouse cells and the 3' and 5' ends of the mRNA have been determined by S1 nuclease mapping. These genes code for a minor fraction of the histone mRNAs expressed in cultured mouse cells. They comprise at most 5-8% of the total histone mRNA of each type. The two H3 genes code for H3.2 and H3.1 histone proteins, while the H2b gene codes for an H2b.1 protein with a single amino acid change (val-leu) at position 18. Only the 3' portion of the H2a gene is contained in the clone and there is an amino acid change (alanine-proline) at position 126. Comparison of the 5' and 3' flanking sequences reveals a conserved sequence at the 3' end of the mRNA which forms a hairpin loop structure. The codon usage in the genes is non-random and there has been no discrimination against CG doublets in the coding region of the genes. Images PMID:6314253

  3. Database for exchangeable gene trap clones: pathway and gene ontology analysis of exchangeable gene trap clone mouse lines.

    PubMed

    Araki, Masatake; Nakahara, Mai; Muta, Mayumi; Itou, Miharu; Yanai, Chika; Yamazoe, Fumika; Miyake, Mikiko; Morita, Ayaka; Araki, Miyuki; Okamoto, Yoshiyuki; Nakagata, Naomi; Yoshinobu, Kumiko; Yamamura, Ken-ichi; Araki, Kimi

    2014-02-01

    Gene trapping in embryonic stem (ES) cells is a proven method for large-scale random insertional mutagenesis in the mouse genome. We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox-mediated recombination. We isolated trap clones, analyzed trapped genes, and constructed the database for Exchangeable Gene Trap Clones (EGTC) [http://egtc.jp]. The number of registered ES cell lines was 1162 on 31 August 2013. We also established 454 mouse lines from trap ES clones and deposited them in the mouse embryo bank at the Center for Animal Resources and Development, Kumamoto University, Japan. The EGTC database is the most extensive academic resource for gene-trap mouse lines. Because we used a promoter-trap strategy, all trapped genes were expressed in ES cells. To understand the general characteristics of the trapped genes in the EGTC library, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analysis and found that the EGTC ES clones covered a broad range of pathways. We also used Gene Ontology (GO) classification data provided by Mouse Genome Informatics (MGI) to compare the functional distribution of genes in each GO term between trapped genes in the EGTC mouse lines and total genes annotated in MGI. We found the functional distributions for the trapped genes in the EGTC mouse lines and for the RefSeq genes for the whole mouse genome were similar, indicating that the EGTC mouse lines had trapped a wide range of mouse genes. PMID:24444128

  4. Clustering of cytokine genes on mouse chromosome 11.

    PubMed

    Wilson, S D; Billings, P R; D'Eustachio, P; Fournier, R E; Geissler, E; Lalley, P A; Burd, P R; Housman, D E; Taylor, B A; Dorf, M E

    1990-04-01

    The presence of positionally conserved amino acid residues suggests that the mouse proteins TCA3, P500, MIP1-alpha, MIP1-beta, and JE are members of a single gene family. These proteins are activation specific and can be expressed by both myeloid and lymphoid cells. MIP1-alpha/MIP1-beta and MCAF (the putative human homologue of JE) act as chemotactic and activating agents for neutrophils and macrophages, respectively. The functions of TCA3 and P500 are unknown. We have used interspecies somatic cell hybrids and recombinant inbred mouse strains to show that the genes encoding TCA3, MIP1-alpha, MIP1-beta, and JE (provisionally termed Tca3, Mip-1a, Mip-1b, and Sigje, respectively) map as a cluster on the distal portion of mouse chromosome 11 near the Hox-2 gene complex. DNA sequence analysis indicates that the P500 and TCA3 proteins are encoded by alternative splicing products of one genomic gene. Additionally, the genes encoding TCA3 and JE are found to be strikingly similar with respect to the positions of intron-exon boundaries. Together, these data support the model that the cytokines TCA3, P500, MIP1-alpha, MIP1-beta, and JE are encoded by a single cluster of related genes. The gene encoding IL-5 (Il-5), which acts as a T cell-replacing factor, a B cell growth factor, and an eosinophil differentiation factor, is also mapped to mouse chromosome 11.Il-5 maps approximately 25 cM proximal to the Tca-3 gene and appears tightly linked to a previously described gene cluster that includes Il-3, Il-4, and Csfgm. We discuss the potential relevance of the two cytokine gene clusters described here with particular attention to specific human hematologic malignancies associated with chromosomal aberrations at corresponding locations on human chromosomes 5 and 17. PMID:1969921

  5. Genomic Sequence Analysis of the Mouse Naip Gene Array

    PubMed Central

    Endrizzi, Matthew G.; Hadinoto, Vey; Growney, Joseph D.; Miller, Webb; Dietrich, William F.

    2000-01-01

    A mouse locus called Lgn1 determines differences in macrophage permissiveness for the intracellular replication of Legionella pneumophila. The only regional candidate genes for this phenotype difference lie within a cluster of closely linked paralogs of the Neuronal Apoptosis Inhibitory Protein (Naip) gene. Previous genetic and physical mapping of the Lgn1 phenotype narrowed it to an interval containing only Naip2 and Naip5, suggesting that there is not complete functional overlap among the mouse Naip loci. In order to gather more information about polymorphisms among the Naip genes of the 129 mouse haplotype, we have determined the genomic sequence of a substantial portion of the 129 Naip gene array. We have constructed an evolutionary model for the expansion of the Naip gene array from a single progenitor Naip gene. This model predicts the presence of two distinct families of Naip paralogs: Naip1/2/3 and Naip4/5/6/7. Unlike the divergences among all the other Naip paralogs, the splits among Naip4, Naip5, Naip6, and Naip7 occurred relatively recently. The high degree of sequence conservation within the Naip4/5/6/7 family increases the likelihood of functional overlap among these genes. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF242431-AF242435.] PMID:10958627

  6. ECOLOGICAL RISK ASSESSMENT OF ALFALFA (MEDICAGO VARIA L.) GENETICLALY ENGINEERED TO EXPRESS A HUMAN METALLOTHIONEIN (HMT) GENE

    EPA Science Inventory

    The objectives of these studies were two-fold: (1) to determine efficacy of low and high expression hMT gene constructs by assessing accumulation of Cu in shoots of parental and transgenic plants of alfalfa (Medicago varia L.) exposed to different concentrations of CuSO4 by addit...

  7. Characterization of the mouse Kid1 gene and identification of a highly related gene, Kid2.

    PubMed

    Tekki-Kessaris, N; Bonventre, J V; Boulter, C A

    1999-11-15

    Kid1 encodes a zinc finger protein that has been implicated in renal cell differentiation. Levels of Kid1 mRNA correlate with maturation of kidney tubule epithelia in rat post-natal kidney development and during kidney regeneration following injury. KID1 is a putative transcriptional repressor, containing a KRAB domain at its amino terminus that mediates transcriptional repression in transient cell transfection assays when fused to a heterologous DNA-binding domain. In this paper, we describe the isolation and characterization of the mouse homologue of Kid1 and the identification of a novel highly related mouse gene, Kid2, Kid1 and Kid2 are tightly linked on mouse chromosome 11 and show conservation across mammals. Both genes are expressed predominantly in the mouse adult kidney and brain, but transcripts are also detected in embryonic brain, kidney, gut and lung, suggesting an additional role for these genes during mouse development. PMID:10564808

  8. Metallothionein prolongs survival and antagonizes senescence-associated cardiomyocyte diastolic dysfunction: role of oxidative stress.

    PubMed

    Yang, Xiaoping; Doser, Thomas A; Fang, Cindy X; Nunn, Jennifer M; Janardhanan, Rajiv; Zhu, Meijun; Sreejayan, Nair; Quinn, Mark T; Ren, Jun

    2006-05-01

    Senescence is accompanied by oxidative stress and cardiac dysfunction, although the link between the two remains unclear. This study examined the role of antioxidant metallothionein on cardiomyocyte function, superoxide generation, the oxidative stress biomarker aconitase activity, cytochrome c release, and expression of oxidative stress-related proteins, such as the GTPase RhoA and NADPH oxidase protein p47phox in young (5-6 mo) and aged (26-28 mo) FVB wild-type (WT) and cardiac-specific metallothionein transgenic mice. Metallothionein mice showed a longer life span (by approximately 4 mo) than FVB mice evaluated by the Kaplan-Meier survival curve. Compared with young cardiomyocytes, aged myocytes displayed prolonged TR(90), reduced tolerance to high stimulus frequency, and slowed intracellular Ca2+ decay, all of which were nullified by metallothionein. Aging increased superoxide generation, active RhoA abundance, cytochrome c release, and p47phox expression and suppressed aconitase activity without affecting protein nitrotyrosine formation in the hearts. These aging-induced changes in oxidative stress and related protein biomarkers were attenuated by metallothionein. Aged metallothionein mouse myocytes were more resistant to the superoxide donor pyrogallol-induced superoxide generation and apoptosis. In addition, aging-associated prolongation in TR90 was blunted by the Rho kinase inhibitor Y-27632. Collectively, our data demonstrated that metallothionein may alleviate aging-induced cardiac contractile defects and oxidative stress, which may contribute to prolonged life span in metallothionein transgenic mice. PMID:16585059

  9. Increased metallothionein gene expression, zinc, and zinc-dependent resistance to apoptosis in circulating monocytes during HIV viremia.

    PubMed

    Raymond, Andrea D; Gekonge, Bethsebah; Giri, Malavika S; Hancock, Aidan; Papasavvas, Emmanouil; Chehimi, Jihed; Kossenkov, Andrew V; Kossevkov, Andrew V; Nicols, Calen; Yousef, Malik; Mounzer, Karam; Shull, Jane; Kostman, Jay; Showe, Louise; Montaner, Luis J

    2010-09-01

    Circulating monocytes exhibit an apoptotic resistance phenotype during HIV viremia in association with increased MT expression. MTs are known to play an important role in zinc metabolism and immune function. We now show, in a cross-sectional study using peripheral monocytes, that expression of MT1 isoforms E, G, H, and X is increased significantly in circulating monocyte cells from HIV+ subjects during chronic viremic episodes as compared with uninfected subjects. This increase in expression is also observed during acute viremia following interruption of suppressive ART. Circulating monocytes from HIV+ donors were also found to have elevated zinc importer gene Zip8 expression in conjunction with elevated intracellular zinc levels in contrast to CD4(+)T-lymphocytes. In vitro HIV-1 infection studies with elutriated MDM confirm a direct relation between HIV-1 infection and increased MDM MT1 (isoform G) gene expression and increased intracellular zinc levels. A direct link between elevated zinc levels and apoptosis resistance was established using a cell-permeable zinc chelator TPEN, which reversed apoptosis resistance effectively in monocytes from HIV-infected to levels comparable with uninfected controls. Taken together, increases in MT gene expression and intracellular zinc levels may contribute directly to maintenance of an immune-activated monocyte by mediating an increased resistance to apoptosis during active HIV-1 viremia. PMID:20551211

  10. Identification and characterisation of imprinted genes in the mouse.

    PubMed

    Peters, Jo; Beechey, Colin

    2004-02-01

    Imprinted genes are expressed specifically from one or other parental allele. Over 70 are now known, and about one-half of these are expressed from the paternal allele and one-half from the maternal allele. Most imprinted genes are clustered within imprinting regions of the mouse genome, regions which are associated with abnormal phenotypes when inherited uniparentally. Imprinted genes have been identified from surveys based on differential expression or differential methylation according to parental origin, as well as analyses of candidate genes, mutants and imprinted gene clusters. Many imprinted genes affect growth and development, and more than 25 per cent determine non-coding RNAs that may have a function in controlling imprinted gene expression. PMID:15163367

  11. Differences in gene expression between mouse and human for dynamically regulated genes in early embryo.

    PubMed

    Madissoon, Elo; Töhönen, Virpi; Vesterlund, Liselotte; Katayama, Shintaro; Unneberg, Per; Inzunza, Jose; Hovatta, Outi; Kere, Juha

    2014-01-01

    Infertility is a worldwide concern that can be treated with in vitro fertilization (IVF). Improvements in IVF and infertility treatment depend largely on better understanding of the molecular mechanisms for human preimplantation development. Several large-scale studies have been conducted to identify gene expression patterns for the first five days of human development, and many functional studies utilize mouse as a model system. We have identified genes of possible importance for this time period by analyzing human microarray data and available data from online databases. We selected 70 candidate genes for human preimplantation development and investigated their expression in the early mouse development from oocyte to the 8-cell stage. Maternally loaded genes expectedly decreased in expression during development both in human and mouse. We discovered that 25 significantly upregulated genes after fertilization in human included 13 genes whose orthologs in mouse behaved differently and mimicked the expression profile of maternally expressed genes. Our findings highlight many significant differences in gene expression patterns during mouse and human preimplantation development. We also describe four cancer-testis antigen families that are also highly expressed in human embryos: PRAME, SSX, GAGE and MAGEA. PMID:25089626

  12. MouseNet v2: a database of gene networks for studying the laboratory mouse and eight other model vertebrates

    PubMed Central

    Kim, Eiru; Hwang, Sohyun; Kim, Hyojin; Shim, Hongseok; Kang, Byunghee; Yang, Sunmo; Shim, Jae Ho; Shin, Seung Yeon; Marcotte, Edward M.; Lee, Insuk

    2016-01-01

    Laboratory mouse, Mus musculus, is one of the most important animal tools in biomedical research. Functional characterization of the mouse genes, hence, has been a long-standing goal in mammalian and human genetics. Although large-scale knockout phenotyping is under progress by international collaborative efforts, a large portion of mouse genome is still poorly characterized for cellular functions and associations with disease phenotypes. A genome-scale functional network of mouse genes, MouseNet, was previously developed in context of MouseFunc competition, which allowed only limited input data for network inferences. Here, we present an improved mouse co-functional network, MouseNet v2 (available at http://www.inetbio.org/mousenet), which covers 17 714 genes (>88% of coding genome) with 788 080 links, along with a companion web server for network-assisted functional hypothesis generation. The network database has been substantially improved by large expansion of genomics data. For example, MouseNet v2 database contains 183 co-expression networks inferred from 8154 public microarray samples. We demonstrated that MouseNet v2 is predictive for mammalian phenotypes as well as human diseases, which suggests its usefulness in discovery of novel disease genes and dissection of disease pathways. Furthermore, MouseNet v2 database provides functional networks for eight other vertebrate models used in various research fields. PMID:26527726

  13. MouseNet v2: a database of gene networks for studying the laboratory mouse and eight other model vertebrates.

    PubMed

    Kim, Eiru; Hwang, Sohyun; Kim, Hyojin; Shim, Hongseok; Kang, Byunghee; Yang, Sunmo; Shim, Jae Ho; Shin, Seung Yeon; Marcotte, Edward M; Lee, Insuk

    2016-01-01

    Laboratory mouse, Mus musculus, is one of the most important animal tools in biomedical research. Functional characterization of the mouse genes, hence, has been a long-standing goal in mammalian and human genetics. Although large-scale knockout phenotyping is under progress by international collaborative efforts, a large portion of mouse genome is still poorly characterized for cellular functions and associations with disease phenotypes. A genome-scale functional network of mouse genes, MouseNet, was previously developed in context of MouseFunc competition, which allowed only limited input data for network inferences. Here, we present an improved mouse co-functional network, MouseNet v2 (available at http://www.inetbio.org/mousenet), which covers 17 714 genes (>88% of coding genome) with 788 080 links, along with a companion web server for network-assisted functional hypothesis generation. The network database has been substantially improved by large expansion of genomics data. For example, MouseNet v2 database contains 183 co-expression networks inferred from 8154 public microarray samples. We demonstrated that MouseNet v2 is predictive for mammalian phenotypes as well as human diseases, which suggests its usefulness in discovery of novel disease genes and dissection of disease pathways. Furthermore, MouseNet v2 database provides functional networks for eight other vertebrate models used in various research fields. PMID:26527726

  14. Structure of mammalian metallothionein

    SciTech Connect

    Kaegi, J.H.R.; Vasak, M.; Lerch, K.; Gilg, D.E.O.; Hunziker, P.; Bernhard, W.R.; Good, M.

    1984-03-01

    All mammalian metallothioneins characterized contain a single polypeptide chain of 61 amino acid residues, among them 20 cysteines providing the ligands for seven metal-binding sites. Native metallothioneins are usually heterogeneous in metal composition, with Zn, Cd, and Cu occurring in varying proportions. However, forms containing only a single metal species, i.e., Zn, Cd, Ni, Co, Hg, Pb, Bi, have now been prepared by in vitro reconstitution from the metal-free apoprotein. By spectroscopic analysis of such derivatives it was established that all cysteine residues participate in metal binding, that each metal ion is bound to four thiolate ligands, and that the symmetry of each complex is close to that of a tetrahedron. To satisfy the requirements of the overall Me/sub 7/(Cys/sup -/)/sub 20/ stoichiometry, the complexes must be combined to form metal-thiolate cluster structures. The actual spatial organization of the clusters and the polypeptide chain remains to be established. An attractive possibility is the arrangement of the tetrahedral metal-thiolates in adamantane-like structures surrounded by properly folded segments of the chain providing the ligands. /sup 1/H-NMR data and infrared absorption measurements are consistent with a tightly folded structure rich in ..beta..-type conformation. 79 references, 11 figures, 4 tables.

  15. Comparing the evolutionary conservation between human essential genes, human orthologs of mouse essential genes and human housekeeping genes.

    PubMed

    Lv, Wenhua; Zheng, Jiajia; Luan, Meiwei; Shi, Miao; Zhu, Hongjie; Zhang, Mingming; Lv, Hongchao; Shang, Zhenwei; Duan, Lian; Zhang, Ruijie; Jiang, Yongshuai

    2015-11-01

    Human housekeeping genes are often confused with essential human genes, and several studies regard both types of genes as having the same level of evolutionary conservation. However, this is not necessarily the case. To clarify this, we compared the differences between human housekeeping genes and essential human genes with respect to four aspects: the evolutionary rate (dN/dS), protein sequence identity, single-nucleotide polymorphism (SNP) density and level of linkage disequilibrium (LD). The results showed that housekeeping genes had lower evolutionary rates, higher sequence identities, lower SNP densities and higher levels of LD compared with essential genes. Together, these findings indicate that housekeeping and essential genes are two distinct types of genes, and that housekeeping genes have a higher level of evolutionary conservation. Therefore, we suggest that researchers should pay careful attention to the distinctions between housekeeping genes and essential genes. Moreover, it is still controversial whether we should substitute human orthologs of mouse essential genes for human essential genes. Therefore, we compared the evolutionary features between human orthologs of mouse essential genes and human housekeeping genes and we got inconsistent results in long-term and short-term evolutionary characteristics implying the irrationality of simply replacing human essential genes with human orthologs of mouse essential genes. PMID:25911641

  16. Genomic organization of mouse Fc gamma receptor genes.

    PubMed

    Kulczycki, A; Webber, J; Soares, H A; Onken, M D; Thompson, J A; Chaplin, D D; Loh, D Y; Tillinghast, J P

    1990-04-01

    We have isolated and characterized the gene coding for the mouse Fc receptor that is termed Fc gamma RIIa. The gene contains five exons and spans approximately 9 kilobases. Unlike most members of the immunoglobulin gene superfamily, this gene utilizes multiple exons to encode its leader peptide. The first exon encodes the hydrophobic region of the signal sequence; the second exon, which contains only 21 base pairs, encodes a segment of the signal peptidase recognition site; and the beginning of the third exon encodes the predicted site of peptidase cleavage. The third and fourth exons each code for immunoglobulin-like extracellular domains. The fifth exon encodes the hydrophobic transmembrane domain and the cytoplasmic tail. Partial characterization of the Fc gamma RIIb gene indicates that it also contains multiple leader exons, including a 21-base-pair exon and two exons coding for homologous immunoglobulin-like extracellular domains. However, the Fc gamma RIIb gene uses four exons to encode its intracytoplasmic region. Analysis using contour-clamped homogeneous electric field (CHEF) gels indicates that the Fc gamma RIIa and Fc gamma RIIb genes are linked within 160 kilobases on mouse chromosome 1. PMID:2138787

  17. Structure and chromosomal localization of the mouse oncomodulin gene.

    PubMed

    Staubli, F; Klein, A; Rentsch, J M; Hameister, H; Berchtold, M W

    1995-11-01

    The rat gene encoding oncomodulin (OM), a small calcium-binding protein, is under the control of a solo LTR derived from an endogenous intracisternal A-particle. The latter sequence is the only OM promoter analyzed so far. In order to study cell type-specific OM expression in a species lacking LTR sequences in the OM locus, we initially synthesized an OM cDNA from mouse placenta. By sequencing, we found a 137-bp-long 5'leader region that differed markedly from its rat counterpart but had high similarity to several mouse genomic sequences. Primers specific to this sequence in addition with primers specific for an exon 2/intron 2 sequence were used to screen a mouse ES cell line genomic P1 library. One positive clone contained the whole OM gene, including intron 1 of 25kb and a 5' flanking region of 27 kb lacking an LTR. The region upstream of exon 1 contains no TATA or CCAAT boxes but has a homopurine/homopyrimidine stretch of 102 bp as well as a (CA)22 repeat. The latter sequence is polymorphic and was therefore, used to map the OM gene to the distal end of the long arm of mouse Chromosome (Chr) 5 by interspecific backcross analysis. Additionally we localized the OM gene by in situ hybridization to the region G1-3 on Chr 5, confirming the genetic linkage results. Finally, the OM gene was found to be structurally conserved and to exist in a single copy in mammals. PMID:8597631

  18. A new spontaneous mouse mutation in the Kcne1 gene.

    PubMed

    Letts, V A; Valenzuela, A; Dunbar, C; Zheng, Q Y; Johnson, K R; Frankel, W N

    2000-10-01

    A new mouse mutant, punk rocker (allele symbol Kcne1(pkr)), arose spontaneously on a C57BL/10J inbred strain background and is characterized by a distinctive head-tossing, circling, and ataxic phenotype. It is also profoundly and bilaterally deaf. The mutation resides in the Kcne1 gene on Chromosome (Chr) 16 and has been identified as a single base change within the coding region of the third exon. The C to T nucleotide substitution causes an arginine to be altered to a termination codon at amino acid position 67, and predictably this will result in a significantly truncated protein product. The Kcne1(pkr) mutant represents the first spontaneous mouse model for the human disorder, Jervell and Lange-Nielsen syndrome, associated with mutations in the homologous KCNE1 gene on human Chr 21. PMID:11003695

  19. Cloning and chromosome localization of the mouse Ews gene

    SciTech Connect

    Plougastel, B.; Thomas, G.; Delattre, O.; Mattei, M.G.

    1994-09-01

    The human EWS gene encodes a putative RNA binding protein. As a result of acquired chromosome rearrangement, the N-terminal portion of the EWS protein is fused to the DNA binding domain of either FLI1-or ERG in the Ewing family of tumors and to the DNA binding domain of ATF1 in malignant melanoma of soft parts. We have determined the cDNA sequence of the mouse Ews gene. Its nucleotide sequence and its translation product demonstrate 93 and 98% homology with the human EWS cDNA and protein, respectively. The murine Ews locus lies within a conserved synteny segment between human chromosome 22q12 and mouse chromosome 11A1-A3.

  20. DEVELOPMENT OF A 950-GENE DNA ARRAY FOR EXAMINING GENE EXPRESSION PATTERNS IN MOUSE TESTIS

    EPA Science Inventory

    Development of a 950-gene DNA array for examining gene expression patterns in mouse testis.

    Rockett JC, Christopher Luft J, Brian Garges J, Krawetz SA, Hughes MR, Hee Kirn K, Oudes AJ, Dix DJ.

    Reproductive Toxicology Division, National Health and Environmental Effec...

  1. An oligonucleotide microarray for mouse imprinted genes profiling.

    PubMed

    Vig, A; Gallou-Kabani, C; Gross, M S; Fabre, A; Junien, C; Jais, J P

    2006-01-01

    Genomic imprinting is an epigenetic phenomenon unique to mammals that causes some genes to be expressed according to their parental origin. It results in developmental asymmetry in the function of the parental genomes. We describe here a method for the profiling of imprinted genes based on the development of a mouse imprinting microchip containing oligonucleotides corresponding to 493 genes, including most of the known imprinted genes (IG = 63), genes involved in epigenetic processes (EPI = 15), in metabolism (= 147), in obesity (= 10) and in neurotransmission (= 256) and housekeeping reference genes (= 2). This custom oligonucleotide microarray has been constructed to make data analysis and handling more manageable than pangenomic microarrays. As a proof of concept we present the differential expression of these 493 genes in different tissues (liver, placenta, embryo) of C57BL6/J mice fed different diets. Appropriate experimental strategies and statistical tools were defined at each step of the data analysis process with regard to the different sources of constraints. Data were confirmed by expression analyses based on quantitative real-time PCR. These oligochips should make it possible to increase our understanding of the involvement of imprinted genes in the timing of expression programs, tissue by tissue, stage by stage, in response to nutrients, lifestyles and other as yet unknown critical environmental factors in a variety of physiopathological situations, and in animals of different strains, ages and sexes. The use of oligonucleotides makes it possible to expand this microchip to include the increasing number of imprinted genes discovered. PMID:16575188

  2. Gene expression profiling of mouse embryos with microarrays

    PubMed Central

    Sharov, Alexei A.; Piao, Yulan; Ko, Minoru S. H.

    2011-01-01

    Global expression profiling by DNA microarrays provides a snapshot of cell and tissue status and becomes an essential tool in biological and medical sciences. Typical questions that can be addressed by microarray analysis in developmental biology include: (1) to find a set of genes expressed in a specific cell type; (2) to identify genes expressed commonly in multiple cell types; (3) to follow the time-course changes of gene expression patterns; (4) to demonstrate cell’s identity by showing similarities or differences among two or multiple cell types; (5) to find regulatory pathways and/or networks affected by gene manipulations, such as overexpression or repression of gene expression; (6) to find downstream target genes of transcription factors; (7) to find downstream target genes of cell signaling; (8) to examine the effects of environmental manipulation of cells on gene expression patterns; and (9) to find the effects of genetic manipulation in embryos and adults. Here we describe strategies for executing these experiments and monitoring changes of cell state with gene expression microarrays in application to mouse embryology. Both statistical assessment and interpretation of data are discussed. We also present a protocol for performing microarray analysis on a small amount of embryonic materials. PMID:20699157

  3. MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY MUTAGENS IN THE TK GENE OF MOUSE LYMPHOMA CELLS

    EPA Science Inventory

    MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY BROMATE AND N- ETHYL-N-NITROSOUREA IN THE TK GENE OF MOUSE L YMPHOMA CELLS

    The mouse lymphoma assay is widely used to identify chemical mutagens The Tk +1- gene located on an autosome in mouse lymphoma cells may recover a wide ra...

  4. Functional and evolutionary analyses on expressed intronless genes in the mouse genome

    SciTech Connect

    Sakharkar, K R; Sakharkar, M K; Culiat, Cymbeline T; Chow, V T K; Pervaiz, S

    2006-01-01

    Using computational approaches we have identified 2017 expressed intronless genes in the mouse genome. Evolutionary analysis reveals that 56 intronless genes are conserved among the three domains of life - bacteria, archea and eukaryotes. These highly conserved intronless genes were found to be involved in essential housekeeping functions. About 80% of expressed mouse intronless genes have orthologs in eukaryotic genomes only, and thus are specific to eukaryotic organisms. 608 of these genes have intronless human orthologs and 302 of these orthologs have a match in OMIM database. Investigation into these mouse genes will be important in generating mouse models for understanding human diseases.

  5. A Mouse Model for Imprinting of the Human Retinoblastoma Gene

    PubMed Central

    Tasiou, Vasiliki; Hiber, Michaela; Steenpass, Laura

    2015-01-01

    The human RB1 gene is imprinted due to integration of the PPP1R26P1 pseudogene into intron 2. PPP1R26P1 harbors the gametic differentially methylated region of the RB1 gene, CpG85, which is methylated in the female germ line. The paternally unmethylated CpG85 acts as promoter for the alternative transcript 2B of RB1, which interferes with expression of full-length RB1 in cis. In mice, PPP1R26P1 is not present in the Rb1 gene and Rb1 is not imprinted. Assuming that the mechanisms responsible for genomic imprinting are conserved, we investigated if imprinting of mouse Rb1 can be induced by transferring human PPP1R26P1 into mouse Rb1. We generated humanized Rb1_PPP1R26P1 knock-in mice that pass human PPP1R26P1 through the mouse germ line. We found that the function of unmethylated CpG85 as promoter for an alternative Rb1 transcript and as cis-repressor of the main Rb1 transcript is maintained in mouse tissues. However, CpG85 is not recognized as a gametic differentially methylated region in the mouse germ line. DNA methylation at CpG85 is acquired only in tissues of neuroectodermal origin, independent of parental transmission of PPP1R26P1. Absence of CpG85 methylation in oocytes and sperm implies a failure of imprint methylation establishment in the germ line. Our results indicate that site-specific integration of a proven human gametic differentially methylated region is not sufficient for acquisition of DNA methylation in the mouse germ line, even if promoter function of the element is maintained. This suggests a considerable dependency of DNA methylation induction on the surrounding sequence. However, our model is suited to determine the cellular function of the alternative Rb1 transcript. PMID:26275142

  6. Gene structure and expression of phospholemman in mouse.

    PubMed

    Bogaev, R C; Jia, L G; Kobayashi, Y M; Palmer, C J; Mounsey, J P; Moorman, J R; Jones, L R; Tucker, A L

    2001-06-13

    Phospholemman (PLM) is a small transmembrane cardiac protein that is the major sarcolemmal substrate for phosphorylation in response to adrenergic stimulation. PLM likely plays a role in muscle contractility and cell volume regulation through its function as a channel or a channel regulator. We are the first to describe the structure of the PLM gene and to demonstrate PLM cDNA splice variants. We cloned the murine PLM cDNA and used it as a probe to isolate the gene from a 129/SvJ genomic library. The gene contains seven introns and eight exons. The coding sequence is interrupted by five introns; the 5' untranslated region by two. Using rapid amplification of 5' cDNA ends we identified transcription start sites and four splice variants of the 5' untranslated domain. There was no TATA box or CAAT box in the putative promoter regions. The gene has several stretches of dinucleotide repeats. The 3' untranslated domains of mouse PLM cDNA clones show sequence differences not accounted for by alternative splicing. Mouse PLM shares 93, 83 and 80% amino acid identity with rat, dog, and human PLMs, respectively. Tissue expression of murine PLM parallels that in other species, being highest in heart, skeletal muscle, and liver. PMID:11410367

  7. Genetic mapping of tumor susceptibility genes involved in mouse plasmacytomagenesis.

    PubMed Central

    Mock, B A; Krall, M M; Dosik, J K

    1993-01-01

    Plasmacytomas (PCTs) were induced in 47% of BALB/cAnPt mice by the intraperitoneal injection of pristane, in 2% of (BALB/c x DBA/2N)F1, and in 11% of 773 BALB/cAnPt x (BALB/cAnPt x DBA/2N)F1 N2 backcross mice. This result indicates a multigenic mode of inheritance for PCT susceptibility. To locate genes controlling this complex genetic trait, tumor susceptibility in backcross progeny generated from BALB/c and DBA/2N (resistant) mice was correlated with alleles of 83 marker loci. The genotypes of the PCT-susceptible progeny displayed an excess homozygosity for BALB/c alleles within a 32-centimorgan stretch of mouse chromosome 4 (> 95% probability of linkage) with minimal recombination (12%) near Gt10. Another susceptibility gene on mouse chromosome 1 may be linked to Fcgr2 (90% probability of linkage); there were excess heterozygotes for Fcgr2 among the susceptible progeny and excess homozygotes among the resistant progeny. Regions of mouse chromosomes 4 and 1 that are correlated with PCT susceptibility share extensive linkage homology with regions of human chromosome 1 that have been associated with cytogenetic abnormalities in multiple myeloma and lymphoid, breast, and endocrine tumors. PMID:8105477

  8. Genetic mapping of tumor susceptibility genes involved in mouse plasmacytomagenesis

    SciTech Connect

    Mock, B.A.; Krall, M.M.; Dosik, J.K. )

    1993-10-15

    Plasmacytomas (PCTs) were induced in 47% of BALB/cAnPt mice by the intraperitoneal injection of pristane, in 2% of (BALB/c [times] DBA/2N)F[sub 1], and in 11% of 773 BALB/cAnPt [times] (BALB/cAnPt [times] DBA/2N)F[sub 1]N[sub 2] backcross mice. This result indicates a multigenic mode of inheritance for PCT susceptibility. To locate genes controlling this complex genetic trait, tumor susceptibility in backcross progeny generated from BALB/c and DBA/2N (resistant) mice was correlated with alleles of 83 marker loci. The genotypes of the PCT-susceptible progeny displayed an excess homozygosity for BALB/c alleles with a 32-centimorgan stretch of mouse chromosome 4 (>95% probability of linkage) with minimal recombination (12%) near Gt10. Another susceptibility gene on mouse chromosome 1 may be linked to Fcgr2 (90% probability of linkage); there were excess heterozygotes for Fcgr2 among the susceptible progeny and excess homozygotes among the resistant progeny. Regions of mouse chromosomes 4 and 1 that are correlated with PCT susceptibility share extensive linkage homology with regions of human chromosome 1 that have been associated with cytogenetic abnormalities in multiple myeloma and lymphoid, breast, and endocrine tumors. 68 refs., 2 figs., 1 tab.

  9. Role of metallothionein in murine experimental colitis.

    PubMed

    Tsuji, Toshifumi; Naito, Yuji; Takagi, Tomohisa; Kugai, Munehiro; Yoriki, Hiroyuki; Horie, Ryusuke; Fukui, Akifumi; Mizushima, Katsura; Hirai, Yasuko; Katada, Kazuhiro; Kamada, Kazuhiro; Uchiyama, Kazuhiko; Handa, Osamu; Konishi, Hideyuki; Yagi, Nobuaki; Ichikawa, Hiroshi; Yanagisawa, Rie; Suzuki, Junko S; Takano, Hirohisa; Satoh, Masahiko; Yoshikawa, Toshikazu

    2013-05-01

    Metallothioneins (MTs) are a family of cysteine-rich low molecular-weight proteins that can act as reactive oxygen species scavengers. Although it is known that the induction of MT expression suppresses various inflammatory disorders, the role of MTs in intestinal inflammation remains unclear. In this study, we investigated the effects of dextran sulfate sodium (DSS) administration in mice with targeted deletions of the MT-I/II genes. Acute colitis was induced by 2% DSS in male MT-I/II double knockout (MT-null) and C57BL/6 (wild-type) mice. The disease activity index (DAI) was determined on a daily basis for each animal, and consisted of a calculated score based on changes in body weight, stool consistency and intestinal bleeding. Histology, colon length, myeloperoxidase (MPO) activity and colonic mRNA expression and the concentration of inflammatory cytokines were evaluated by real-time-PCR and enzyme-linked immunosorbent assay (ELISA). The localization of MTs and macrophages was determined by immunohistological and immunofluorescence staining. To investigate the role of MTs in macrophages, peritoneal macrophages were isolated and their responses to lipopolysaccharide were measured. Following DSS administration, the DAI score increased in a time-dependent manner and was significantly enhanced in the MT-I/II knockout mice. Colonic MPO activity levels and inflammatory cytokines [tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-17] production increased following DSS administration, and these increases were significantly enhanced in the MT-I/II knockout mice compared with the wild-type mice. MT-positive cells were detected in the lamina propria and submucosal layer by immunohistochemical and immunofluorescence staining, and were mainly co-localized in F4/80-positive macrophages. The production of inflammatory cytokines (TNF-α, IFN-γ and IL-17) from isolated peritoneal macrophages increased following lipopolysaccharide stimulation, and these increases were significantly enhanced in the macrophages obtained from the MT-I/II knockout mice. These data indicate that MTs play an important role in the prevention of colonic mucosal inflammation in a mouse model of DSS-induced colitis, thus suggesting that endogenous MTs play a protective role against intestinal inflammation. PMID:23467591

  10. Overlapping gene expression profiles of cell migration and tumor invasion in human bladder cancer identify metallothionein 1E and nicotinamide N-methyltransferase as novel regulators of cell migration.

    PubMed

    Wu, Y; Siadaty, M S; Berens, M E; Hampton, G M; Theodorescu, D

    2008-11-01

    Cell migration is essential to cancer invasion and metastasis and is spatially and temporally integrated through transcriptionally dependent and independent mechanisms. As cell migration is studied in vitro, it is important to identify genes that both drive cell migration and are biologically relevant in promoting invasion and metastasis in patients with cancer. Here, gene expression profiling and a high-throughput cell migration system answers this question in human bladder cancer. In vitro migration rates of 40 microarray-profiled human bladder cancer cell lines were measured by radial migration assay. Genes whose expression was either directly or inversely associated with cell migration rate were identified and subsequently evaluated for their association with cancer stage in 61 patients. This analysis identified genes known to be associated with cell invasion such as versican, and novel ones, including metallothionein 1E (MT1E) and nicotinamide N-methyltransferase (NNMT), whose expression correlated positively with cancer cell migration and tumor stage. Using loss of function analysis, we show that MT1E and NNMT are necessary for cancer cell migration. These studies provide a general approach to identify the clinically relevant genes in cancer cell migration and mechanistically implicate two novel genes in this process in human bladder cancer. PMID:18724390

  11. Cloning and expression of metallothionein mutant alpha-KKS-alpha in Anabaena sp. PCC 7120.

    PubMed

    Shao, Qiang; Shi, Ding-Ji; Hao, Fu-Ying; Ma, Li-Na; Chen, Zhen-Jia; Yu, Mei-Min; Ru, Bing-Gen

    2002-01-01

    The mouse metallothionein (mMT) mutant alpha-KKS-alpha has a higher capacity for binding heavy metals than wild type mMT. The mMT mutant alpha-KKS-alpha gene was placed under the control of the strong promoter PpbsA to generate the intermediate vector pRL-alpha-KKS-alpha. pRLalpha-KKS-alpha was then linked with the plasmid pDC-08 to construct shuttle expression vector pDC-alphaKKS-alpha. This expression vector was transformed into Anabaena sp. PCC 7120 using triparental conjugative transfer. After antibiotic selection (ampicillin and kanamycin), transgenic Anabaena was identified by PCR and Western blotting. The expression level of the mMT mutation alpha-KKS-alpha reached 7.4 mg/g dry cells weight, as detected by ELISA, and heavy metal resistance of the transgenic Anabaena was significantly improved. PMID:12398381

  12. Gene Expression by Mouse Inner Ear Hair Cells during Development.

    PubMed

    Scheffer, Dborah I; Shen, Jun; Corey, David P; Chen, Zheng-Yi

    2015-04-22

    Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from E16 to P7, we performed a comprehensive cell type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes. PMID:25904789

  13. Identification of gene signatures regulated by carvedilol in mouse heart.

    PubMed

    Teoh, Jian-Peng; Park, Kyoung-Mi; Broskova, Zuzana; Jimenez, Felix R; Bayoumi, Ahmed S; Archer, Krystal; Su, Huabo; Johnson, John; Weintraub, Neal L; Tang, Yaoliang; Kim, Il-Man

    2015-09-01

    Chronic treatment with the β-blocker carvedilol has been shown to reduce established maladaptive left ventricle (LV) hypertrophy and to improve LV function in experimental heart failure. However, the detailed mechanisms by which carvedilol improves LV failure are incompletely understood. We previously showed that carvedilol is a β-arrestin-biased β1-adrenergic receptor ligand, which activates cellular pathways in the heart independent of G protein-mediated second messenger signaling. More recently, we have demonstrated by microRNA (miR) microarray analysis that carvedilol upregulates a subset of mature and pre-mature miRs, but not their primary miR transcripts in mouse hearts. Here, we next sought to identify the effects of carvedilol on LV gene expression on a genome-wide basis. Adult mice were treated with carvedilol or vehicle for 1 wk. RNA was isolated from LV tissue and hybridized for microarray analysis. Gene expression profiling analysis revealed a small group of genes differentially expressed after carvedilol treatment. Further analysis categorized these genes into pathways involved in tight junction, malaria, viral myocarditis, glycosaminoglycan biosynthesis, and arrhythmogenic right ventricular cardiomyopathy. Genes encoding proteins in the tight junction, malaria, and viral myocarditis pathways were upregulated in the LV by carvedilol, while genes encoding proteins in the glycosaminoglycan biosynthesis and arrhythmogenic right ventricular cardiomyopathy pathways were downregulated by carvedilol. These gene expression changes may reflect the molecular mechanisms that underlie the functional benefits of carvedilol therapy. PMID:26152686

  14. Gene Expression by Mouse Inner Ear Hair Cells during Development

    PubMed Central

    Scheffer, Déborah I.; Shen, Jun

    2015-01-01

    Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from E16 to P7, we performed a comprehensive cell type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes. PMID:25904789

  15. AAV-mediated gene transfer to the mouse CNS

    PubMed Central

    Stoica, Lorelei; Ahmed, Seemin S.

    2013-01-01

    Recombinant adeno associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. Recombinant AAVs have been used at various ages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the CNS, depending on the stage of development of the mouse. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to also target the adult CNS by intravascular injections. rAAVs have been successfully used as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. PMID:23686825

  16. Metallothioneins: Structure and Functions.

    PubMed

    Dziegiel, Piotr; Pula, Bartosz; Kobierzycki, Christopher; Stasiolek, Mariusz; Podhorska-Okolow, Marzenna

    2016-01-01

    All metallothioneins (MTs) possess a highly conserved amino acid sequence and present only a few structural changes even when isolated from different animal species. In mammals, a single MT molecule is made up of 61-68 amino acids, depending on the isoform (the MT-1, MT-2, and MT-4 isoforms consist of 61-62 amino acids, whereas the MT-3 isoform comprises 68 amino acids), and the protein sequence is composed of up to 20 cysteine (Cys) residues (Vasak 2005; Vasak and Meloni 2011). Furthermore, in mammals, no aromatic amino acids are found in the MT molecules. Protein sequencing has revealed that the MT molecule is a single polypeptide chain, in which the Cys residues are organized in the sequences Cys-X-Cys, Cys-X-X-Cys, and Cys-Cys, where "X" denotes an amino acid other than Cys (Kojima et al. 1976; Huang and Yoshida 1977). The Cys residues are the metal-binding domains of the MT molecule, in which they are juxtaposed with lysine (Lys) and arginine (Arg) amino acid residues and arranged in two thiol-rich sites designated domains ? and ? (Fig. 2.1). The two metal-binding domains are separated by a non-cysteine-containing sequence often designated as the spacer or linker (Zangger et al. 2001; Babula et al. 2012). The ?-domain consists of amino acids 31-68 and is located on the C-terminal edge, whereas the N-terminal ?-domain contains amino acids 1-30 (Zangger et al. 2001; Dziegiel 2004). It has been demonstrated that the ?-domain is capable of binding up to four, and the ?-domain up to three, bivalent metal ions such as zinc, cadmium, mercury, or lead (Coyle et al. 2002b; Duncan et al. 2006). The part of the protein with no bound metal ions is termed apo-metallothionein (apo-MT) or thionein (Coyle et al. 2002b). Metallothioneins are also capable of reacting with up to 12 univalent metal ions (Palmiter 1998; Coyle et al. 2002b). Zinc ions, which naturally occur in the organism, are regarded as the main binding partner of apo-MT. However, other nonessential metal ions occurring pathologically in the organism-such as lead, copper, cadmium, mercury, platinum, chromate, bismuth, and silver-often possess higher affinity to the apo-MT-binding sites (Nordberg and Nordberg 2000; Ngu and Stillman 2009; Ngu et al. 2010b; Gumulec et al. 2011; Babula et al. 2012). So far, only iron ions (Fe(2+)) have been identified to possess lower affinity to the metal-binding sites of the apo-MT domains (Foster and Robinson 2011). Interestingly, only a small proportion of MT molecules was found bound to zinc ions in various organisms. In rat tissues, apo-MT has been shown to constitute up to 54% of the total amount of MT, whereas higher apo-MT levels were detected in rat cancer cells (Yang et al. 2001). Recent studies have also identified small amounts of sulfide ligands bound to recombinant MT-1 and MT-4 proteins overexpressed in Escherichia coli (Capdevila et al. 2005; Tio et al. 2006). Nevertheless, studies analyzing MT proteins in the cytoplasm of mammalian cells have failed to detect sulfide ligands bound to their molecules (Mounicou et al. 2010). PMID:26847564

  17. Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability

    PubMed Central

    Ruiz, Oscar N.; Alvarez, Derry; Torres, Cesar; Roman, Laura; Daniell, Henry

    2015-01-01

    Summary Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy. Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183 000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 ?m mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioniens in plants for mercury phytoremediation. Chloroplast genetic engineering approach is useful to express metal-scavenging proteins for phytoremediation. PMID:21518240

  18. The mouse Gene Expression Database (GXD): 2014 update.

    PubMed

    Smith, Constance M; Finger, Jacqueline H; Hayamizu, Terry F; McCright, Ingeborg J; Xu, Jingxia; Berghout, Joanne; Campbell, Jeff; Corbani, Lori E; Forthofer, Kim L; Frost, Pete J; Miers, Dave; Shaw, David R; Stone, Kevin R; Eppig, Janan T; Kadin, James A; Richardson, Joel E; Ringwald, Martin

    2014-01-01

    The Gene Expression Database (GXD; http://www.informatics.jax.org/expression.shtml) is an extensive and well-curated community resource of mouse developmental expression information. GXD collects different types of expression data from studies of wild-type and mutant mice, covering all developmental stages and including data from RNA in situ hybridization, immunohistochemistry, RT-PCR, northern blot and western blot experiments. The data are acquired from the scientific literature and from researchers, including groups doing large-scale expression studies. Integration with the other data in Mouse Genome Informatics (MGI) and interconnections with other databases places GXD's gene expression information in the larger biological and biomedical context. Since the last report, the utility of GXD has been greatly enhanced by the addition of new data and by the implementation of more powerful and versatile search and display features. Web interface enhancements include the capability to search for expression data for genes associated with specific phenotypes and/or human diseases; new, more interactive data summaries; easy downloading of data; direct searches of expression images via associated metadata; and new displays that combine image data and their associated annotations. At present, GXD includes >1.4 million expression results and 250,000 images that are accessible to our search tools. PMID:24163257

  19. EMAGE mouse embryo spatial gene expression database: 2010 update.

    PubMed

    Richardson, Lorna; Venkataraman, Shanmugasundaram; Stevenson, Peter; Yang, Yiya; Burton, Nicholas; Rao, Jianguo; Fisher, Malcolm; Baldock, Richard A; Davidson, Duncan R; Christiansen, Jeffrey H

    2010-01-01

    EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (approximately 19,000 gene) 'EURExpress' dataset into EMAGE. PMID:19767607

  20. ATM localization and gene expression in the adult mouse eye

    PubMed Central

    Leemput, Julia; Masson, Christel; Bigot, Karine; Errachid, Abdelmounaim; Dansault, Anouk; Provost, Alexandra; Gadin, Stphanie; Aoufouchi, Said; Menasche, Maurice

    2009-01-01

    Purpose High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. Methods Atm gene expression was analyzed by RTPCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue sections, with a special focus on retinal cells. Results Using RTPCR, we detected a band of the expected size, with its sequence matching the amplified Atm cDNA sequence. Atm mRNA was detected in most cell bodies of the adult mouse eye by in situ hybridization of ocular tissue sections with specific digoxigenin-labeled PCR-amplified cDNA probes. Western blotting with different specific antibodies revealed bands corresponding to the expected sizes of ATM and its active forms (ATMp). These bands were not observed in the analysis of protein homogenates from Atm-deficient mouse tissues. ATM immunoreactivity was detected in the nucleus of all adult mice retinal cells and in most non-neuronal ocular cell types. The active phosphorylated form of ATM was also present in the retina as well as in non-neuronal cells of the adult mouse eye. However, its subcellular localization differed as a function of the cell type examined. A major finding of this study was that ATMp immunostaining in photoreceptor cells was exclusively in the cytoplasm, whereas ATM immunostaining was only in the nucleus of these cells. Furthermore, the specific and distinct ATM and ATMp immunolabeling patterns in photoreceptor cells were identical to those observed in the adult mouse cerebellar granule cells. Conclusions We report the expression profile of Atm gene and protein in the adult mouse eye. In particular, we observed a difference between the localization patterns of the active and inactive forms of ATM in photoreceptor cells. These localization patterns suggest that ATM and its phosphorylated activated form may be involved in both the protection of cells from oxidative damage and the maintenance of ocular cell structure and function. The protection mechanisms mediated by the two forms of ATM appear to be particularly important in maintaining photoreceptor integrity. PMID:19234633

  1. Rearrangement and expression of erythropoietin genes in transformed mouse cells.

    PubMed Central

    McDonald, J; Beru, N; Goldwasser, E

    1987-01-01

    The erythroleukemia cell line IW32, derived by transformation with the Friend murine leukemia virus, has been shown previously to produce erythropoietin (EPO) constitutively. Here we demonstrate that, in addition to the normal mouse EPO locus, this cell line has another EPO locus which has undergone rearrangement and amplification. Both loci were cloned, and the rearrangement breakpoint of the second EPO locus was located within a 1.1-kilobase region upstream of an otherwise apparently normal EPO gene. There are no viral sequences present in the immediate vicinity of the rearranged EPO gene. DNase I digestion studies suggest that the rearranged gene is in a region where the chromatin is more sensitive to DNase hydrolysis than is the site of the normal gene. We conclude, tentatively, that the rearranged EPO locus is probably the transcriptionally active one and that either proviral sequences are acting at a distance to activate the EPO gene or the rearrangement itself has served to activate the gene. Images PMID:3561395

  2. Mouse Genetic Nomenclature: Standardization of Strain, Gene, and Protein Symbols

    PubMed Central

    Sundberg, John P.; Schofield, Paul N

    2011-01-01

    The use of standard nomenclatures for describing the strains, genes, and proteins of species is vital for the interpretation, archiving, analysis, and recovery of experimental data on the laboratory mouse. At a time when sharing of data and meta- analysis of experimental results is becoming a dominant mode of scientific investigation, failure to respect formal nomenclatures can cause confusion, errors, and in some cases contribute to poor science. Here we present the basic nomenclature rules for laboratory mice and explain how these rules should be applied to complex genetic manipulations and crosses. PMID:20685919

  3. Mouse models of gene-environment interactions in schizophrenia

    PubMed Central

    Kannan, Geetha; Sawa, Akira; Pletnikov, Mikhail V.

    2013-01-01

    Gene-environment interactions (GEI) likely play significant roles in the pathogenesis of schizophrenia and underlie differences in pathological, behavioral, and clinical presentations of the disease. Findings from epidemiology and psychiatric genetics have assisted in the generation of animal models of GEI relevant to schizophrenia. These models may provide a foundation for elucidating the molecular, cellular, and circuitry mechanisms that mediate GEI in schizophrenia. Here we critically review current mouse models of GEI related to schizophrenia, describe directions for their improvement, and propose endophenotypes provide a more tangible basis for molecular studies of pathways of GEI and facilitate the identification of novel therapeutic targets. PMID:23748077

  4. Genetic mapping of the mouse stromal cell-derived factor gene (Sdf1) to mouse and rat chromosomes.

    PubMed

    Nomura, M; Matsuda, Y; Itoh, H; Hori, T; Suzuki, G

    1996-01-01

    Stromal cell-derived factor 1 (SDF1) is a new member of the Cys-X-Cys chemokine family. The chromosomal location of Sdf1, the gene coding mouse SDF1, was determined by fluorescence in situ hybridization (FISH) and molecular linkage analysis. The mouse Sdf1 gene was localized to the R-band-positive F1 band of chromosome 6 by direct R-banding FISH. Interspecific backcross analysis identified the mouse Sdf1 gene locus at 0.8 cM terminal to D6Nit55 and 3.0 cM proximal to D6Mit12. With in situ hybridization using a mouse cDNA clone as a probe, the rat Sdf1 gene was localized to the R-band-positive band 4q42.1, where conserved linkage homology to mouse chromosome 6 has been identified. Although other Cys-X-Cys chemokine genes have been mapped on human chromosome 4, the chromosomal segment where the mouse and rat Sdf1 gene reside have no conserved linkage homology to human chromosome 4. This result suggests that SDF1 is a new chemokine class. PMID:8751377

  5. A Crystallin Gene Network in the Mouse Retina

    PubMed Central

    Templeton, Justin P.; Wang, XiangDi; Freeman, Natalie E.; Ma, Zhiwei; Lu, Anna; Hejtmancik, Fielding; Geisert, Eldon E.

    2013-01-01

    The present study was designed to examine the regulation of crystallin genes and protein in the mouse retina using the BXD recombinant inbred (RI) strains. Illumina Sentrix BeadChip Arrays (MouseWG-6v2) were used to analyze mRNA levels in 75 BXD RI strains along with the parental strains (C57Bl/6J and DBA/2J), and the reciprocal crosses in the Hamilton Eye Institute (HEI) Retina Dataset (www.genenetwork.org). Protein levels were investigated using immunoblots to quantify levels of proteins and indirect immunohistochemistry to define the distribution of protein. Algorithms in the Genomatix program were used to identify transcription factor binding sites common to the regulatory sequences in the 5′ regions of co-regulated set of crystallin and other genes as compared to a set of control genes. As subset of genes, including many encoding lens crystallins is part of a tightly co-regulated network that is active in the retina. Expression of this crystallin network appears to be binary in nature, being expressed either at relatively low levels or being highly upregulated. Relative to a control set of genes, the 5′ regulatory sequences of the crystallin network genes show an increased frequency of a set of common transcription factor-binding sites, the most common being those of the Maf family. Chromatin immunoprecipitation of human lens epithelial cells (HLEC) and rat retinal ganglion cells (RGC) confirmed the functionality of these sites, showing that MafA binds the predicted sites of CRYGA and CRYGD in HLE and CRYAB, CRYGA, CRYBA1, and CRYBB3 in RGC cells. In the retina there is a highly correlated group of genes containing many members of the α- β- and γ-crystallin families. These genes can be dramatically upregulated in the retina. One transcription factor that appears to be involved in this coordinated expression is the MAF family transcription of factors associated with both lens and extralenticular expression of crystallin genes. PMID:23978599

  6. In vitro transcription of a cloned mouse ribosomal RNA gene.

    PubMed Central

    Mishima, Y; Yamamoto, O; Kominami, R; Muramatsu, M

    1981-01-01

    An in vitro transcription system which utilizes cloned mouse ribosomal RNA gene (rDNA) fragments and a mouse cell extract has been developed. RNA polymerases I is apparently responsible for this transcription as evidenced by the complete resistance to a high concentration (200 micrograms/ml) of alpha-amanitin. Run-off products obtained with three different truncated rDNA fragments indicated that RNA was transcribed from a unique site of rDNA. The S1 nuclease protection mapping of the in vitro product and of in vivo 45S RNA confirmed this site, indicating that, in this in vitro system, transcription of rDNA started from the same site as in vivo. This site is located at several hundred nucleotides upstream from the putative initiation site reported by us (1) and by others (2). Some sequence homology surrounding this region was noted among mouse, Xenopus laevis and Drosophila melanogaster. The data also suggest that some processing of the primary transcript occurs in this in vitro system. Images PMID:6278446

  7. Characterization of the p16 gene in the mouse: Evidence for a large gene family

    SciTech Connect

    Fountain, J.W.; Giendening, J.M.; Flores, J.F.

    1994-09-01

    The p16 gene product is an inhibitor of the cyclin-dependent kinase 4 (CDK4)/cyclin D complex. When uninhibited, the CDK4/cyclin D complex participates in the phosphorylation of the retinoblastoma (RB) protein and renders it inactive. Upon inactivation of the RB protein, transition from the G{sub 1} to the S phase of mitosis occurs and results in cellular proliferation. Thus, p16 is presumed to act as a negative regulator of cell growth by preventing the phosphorylation, and thereby subsequent inactivation, of RB by CDK4/cyclin D. Recently, the p16 gene (also known as the multiple tumor suppressor 1 (MTS1) gene) has been mapped to chromosome 9p21 and found to be deleted or mutated in a number of tumor cell lines. These findings support the role of p16 as a growth inhibitor or tumor suppressor gene and suggest that the mutation of this gene may have global implications in carcinogenesis. We have chosen to test the functional significance of p16 mutations in vivo through the generation of a mouse mutant for p16. In preparation for this undertaking, eight apparently independent (as judged by restriction enzyme digestion and differential hybridization) mouse genomic embryonic stem cell clones have been identified using exon 2 from the human p16 gene as a probe. The identification of these multiple nonoverlapping clones was not entirely surprising since the reduced stringency hybridization of a zoo blot with the same probe also revealed 10-15 positive EcoRI fragments in all species tested, including human, monkey, cow, dog, cat, rabbit, hamster, mouse, chicken and D. melanogaster. Taken together, these findings suggest that the p16 gene is a member of a large gene family. The location of these genomic clones, as well as their potential expression in the mouse, is currently under investigation.

  8. Screening Helicobacter pylori genes induced during infection of mouse stomachs

    PubMed Central

    Singh, Aparna; Hodgson, Nathaniel; Yan, Ming; Joo, Jungsoo; Gu, Lei; Sang, Hong; Gregory-Bryson, Emmalena; Wood, William G; Ni, Yisheng; Smith, Kimberly; Jackson, Sharon H; Coleman, William G

    2012-01-01

    AIM: To investigate the effect of in vivo environment on gene expression in Helicobacter pylori (H. pylori) as it relates to its survival in the host. METHODS: In vivo expression technology (IVET) systems are used to identify microbial virulence genes. We modified the IVET-transcriptional fusion vector, pIVET8, which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors, pIVET11 and pIVET12. Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H. pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase. Additionally, each vector contains a kanamycin resistance gene. We used a mouse macrophage cell line, RAW 264.7 and mice, as selective media to identify specific genes that H. pylori expresses in vivo. Gene expression studies were conducted by infecting RAW 264.7 cells with H. pylori. This was followed by real time polymerase chain reaction (PCR) analysis to determine the relative expression levels of in vivo induced genes. RESULTS: In this study, we have identified 31 in vivo induced (ivi) genes in the initial screens. These 31 genes belong to several functional gene families, including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs. Virulence factors, vacA and cagA, were found in this screen and are known to play important roles in H. pylori infection, colonization and pathogenesis. Their detection validates the efficacy of these screening systems. Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H. pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae. Transcription profiles of all ivi genes were confirmed by real time PCR analysis of H. pylori RNA isolated from H. pylori infected RAW 264.7 macrophages. We compared the expression profile of H. pylori and RAW 264.7 coculture with that of H. pylori only. Some genes such as cagA, vacA, lpxC, murI, tlpC, trxB, sodB, tnpB, pgi, rbfA and infB showed a 2-20 fold upregulation. Statistically significant upregulation was obtained for all the above mentioned genes (P < 0.05). tlpC, cagA, vacA, sodB, rbfA, infB, tnpB, lpxC and murI were also significantly upregulated (P < 0.01). These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal. CONCLUSION: The positive identification of these genes demonstrates that our IVET systems are powerful tools for studying H. pylori gene expression in the host environment. PMID:22969195

  9. The Role of Zic Genes in Inner Ear Development in the Mouse: Exploring Mutant Mouse Phenotypes

    PubMed Central

    Chervenak, Andrew P.; Bank, Lisa M.; Thomsen, Nicole; Glanville-Jones, Hannah C; Skibo, Jonathan; Millen, Kathleen J.; Arkell, Ruth M.; Barald, Kate F.

    2014-01-01

    Background Murine Zic genes (Zic1-5) are expressed in the dorsal hindbrain and in periotic mesenchyme (POM) adjacent to the developing inner ear. Zic genes are involved in developmental signaling pathways in many organ systems, including the ear, although their exact roles haven't been fully elucidated. This report examines the role of Zic1, Zic2, and Zic4 during inner ear development in mouse mutants in which these Zic genes are affected Results Zic1/Zic4 double mutants don't exhibit any apparent defects in inner ear morphology. By contrast, inner ears from Zic2kd/kd and Zic2Ku/Ku mutants have severe but variable morphological defects in endolymphatic duct/sac and semicircular canal formation and in cochlear extension in the inner ear. Analysis of otocyst patterning in the Zic2Ku/Ku mutants by in situ hybridization showed changes in the expression patterns of Gbx2 and Pax2. Conclusions The experiments provide the first genetic evidence that the Zic genes are required for morphogenesis of the inner ear. Zic2 loss-of-function doesn't prevent initial otocyst patterning but leads to molecular abnormalities concomitant with morphogenesis of the endolymphatic duct. Functional hearing deficits often accompany inner ear dysmorphologies, making Zic2 a novel candidate gene for ongoing efforts to identify the genetic basis of human hearing loss. PMID:25178196

  10. Zeptomole Electrochemical Detection of Metallothioneins

    PubMed Central

    Adam, Vojtech; Petrlova, Jitka; Wang, Joseph; Eckschlager, Tomas; Trnkova, Libuse; Kizek, Rene

    2010-01-01

    Background Thiol-rich peptides and proteins possess a large number of biological activities and may serve as markers for numerous health problems including cancer. Metallothionein (MT), a small molecular mass protein rich in cysteine, may be considered as one of the promising tumour markers. The aim of this paper was to employ chronopotentiometric stripping analysis (CPSA) for highly sensitive detection of MT. Methodology/Principal Findings In this study, we used adsorptive transfer stripping technique coupled with CPSA for detection of cysteine, glutathione oxidized and reduced, phytochelatin, bovine serum albumin, and metallothionein. Under the optimal conditions, we were able to estimate detection limits down to tens of fg per ml. Further, this method was applied to detect metallothioneins in blood serum obtained from patients with breast cancer and in neuroblastoma cells resistant and sensitive to cisplatin in order to show the possible role of metallothioneins in carcinogenesis. It was found that MT level in blood serum was almost twice higher as compared to the level determined in healthy individuals. Conclusions/Significance This paper brings unique results on the application of ultra-sensitive electroanalytical method for metallothionein detection. The detection limit and other analytical parameters are the best among the parameters of other techniques. In spite of the fact that the paper is mainly focused on metallothionein, it is worth mentioning that successful detection of other biologically important molecules is possible by this method. Coupling of this method with simple isolation methods such as antibody-modified paramagnetic particles may be implemented to lab–on-chip instrument. PMID:20625429

  11. Genetics of gene expression surveyed in maize, mouse and man.

    PubMed

    Schadt, Eric E; Monks, Stephanie A; Drake, Thomas A; Lusis, Aldons J; Che, Nam; Colinayo, Veronica; Ruff, Thomas G; Milligan, Stephen B; Lamb, John R; Cavet, Guy; Linsley, Peter S; Mao, Mao; Stoughton, Roland B; Friend, Stephen H

    2003-03-20

    Treating messenger RNA transcript abundances as quantitative traits and mapping gene expression quantitative trait loci for these traits has been pursued in gene-specific ways. Transcript abundances often serve as a surrogate for classical quantitative traits in that the levels of expression are significantly correlated with the classical traits across members of a segregating population. The correlation structure between transcript abundances and classical traits has been used to identify susceptibility loci for complex diseases such as diabetes and allergic asthma. One study recently completed the first comprehensive dissection of transcriptional regulation in budding yeast, giving a detailed glimpse of a genome-wide survey of the genetics of gene expression. Unlike classical quantitative traits, which often represent gross clinical measurements that may be far removed from the biological processes giving rise to them, the genetic linkages associated with transcript abundance affords a closer look at cellular biochemical processes. Here we describe comprehensive genetic screens of mouse, plant and human transcriptomes by considering gene expression values as quantitative traits. We identify a gene expression pattern strongly associated with obesity in a murine cross, and observe two distinct obesity subtypes. Furthermore, we find that these obesity subtypes are under the control of different loci. PMID:12646919

  12. Mouse models for the discovery of colorectal cancer driver genes.

    PubMed

    Clark, Christopher R; Starr, Timothy K

    2016-01-14

    Colorectal cancer (CRC) constitutes a major public health problem as the third most commonly diagnosed and third most lethal malignancy worldwide. The prevalence and the physical accessibility to colorectal tumors have made CRC an ideal model for the study of tumor genetics. Early research efforts using patient derived CRC samples led to the discovery of several highly penetrant mutations (e.g., APC, KRAS, MMR genes) in both hereditary and sporadic CRC tumors. This knowledge has enabled researchers to develop genetically engineered and chemically induced tumor models of CRC, both of which have had a substantial impact on our understanding of the molecular basis of CRC. Despite these advances, the morbidity and mortality of CRC remains a cause for concern and highlight the need to uncover novel genetic drivers of CRC. This review focuses on mouse models of CRC with particular emphasis on a newly developed cancer gene discovery tool, the Sleeping Beauty transposon-based mutagenesis model of CRC. PMID:26811627

  13. Mouse models for the discovery of colorectal cancer driver genes

    PubMed Central

    Clark, Christopher R; Starr, Timothy K

    2016-01-01

    Colorectal cancer (CRC) constitutes a major public health problem as the third most commonly diagnosed and third most lethal malignancy worldwide. The prevalence and the physical accessibility to colorectal tumors have made CRC an ideal model for the study of tumor genetics. Early research efforts using patient derived CRC samples led to the discovery of several highly penetrant mutations (e.g., APC, KRAS, MMR genes) in both hereditary and sporadic CRC tumors. This knowledge has enabled researchers to develop genetically engineered and chemically induced tumor models of CRC, both of which have had a substantial impact on our understanding of the molecular basis of CRC. Despite these advances, the morbidity and mortality of CRC remains a cause for concern and highlight the need to uncover novel genetic drivers of CRC. This review focuses on mouse models of CRC with particular emphasis on a newly developed cancer gene discovery tool, the Sleeping Beauty transposon-based mutagenesis model of CRC. PMID:26811627

  14. Mammalian metallothioneins: properties and functions.

    PubMed

    Babula, Petr; Masarik, Michal; Adam, Vojtech; Eckschlager, Tomas; Stiborova, Marie; Trnkova, Libuse; Skutkova, Helena; Provaznik, Ivo; Hubalek, Jaromir; Kizek, Rene

    2012-08-01

    Metallothioneins (MT) are a family of ubiquitous proteins, whose role is still discussed in numerous papers, but their affinity to some metal ions is undisputable. These cysteine-rich proteins are connected with antioxidant activity and protective effects on biomolecules against free radicals, especially reactive oxygen species. In this review, the connection between zinc(II) ions, reactive oxygen species, heavy metal ions and metallothioneins is demonstrated with respect to effect of these proteins on cell proliferation and a possible negative role in resistance to heavy metal-based and non-heavy metal-based drugs. PMID:22791193

  15. Genomic organization and characterization of the mouse ELYS gene.

    PubMed

    Okita, Keisuke; Nobuhisa, Ikuo; Takizawa, Makiko; Ueno, Masaya; Kimura, Naoki; Taga, Tetsuya

    2003-05-30

    Differentiation of hematopoietic stem cells into blood cells is controlled by several transcription factors. Recently, we identified a putative transcription factor, ELYS (for embryonic large molecule derived from yolk sac), using a subtraction strategy. During mouse embryogenesis, ELYS transcripts were predominantly expressed in hematopoietic tissues, such as the yolk sac, aorta-gonad-mesonephros (AGM), and liver. Here, we report the cloning and characterization of the mouse ELYS gene. The ELYS gene spanned approximately 60kb encoding 36 exons, and was assigned between D1Mit315 and D1Mit458 markers in chromosome 1. The transcription initiation site was identified as the G residue located 670bp upstream of the translation start codon. A region downstream of the transcriptional start site contributed to high promoter activity. This region contained potential DNA elements for transcription factors such as GATA-1, -2, -3, heat shock factor (HSF) 2, and NF-kappaB, which are known to play important roles in hematopoietic events. PMID:12745078

  16. Sleeping Beauty Mouse Models Identify Candidate Genes Involved in Gliomagenesis

    PubMed Central

    Vyazunova, Irina; Maklakova, Vilena I.; Berman, Samuel; De, Ishani; Steffen, Megan D.; Hong, Won; Lincoln, Hayley; Morrissy, A. Sorana; Taylor, Michael D.; Akagi, Keiko; Brennan, Cameron W.; Rodriguez, Fausto J.; Collier, Lara S.

    2014-01-01

    Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma. PMID:25423036

  17. Sleeping Beauty mouse models identify candidate genes involved in gliomagenesis.

    PubMed

    Vyazunova, Irina; Maklakova, Vilena I; Berman, Samuel; De, Ishani; Steffen, Megan D; Hong, Won; Lincoln, Hayley; Morrissy, A Sorana; Taylor, Michael D; Akagi, Keiko; Brennan, Cameron W; Rodriguez, Fausto J; Collier, Lara S

    2014-01-01

    Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma. PMID:25423036

  18. Brain Gene Expression of a Sporadic (icv-STZ Mouse) and a Familial Mouse Model (3xTg-AD Mouse) of Alzheimers Disease

    PubMed Central

    Liang, Zhihou; Sun, Shenggang; Dai, Chun-ling; Lee, Moon H.; LaFerla, Frank M.; Grundke-Iqbal, Inge; Iqbal, Khalid; Liu, Fei; Gong, Cheng-Xin

    2012-01-01

    Alzheimers disease (AD) can be divided into sporadic AD (SAD) and familial AD (FAD). Most AD cases are sporadic and may result from multiple etiologic factors, including environmental, genetic and metabolic factors, whereas FAD is caused by mutations of presenilins or amyloid-? (A?) precursor protein (APP). A commonly used mouse model for AD is 3xTg-AD mouse, which is generated by over-expression of mutated presenilin 1, APP and tau in the brain and thus represents a mouse model of FAD. A mouse model generated by intracerebroventricular (icv) administration of streptozocin (STZ), icv-STZ mouse, shows many aspects of SAD. Despite the wide use of these two models for AD research, differences in gene expression between them are not known. Here, we compared the expression of 84 AD-related genes in the hippocampus and the cerebral cortex between icv-STZ mice and 3xTg-AD mice using a custom-designed qPCR array. These genes are involved in APP processing, tau/cytoskeleton, synapse function, apoptosis and autophagy, AD-related protein kinases, glucose metabolism, insulin signaling, and mTOR pathway. We found altered expression of around 20 genes in both mouse models, which affected each of above categories. Many of these gene alterations were consistent with what was observed in AD brain previously. The expression of most of these altered genes was decreased or tended to be decreased in the hippocampus of both mouse models. Significant diversity in gene expression was found in the cerebral cortex between these two AD mouse models. More genes related to synaptic function were dysregulated in the 3xTg-AD mice, whereas more genes related to insulin signaling and glucose metabolism were down-regulated in the icv-STZ mice. The present study provides important fundamental knowledge of these two AD mouse models and will help guide future studies using these two mouse models for the development of AD drugs. PMID:23236499

  19. Mouse histone H2A and H2B genes: four functional genes and a pseudogene undergoing gene conversion with a closely linked functional gene.

    PubMed Central

    Liu, T J; Liu, L; Marzluff, W F

    1987-01-01

    The sequence of five mouse histone genes, two H2a and three H2b genes on chromosome 13 has been determined. The three H2b genes all code for different proteins, each differing in two amino acids from the others. The H2b specific elements present 5' to H2b genes from other species are present in all three mouse H2b genes. All three H2b genes are expressed in the same relative amounts in three different mouse cell lines and fetal mice. The H2b gene with the H2b specific sequence closest to the TATAA sequence is expressed in the highest amount. One of the H2a genes lacks the first 9 amino acids, the promoter region, the last 3 amino acids and contains an altered 3' end sequence. Despite these multiple defects, there is only one nucleotide change between the two H2a genes from codon 9 to 126. This indicates that a recent gene conversion has occurred between these two genes. The similarity of the nucleotide sequences in the coding regions of mouse histone genes is probably due to gene conversion events targeted precisely at the coding region. Images PMID:3562244

  20. Diversity and Complexity of the Mouse Saa1 and Saa2 genes

    PubMed Central

    Mori, Masayuki; Tian, Geng; Ishikawa, Akira; Higuchi, Keiichi

    2014-01-01

    Mouse strains show polymorphisms in the amino acid sequences of serum amyloid A 1 (SAA1) and serum amyloid A 2 (SAA2). Major laboratory mouse strains are classified based on the sequence as carrying the A haplotype (e.g., BALB/c) or B haplotype (e.g., SJL/J) of the Saa1 and Saa2 gene unit. We attempted to elucidate the diversity of the mouse Saa1 and Saa2 family genes at the nucleotide sequence level by a systematic survey of 6 inbred mouse strains from 4 Mus subspecies, including Mus musculus domesticus, Mus musculus musculus, Mus musculus castaneus, and Mus spretus. Saa1 and Saa2 genes were obtained from the mouse genome by PCR amplification, and each full-length nucleotide sequence was determined. We found that Mus musculus castaneus mice uniquely possess 2 divergent Saa1 genes linked on chromosome 7. Overall, the mouse strains had distinct composite patterns of amino acid substitutions at 9 positions in SAA1 and SAA2 isoforms. The mouse strains also had distinct composite patterns of 2 polymorphic upstream regulatory elements that influenced gene transcription in in vitro reporter assays. B haplotype mice were revealed to possess an LTR insertion in the downstream region of Saa1. Collectively, these results indicate that the mouse Saa genes hold broader diversity and greater complexity than previously known, and these characteristics were likely attained through gene duplication and repeated gene conversion events in the Mus lineage. PMID:24521869

  1. Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging

    PubMed Central

    2012-01-01

    Background Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented. Results We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P. Conclusion We present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species. PMID:22780875

  2. Identification of Four Mouse Diabetes Candidate Genes Altering ?-Cell Proliferation.

    PubMed

    Kluth, Oliver; Matzke, Daniela; Kamitz, Anne; Jhnert, Markus; Vogel, Heike; Scherneck, Stephan; Schulze, Matthias; Staiger, Harald; Machicao, Fausto; Hring, Hans-Ulrich; Joost, Hans-Georg; Schrmann, Annette

    2015-09-01

    Beta-cell apoptosis and failure to induce beta-cell regeneration are hallmarks of type 2-like diabetes in mouse models. Here we show that islets from obese, diabetes-susceptible New Zealand Obese (NZO) mice, in contrast to diabetes-resistant C57BL/6J (B6)-ob/ob mice, do not proliferate in response to an in-vivo glucose challenge but lose their beta-cells. Genome-wide RNAseq based transcriptomics indicated an induction of 22 cell cycle-associated genes in B6-ob/ob islets that did not respond in NZO islets. Of all genes differentially expressed in islets of the two strains, seven mapped to the diabesity QTL Nob3, and were hypomorphic in either NZO (Lefty1, Apoa2, Pcp4l1, Mndal, Slamf7, Pydc3) or B6 (Ifi202b). Adenoviral overexpression of Lefty1, Apoa2, and Pcp4l1 in primary islet cells increased proliferation, whereas overexpression of Ifi202b suppressed it. We conclude that the identified genes in synergy with obesity and insulin resistance participate in adaptive islet hyperplasia and prevention from severe diabetes in B6-ob/ob mice. PMID:26348837

  3. Hox-5.1 defines a homeobox-containing gene locus on mouse chromosome 2.

    PubMed Central

    Featherstone, M S; Baron, A; Gaunt, S J; Mattei, M G; Duboule, D

    1988-01-01

    We have isolated a murine homeobox-containing gene, Hox-5.1, by virtue of its relatedness to the Hox-1.4 gene. In situ hybridization to metaphase spreads mapped Hox-5.1 to band D of mouse chromosome 2. Sequence comparisons indicate that Hox-5.1 is the murine homolog of the human C13 homeobox-containing gene. Hox-5.1 also bears significant similarity to the Xenopus Xhox-1A homeobox-containing gene and the Drosophila deformed homeotic gene at N-terminal and homeobox regions. Hox-5.1 transcripts were detected in mouse embryos, in adult mouse testis, kidney, heart, and intestine, and in mouse embryonal carcinoma cells treated with retinoic acid. In situ hybridization to sections from whole mouse embryos revealed Hox-5.1 expression in spinal cord and prevertebrae. Images PMID:2898782

  4. Targeted disruption of the mouse Lipoma Preferred Partner gene

    SciTech Connect

    Vervenne, Hilke B.V.K.; Crombez, Koen R.M.O.; Delvaux, Els L.; Janssens, Veerle; Ven, Wim J.M. van de Petit, Marleen M.R.

    2009-02-06

    LPP (Lipoma Preferred Partner) is a zyxin-related cell adhesion protein that is involved in the regulation of cell migration. We generated mice with a targeted disruption of the Lpp gene and analysed the importance of Lpp for embryonic development and adult functions. Aberrant Mendelian inheritance in heterozygous crosses suggested partial embryonic lethality of Lpp{sup -/-} females. Fertility of Lpp{sup -/-} males was proven to be normal, however, females from Lpp{sup -/-} x Lpp{sup -/-} crosses produced a strongly reduced number of offspring, probably due to a combination of female embryonic lethality and aberrant pregnancies. Apart from these developmental and reproductive abnormalities, Lpp{sup -/-} mice that were born reached adulthood without displaying any additional macroscopic defects. On the other hand, Lpp{sup -/-} mouse embryonic fibroblasts exhibited reduced migration capacity, reduced viability, and reduced expression of some Lpp interaction partners. Finally, we discovered a short nuclear form of Lpp, expressed mainly in testis via an alternative promoter.

  5. Conditional Gene Targeting in Mouse High Endothelial Venules

    PubMed Central

    Kawashima, Hiroto; Hirakawa, Jotaro; Tobisawa, Yuki; Fukuda, Minoru; Saga, Yumiko

    2009-01-01

    High endothelial venules (HEVs) are specialized blood vessels of secondary lymphoid organs composed of endothelial cells with a characteristic cuboidal morphology. Lymphocytes selectively adhere to and migrate across HEVs to initiate immune responses. In this study, we established a novel transgenic mouse line expressing Cre recombinase under the transcriptional control of the gene encoding HEV-expressed sulfotransferase, N-acetylglucosamine-6-O-sulfotransferase 2 (GlcNAc6ST-2), using bacterial artificial chromosome recombineering. Crossing these transgenic mice with the ROSA26 reporter strain, which expresses lacZ following Cre-mediated recombination, and staining the resulting progeny with 5-bromo-4-chloro-5-indolyl-?-D-galactoside indicated that Cre recombinase was specifically expressed in mAb MECA79-reactive HEVs in secondary lymphoid organs but not in any other blood vessels of the transgenic mice. The expression of Cre recombinase correlated with a developmental switch, from immature, mAb MECA367-reactive HEVs to mature, mAb MECA79-reactive HEVs in neonatal lymph nodes. In addition to the HEVs, Cre recombinase was also strongly expressed in the colonic villi, which recapitulated the intrinsic expression of GlcNAc6ST-2 as confirmed in GlcNAc6ST-2GFP/GFP knock-in mice and by RT-PCR. Furthermore, treatment with an antimicrobial agent revealed that the colonic expression of Cre recombinase in the transgenic mice was regulated by commensal bacteria in the colon. In addition, Cre recombinase was expressed in a small subset of cells in the brain, testis, stomach, small intestine, and lung. In view of the restricted expression of Cre recombinase, this transgenic mouse line should be useful for elucidating tissue-specific gene functions using the Cre/loxP system. PMID:19380794

  6. Genomic cloning of mouse MIF (macrophage inhibitory factor) and genetic mapping of the human and mouse expressed gene and nine mouse pseudogenes

    SciTech Connect

    Kozak, C.A.; Adamson, M.C.; Buckler, C.E.

    1995-06-10

    The single functional mouse gene for MIF (macrophage migration inhibitory factor) has been cloned from a P1 library, and its exon/intron structure determined and shown to resemble that of the human gene. The gene was mapped to chromosome 10 using two multilocus crosses between laboratory strains and either Mus musculus or Mus spretus. Nine additional loci containing related sequences, apparently all processed pseudogenes, were also mapped to chromosomes 1, 2, 3, 7, 8, 9, 12, 17, and 19. While most of these pseudogenes were also found in inbred mice and M. spretus, some are species specific. This suggests that there have been active phases of pseudogene formation in Mus both before and after the separation of musculus and spretus. The human gene contains no pseudogene; we assigned the human gene to chromosome 19, consistent with the location of mouse and human functional genes for MIF in a region of conserved linkage. 43 refs., 4 figs., 1 tab.

  7. Genomic structure of the mouse gene (Lmnb1) encoding nuclear lamin B1

    SciTech Connect

    Maeno, Hideki; Sugimoto, Kazunori; Nakajima, Noboru

    1995-11-20

    The mouse gene (Lmnb1) that encodes nuclear lamin B1 was isolated. Structural analyses revealed that the lamin B1 gene spans about 43 kb of the genome and consists of 11 exons and 10 introns. Exon/intron structure of the B1 gene clearly showed the conserved organization shared among the intermediate filament protein family genes. The presumptive promoter region has high GC content and contains a CAAT box and multiple SP1 sites but no classical TATA box, suggesting that the lamin B1 gene has a typical housekeeping gene promoter with a CpG island. These data reveal the gene structure of the only remaining unanalyzed mouse somatic lamin gene. Gene structures of all the mouse somatic lamins are compared. 22 refs., 3 figs.

  8. The mouse homologue of the polycystic kidney disease gene (Pkd1) is a single-copy gene

    SciTech Connect

    Olsson, P.G.; Loehning, C.; Frischauf, A.M.

    1996-06-01

    The mouse homologue of the polycystic kidney disease 1 gene (PKD1) was mapped to chromosome 17 using somatic cell hybrid, BXD recombinant inbred strains, and FISH. The gene is located within a previously defined conserved synteny group that includes the mouse homologue of tuberous sclerosis 2 (TSC2) and is linked to the {alpha} globin pseudogene Hba-ps4. Although the human genome contains multiple copies of genes related to PKD1, there is no evidence for more than one copy in the mouse genome. Like their human counterparts, the mouse Tsc2 and Pkd1 genes are arranged in a tail-to-tail orientation with a distance of only 63 bp between the polyadenylation signals of the two genes. 17 refs., 3 figs.

  9. The NEUROD gene maps to human chromosome 2q32 and mouse chromosome 2.

    PubMed

    Tamimi, R; Steingrimsson, E; Copeland, N G; Dyer-Montgomery, K; Lee, J E; Hernandez, R; Jenkins, N A; Tapscott, S J

    1996-06-15

    The Neurod gene is a basic-helix-loop-helix gene that regulates neurogenesis and is identical to the hamster beta2 gene that was cloned as a regulator of insulin transcription. Here we report the cloning of human NEUROD and mapping of the gene to human chromosome 2q32 and to mouse chromosome 2. PMID:8786144

  10. Single and Multiple Gene Manipulations in Mouse Models of Human Cancer

    PubMed Central

    Lehman, Heather L; Stairs, Douglas B

    2015-01-01

    Mouse models of human cancer play a critical role in understanding the molecular and cellular mechanisms of tumorigenesis. Advances continue to be made in modeling human disease in a mouse, though the relevance of a mouse model often relies on how closely it is able to mimic the histologic, molecular, and physiologic characteristics of the respective human cancer. A classic use of a genetically engineered mouse in studying cancer is through the overexpression or deletion of a gene. However, the manipulation of a single gene often falls short of mimicking all the characteristics of the carcinoma in humans; thus a multiple gene approach is needed. Here we review genetic mouse models of cancers and their abilities to recapitulate human carcinoma with single versus combinatorial approaches with genes commonly involved in cancer. PMID:26380553

  11. Arbuscular mycorrhizal fungi restore normal growth in a white poplar clone grown on heavy metal-contaminated soil, and this is associated with upregulation of foliar metallothionein and polyamine biosynthetic gene expression

    PubMed Central

    Cicatelli, Angela; Lingua, Guido; Todeschini, Valeria; Biondi, Stefania; Torrigiani, Patrizia; Castiglione, Stefano

    2010-01-01

    Background and Aims It is increasingly evident that plant tolerance to stress is improved by mycorrhiza. Thus, suitable plant–fungus combinations may also contribute to the success of phytoremediation of heavy metal (HM)-polluted soil. Metallothioneins (MTs) and polyamines (PAs) are implicated in the response to HM stress in several plant species, but whether the response is modulated by arbuscular mycorrhizal fungi (AMF) remains to be clarified. The aim of the present study was to check whether colonization by AMF could modify growth, metal uptake/translocation, and MT and PA gene expression levels in white poplar cuttings grown on HM-contaminated soil, and to compare this with plants grown on non-contaminated soil. Methods In this greenhouse study, plants of a Populus alba clone were pre-inoculated, or not, with either Glomus mosseae or G. intraradices and then grown in pots containing either soil collected from a multimetal- (Cu and Zn) polluted site or non-polluted soil. The expression of MT and PA biosynthetic genes was analysed in leaves using quantitative reverse transcription–PCR. Free and conjugated foliar PA concentrations were determined in parallel. Results On polluted soil, AMF restored plant biomass despite higher Cu and Zn accumulation in plant organs, especially roots. Inoculation with the AMF caused an overall induction of PaMT1, PaMT2, PaMT3, PaSPDS1, PaSPDS2 and PaADC gene expression, together with increased free and conjugated PA levels, in plants grown on polluted soil, but not in those grown on non-polluted soil. Conclusions Mycorrhizal plants of P. alba clone AL35 exhibit increased capacity for stabilization of soil HMs, together with improved growth. Their enhanced stress tolerance may derive from the transcriptional upregulation of several stress-related genes, and the protective role of PAs. PMID:20810743

  12. Differential Divergence of Three Human Pseudoautosomal Genes and Their Mouse Homologs: Implications for Sex Chromosome Evolution

    PubMed Central

    Gianfrancesco, Fernando; Sanges, Remo; Esposito, Teresa; Tempesta, Sergio; Rao, Ercole; Rappold, Gudrun; Archidiacono, Nicoletta; Graves, Jennifer A.M.; Forabosco, Antonino; D'Urso, Michele

    2001-01-01

    The human pseudoautosomal region 1 (PAR1) is essential for meiotic pairing and recombination, and its deletion causes male sterility. Comparative studies of human and mouse pseudoautosomal genes are valuable in charting the evolution of this interesting region, but have been limited by the paucity of genes conserved between the two species. We have cloned a novel human PAR1 gene, DHRSXY, encoding an oxidoreductase of the short-chain dehydrogenase/reductase family, and isolated a mouse ortholog Dhrsxy. We also searched for mouse homologs of recently reported PGPL and TRAMP genes that flank it within PAR1. We recovered a highly conserved mouse ortholog of PGPL by cross-hybridization, but found no mouse homolog of TRAMP. Like Csf2ra and Il3ra, both mouse homologs are autosomal; Pgpl on chromosome 5, and Dhrsxy subtelomeric on chromosome 4. TRAMP, like the human genes within or near PAR1, is probably very divergent or absent in the mouse genome. We interpret the rapid divergence and loss of pseudoautosomal genes in terms of a model of selection for the concentration of repetitive recombinogenic sequences that predispose to high recombination and translocation. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AJ293620, AJ296079, and AJ293619.] PMID:11731500

  13. In vivo selection for metastasis promoting genes in the mouse.

    PubMed

    Gumireddy, Kiranmai; Sun, Fangxian; Klein-Szanto, Andres J; Gibbins, Jonathan M; Gimotty, Phyllis A; Saunders, Aleister J; Schultz, Peter G; Huang, Qihong

    2007-04-17

    Here, we report the identification of a metastasis promoting factor by a forward genetic screen in mice. A retroviral cDNA library was introduced into the nonmetastatic cancer cell line 168FARN, which was then orthotopically transplanted into mouse mammary fat pads, followed by selection for cells that metastasize to the lung. The genes encoding the disulfide isomerase ERp5 and beta-catenin were found to promote breast cancer invasion and metastasis. Disulfide isomerases (thiol isomerases), which catalyze disulfide bond formation, reduction, and isomerization, have not previously been implicated in cancer cell signaling and tumor metastasis. Overexpression of ERp5 promotes both in vitro migration and invasion and in vivo metastasis of breast cancer cells. These effects were shown to involve activation of ErbB2 and phosphoinositide 3-kinase (PI3K) pathways through dimerization of ErbB2. Activation of ErbB2 and PI3K subsequently stimulates RhoA and beta-catenin, which mediate the migration and invasion of tumor cells. Inhibition of ErbB2 and PI3K reverses the phenotypes induced by ERp5. Finally, ERp5 was shown to be up-regulated in human surgical samples of invasive breast cancers. These data identify a link between disulfide isomerases and tumor development, and provide a mechanism that modulates ErbB2 and PI3K signaling in the promotion of cancer progression. PMID:17420453

  14. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

    SciTech Connect

    Ding, J.H.; Yang, B.Z.; Liu, H.M.

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  15. Genomic structure and chromosomal assignment of the mouse Ku70 gene

    SciTech Connect

    Takiguchi, Yuichi; Kurimasa, Akihiro; Chen, Fanqing

    1996-07-01

    DNA-dependent protein kinase (DNA-PK) consists of three polypeptide subunits: Ku70, Ku80, and the DNA-PK catalytic subunit (DNA-PKcs). Mammalian mutants deficient in either Ku80 or DNA-PKcs function have been shown to be lacking in DNA double-strand break repair and V(D)J recombination, respectively. The precise role of the Ku70 gene in this process has not yet been determined, in part because no cell lines, animals, or human diseases involved with deficiencies in this gene have yet been identified. Both the human and the mouse Ku70 cDNAs have been cloned, and the human gene has been mapped to chromosome 22q13. The original mouse cDNA clones, however, lacked a complete 5{prime}-region, and none of the mammalian Ku70 genomic sequences have been characterized. This report contains an analysis of the 5{prime}-region of the mouse cDNA sequence, a characterization of the mouse Ku70 genomic structure, and fluorescence in situ hybridization data that map the mouse gene to chromosome 15. The deduced amino acid sequence of the mouse gene consists of 608 amino acids compared to 609 for the human gene. The genomic sequence is 24 kb and consists of 13 exons, including an untranslated first exon. Sequences form the upstream region of exon 1 revealed four consensus GC box sequences and a strong transcription initiation site at a reasonable location. The assignment of the mouse Ku70 gene to chromosome 15 is consistent with the syntenic relationship of this gene in human (chromosome 22q13) and mouse and adds to the comparative mapping data for the genes involved in the SCID phenotype. 39 refs., 3 figs.

  16. Duplications in ADHD patients harbour neurobehavioural genes that are co-expressed with genes associated with hyperactivity in the mouse.

    PubMed

    Taylor, Avigail; Steinberg, Julia; Webber, Caleb

    2015-03-01

    Attention deficit/hyperactivity disorder (ADHD) is a childhood onset disorder, prevalent in 5.3% of children and 1-4% of adults. ADHD is highly heritable, with a burden of large (>500 Kb) copy number variants (CNVs) identified among individuals with ADHD. However, how such CNVs exert their effects is poorly understood. We examined the genes affected by 71 large, rare, and predominantly inherited CNVs identified among 902 individuals with ADHD. We applied both mouse-knockout functional enrichment analyses, exploiting behavioral phenotypes arising from the determined disruption of 1:1 mouse orthologues, and human brain-specific spatio-temporal expression data to uncover molecular pathways common among genes contributing to enriched phenotypes. Twenty-two percent of genes duplicated in individuals with ADHD that had mouse phenotypic information were associated with abnormal learning/memory/conditioning ("l/m/c") phenotypes. Although not observed in a second ADHD-cohort, we identified a similar enrichment among genes duplicated by eight de novo CNVs present in eight individuals with Hyperactivity and/or Short attention span ("Hyperactivity/SAS", the ontologically-derived phenotypic components of ADHD). In the brain, genes duplicated in patients with ADHD and Hyperactivity/SAS and whose orthologues' disruption yields l/m/c phenotypes in mouse ("candidate-genes"), were co-expressed with one another and with genes whose orthologues' mouse models exhibit hyperactivity. Moreover, genes associated with hyperactivity in the mouse were significantly more co-expressed with ADHD candidate-genes than with similarly identified genes from individuals with intellectual disability. Our findings support an etiology for ADHD distinct from intellectual disability, and mechanistically related to genes associated with hyperactivity phenotypes in other mammalian species. PMID:25656289

  17. The effect of Vdr gene ablation on global gene expression in the mouse placenta

    PubMed Central

    Buckberry, Sam; Spronk, Fleur; Wilson, Rebecca L.; Laurence, Jessica A.; Bianco-Miotto, Tina; Leemaqz, Shalem; O'Leary, Sean; Anderson, Paul H.; Roberts, Claire T.

    2015-01-01

    The effects of vitamin D are mediated through the vitamin D receptor (VDR), a predominantly nuclear receptor, expressed in numerous tissues including the placenta. VDR and the retinoid X receptor (RXR) form a dimer complex which binds to genomic vitamin D responsive elements located primarily in promoter regions and recruit cell-specific transcription factor complexes which regulate the expression of numerous genes. To investigate the role of VDR on regulating placental gene expression, mice heterozygous (+/?) for an ablated Vdr allele (C57Bl6 strain B6.129S4-VDRtm1Mbd/J, Jackson Laboratory) were mated to generate Vdr+/+, Vdr+/? and Vdr ?/? fetuses and placental samples were collected at day 18.5 of pregnancy. RNA was isolated from placental tissue with global gene expression measured using Affymetrix Mouse Gene 2.1 ST Arrays to assess the effects of VDR on global gene expression in the placenta. All raw array data are deposited in Gene Expression Omnibus (GEO) under accession GSE61583.

  18. Localization of the murine activating transcription factor 4 gene to mouse chromosome 15

    SciTech Connect

    Mielnicki, L.M.; Elliott, R.W.; Pruitt, S.C. )

    1993-01-01

    Restriction fragment length variant analysis employing a mouse cDNA probe was used to localize the gene encoding murine activating transcription factor 4 (ATF-4) to mouse chromosome 15 in close proximity to Sis (the cellular homolog of the simian sarcoma virus oncoprotein). Previous studies suggest that conserved linkage relationships exist between this region of mouse chromosome 15 and human chromosome 22q. The chromosomal locations of genes encoding most members of the ATF and cyclic AMP response element binding protein (CREB) subfamilv of b-zip proteins have not been determined. This study demonstrates that the location of the gene for murine ATF-4 is not linked to the genes for JUN family members, CREB1 and CREB2. Further mapping of individual ATF/ CREB subfamily members in the mouse will provide insight into the evolution of this multigene family. 15 refs., 1 fig., 1 tab.

  19. Otitis Media Impacts Hundreds of Mouse Middle and Inner Ear Genes

    PubMed Central

    MacArthur, Carol J.; Hausman, Fran; Kempton, J. Beth; Choi, Dongseok; Trune, Dennis R.

    2013-01-01

    Objective Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. Methods To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. Results Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. Conclusion Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media. PMID:24124478

  20. Persistent Gene Expression in Mouse Nasal Epithelia following Feline Immunodeficiency Virus-Based Vector Gene Transfer

    PubMed Central

    Sinn, Patrick L.; Burnight, Erin R.; Hickey, Melissa A.; Blissard, Gary W.; McCray, Paul B.

    2005-01-01

    Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 106 transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 107 to 109 TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (?109 TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for ?1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity. PMID:16188984

  1. Assignment of the mouse tartrate-resistant acid phosphatase gene (Acp5) to chromosome 9

    SciTech Connect

    Grimes, R.; Reddy, S.V.; Leach, R.J.; Scarcez, T.; Sakaguchi, A.Y. ); Roodman, G.D. Audie Murphy Veterans Administration Hospital, San Antonio, TX ); Lalley, P.A. ); Windle, J.J. )

    1993-02-01

    Tartrate-resistant acid phosphatase is a marker enzyme for osteoclasts, the multinucleated cell responsible for bone resorption. Interspecific somatic whole cell hybrids and karyotypically simple microcell hybrids were used to map the gene encoding tartrate-resistant acid phosphatse (acp5) to mouse Chromosome 9. Acp5 is therefore a member of a syntenic family of genes that map to human chromosome 19p13.1-p13.3 and mouse Chromosome 9. 8 refs., 1 fig., 1 tab.

  2. The MHC class I-like Zn-{alpha}{sub 2}-glycoprotein gene maps to mouse chromosome 5

    SciTech Connect

    Noguchi, Munechika; Ishibashi, Teruo; Kasahara, Masanori

    1995-05-01

    We showed that the mouse Azgp gene is not linked to the MHC and maps to chromosome 5. In mice, Cd1 genes are located on chromosome 3, and the gene encoding the neonatal intestinal Fc receptor maps to chromosome 7. Thus, all of the currently known mouse class I genes encoded outside the MHC are dispersed to separate chromosomes. 16 refs., 2 figs.

  3. Cloning, analysis, and chromosomal localization of myoxin (MYH12), the human homologue to the mouse dilute gene

    SciTech Connect

    Engle, L.J.; Kennett, R.H. )

    1994-02-01

    The mouse dilute gene encodes a novel type of non-muscle myosin that structurally combines elements from both nonmuscle myosin type I and nonmuscle myosin type II. Phenotypically, mutations in the mouse dilute gene result not only in the lightening of coat color, but also in the onset of severe neurological defects shortly after birth. This may indicate that the mouse dilute gene is important in maintaining the normal neuronal function in the mouse. The authors report the isolation and sequencing of [open quotes]myoxin[close quotes] (MYH12), the human homologue of the mouse dilute gene, and its assignment to human chromosome 15. 35 refs., 6 figs.

  4. Human Jk recombination signal binding protein gene (IGKJRB): Comparison with its mouse homologue

    SciTech Connect

    Amakawa, Ryuichi; Jing, Wu; Matsunami, Norisada; Hamaguchi, Yasushi; Matsuda, Fumihiko; Kawaichi, Masashi; Honjo, Tasuku ); Ozawa, Kazuo )

    1993-08-01

    The mouse Igkjrb protein specifically binds to the immunoglobulin Jk recombination signal sequence. The IGKJRB gene is highly conserved among many species such as human, Xenopus, and Drosophila. Using cDNA fragments of the mouse Igkjrb gene, the authors isolated its human counterpart, IGKJRB. The human genome contains one functional IGKJRB gene and two types of processed pseudogenes. In situ chromosome hybridization analysis demonstrated that the functional gene is localized at chromosome 3q25, and the pseudogenes (IGKJRBP1 and IGKJRBP2, respectively) are located at chromosomes 9p13 and 9q13. The functional gene is composed of 13 exons spanning at least 67 kb. Three types of cDNA with different 5[prime] sequences were isolated by rapid amplification of cDNA ends, suggesting the presence of three proteins. The aPCR-1 protein, which possessed the exon 1 sequence, was the counterpart of the mouse RBP-2 type protein. The aPCR-2 and 3 proteins may be specific to human cells because the mouse counterparts were not detected. The amino acid sequences of the human and mouse IGKJRB genes were 98% homologous in exons 2-11, whereas the homology of the human and mouse exon 1 sequences was 75%. 40 refs., 7 figs.

  5. Aup1, a novel gene on mouse Chromosome 6 and human Chromosome 2p13

    SciTech Connect

    Jang, Wonhee; Weber, J.S.; Meisler, M.H.

    1996-09-01

    We have cloned a novel mouse cDNA, Aup1, encoding a predicted protein of 410 amino acid residues. The 1.5-kb Aup1 transcript is ubiquitously expressed in mouse tissues. An evolutionary relationship to the Caenorhabditis elegans predicted protein F44b9.5 is indicated by the 35% identity and 53% conservation of the amino acid sequences. Nineteen related human ESTs spanning 80% of the protein have also been identified, with a predicted amino acid sequence identity of 86% between the human and the mouse proteins. The gene has been mapped to a conserved linkage group on human chromosome 2p13 and mouse Chromosome 6. Aup1 was eliminated as a candidate gene for two closely linked disorders, human LGMD2B and mouse mnd2. 15 refs., 2 figs.

  6. Characterization of the mouse Ron/Stk receptor tyrosine kinase gene.

    PubMed

    Waltz, S E; Toms, C L; McDowell, S A; Clay, L A; Muraoka, R S; Air, E L; Sun, W Y; Thomas, M B; Degen, S J

    1998-01-01

    In an effort to understand the mechanisms governing the regulation of the mouse Ron receptor gene, a mouse genomic library was screened and overlapping clones coding for the Ron gene and flanking DNA were identified. Continuous DNA sequence was obtained for approximately 16.4 kilobases. The gene, from the initiator methionine to the polyadenylation site, is contained within 13 244 basepairs and contains 19 exons. Primer extension analyses were performed to determine the transcription start site of the mouse Ron transcript. Multiple transcription start sites were found which also appear to be used in transfected reporter constructs containing Ron 5' flanking DNA. To determine the location of sites which may be critical for the function of the Ron gene promoter, a series of chimeric genes containing serial deletions of the Ron gene promoter fused to the coding sequences for the chloramphenicol acetyl-transferase gene were constructed. Transient transfection analyses of these hybrid genes into various cell lines demonstrated that two regions of the Ron gene promoter, encompassing nucleotides -585 to -465 and from -465 to -285, are important for expression of this transcript in CMT-93 cells. Further analysis of the Ron promoter utilizing gel mobility shift analyses suggests that regions encompassing nucleotides -585 to - 508 and nucleotides -375 to -285 appear to bind specific proteins which may be involved in the negative and positive regulation, respectively, of the mouse Ron gene. PMID:9467940

  7. c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation.

    PubMed

    Ishibashi, Riki; Kozuki, Satoshi; Kamakura, Sachiko; Sumimoto, Hideki; Toyoshima, Fumiko

    2016-02-12

    Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation. PMID:26694615

  8. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  9. Antiserum specific for the intact isoform-3 of metallothionein.

    PubMed

    Tokheim, Abigail M; Armitage, Ian M; Martin, Bruce L

    2005-04-29

    The recombinant form of isoform-3 of mouse brain metallothionein (MT3) was used as an antigen to immunize rabbits and raise MT3-selective antiserum. The antiserum was essentially specific for MT3 with 100-fold greater sensitivity for MT3 compared to MT1 or MT2. Immunonblot analysis of whole mouse brain homogenates showed that MT3 was present only in the fraction retained by a 30,000-Da cut-off filter. The antiserum was used to immunoprecipitate MT3 from mouse brain extracts of Swiss Webster mice and provided evidence that MT3 was a member of a macromolecular complex of greater than 30,000 Da mass in brain. An ELISA was developed using purified, recombinant mouse brain Cd(7)-MT3 as the antigen and used to quantify MT3 in mouse brain extracts. The concentration of MT3 was found to be 3.0+/-0.8 microg/ml or approximately 3.5 microg/g mouse brain (wet weight). PMID:15892977

  10. Effects of Methylmercury Contained in a Diet Mimicking the Wayana Amerindians Contamination through Fish Consumption: Mercury Accumulation, Metallothionein Induction, Gene Expression Variations, and Role of the Chemokine CCL2

    PubMed Central

    Bourdineaud, Jean-Paul; Laclau, Muriel; Maury-Brachet, Régine; Gonzalez, Patrice; Baudrimont, Magalie; Mesmer-Dudons, Nathalie; Fujimura, Masatake; Marighetto, Aline; Godefroy, David; Rostène, William; Brèthes, Daniel

    2012-01-01

    Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. We addressed the question of whether a diet mimicking the fish consumption of Wayanas Amerindians from French Guiana could result in observable adverse effects in mice. Wayanas adult men are subjected to a mean mercurial dose of 7 g Hg/week/kg of body weight. We decided to supplement a vegetarian-based mice diet with 0.1% of lyophilized Hoplias aimara fish, which Wayanas are fond of and equivalent to the same dose as that afflicting the Wayanas Amerindians. Total mercury contents were 1.4 ± 0.2 and 5.4 ± 0.5 ng Hg/g of food pellets for the control and aimara diets, respectively. After 14 months of exposure, the body parts and tissues displaying the highest mercury concentration on a dry weight (dw) basis were hair (733 ng/g) and kidney (511 ng/g), followed by the liver (77 ng/g). Surprisingly, despite the fact that MeHg is a neurotoxic compound, the brain accumulated low levels of mercury (35 ng/g in the cortex). The metallothionein (MT) protein concentration only increased in those tissues (kidney, muscles) in which MeHg demethylation had occurred. This can be taken as a molecular sign of divalent mercurial contamination since only Hg2+ has been reported yet to induce MT accumulation in contaminated tissues. The suppression of the synthesis of the chemokine CCL2 in the corresponding knockout (KO) mice resulted in important changes in gene expression patterns in the liver and brain. After three months of exposure to an aimara-containing diet, eight of 10 genes selected (Sdhb, Cytb, Cox1, Sod1, Sod2, Mt2, Mdr1a and Bax) were repressed in wild-type mice liver whereas none presented a differential expression in KO Ccl2−/− mice. In the wild-type mice brain, six of 12 genes selected (Cytb, Cox1, Sod1, Sod2, Mdr1a and Bax) presented a stimulated expression, whereas all remained at the basal level of expression in KO Ccl2−/− mice. In the liver of aimara-fed mice, histological alterations were observed for an accumulated mercury concentration as low as 32 ng/g, dw, and metal deposits were observed within the cytoplasm of hepatic cells. PMID:22837723

  11. Effects of methylmercury contained in a diet mimicking the Wayana Amerindians contamination through fish consumption: mercury accumulation, metallothionein induction, gene expression variations, and role of the chemokine CCL2.

    PubMed

    Bourdineaud, Jean-Paul; Laclau, Muriel; Maury-Brachet, Régine; Gonzalez, Patrice; Baudrimont, Magalie; Mesmer-Dudons, Nathalie; Fujimura, Masatake; Marighetto, Aline; Godefroy, David; Rostène, William; Brèthes, Daniel

    2012-01-01

    Methylmercury (MeHg) is a potent neurotoxin, and human beings are mainly exposed to this pollutant through fish consumption. We addressed the question of whether a diet mimicking the fish consumption of Wayanas Amerindians from French Guiana could result in observable adverse effects in mice. Wayanas adult men are subjected to a mean mercurial dose of 7 g Hg/week/kg of body weight. We decided to supplement a vegetarian-based mice diet with 0.1% of lyophilized Hoplias aimara fish, which Wayanas are fond of and equivalent to the same dose as that afflicting the Wayanas Amerindians. Total mercury contents were 1.4 ± 0.2 and 5.4 ± 0.5 ng Hg/g of food pellets for the control and aimara diets, respectively. After 14 months of exposure, the body parts and tissues displaying the highest mercury concentration on a dry weight (dw) basis were hair (733 ng/g) and kidney (511 ng/g), followed by the liver (77 ng/g). Surprisingly, despite the fact that MeHg is a neurotoxic compound, the brain accumulated low levels of mercury (35 ng/g in the cortex). The metallothionein (MT) protein concentration only increased in those tissues (kidney, muscles) in which MeHg demethylation had occurred. This can be taken as a molecular sign of divalent mercurial contamination since only Hg(2+) has been reported yet to induce MT accumulation in contaminated tissues. The suppression of the synthesis of the chemokine CCL2 in the corresponding knockout (KO) mice resulted in important changes in gene expression patterns in the liver and brain. After three months of exposure to an aimara-containing diet, eight of 10 genes selected (Sdhb, Cytb, Cox1, Sod1, Sod2, Mt2, Mdr1a and Bax) were repressed in wild-type mice liver whereas none presented a differential expression in KO Ccl2(-/-) mice. In the wild-type mice brain, six of 12 genes selected (Cytb, Cox1, Sod1, Sod2, Mdr1a and Bax) presented a stimulated expression, whereas all remained at the basal level of expression in KO Ccl2(-/-) mice. In the liver of aimara-fed mice, histological alterations were observed for an accumulated mercury concentration as low as 32 ng/g, dw, and metal deposits were observed within the cytoplasm of hepatic cells. PMID:22837723

  12. Genomic characterization of the human and mouse protein tyrosine phosphatase-1B genes.

    PubMed

    Forsell, P A; Boie, Y; Montalibet, J; Collins, S; Kennedy, B P

    2000-12-30

    PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and a resistance to diet-induced obesity. Interestingly, the human PTP-1B gene maps to chromosome 20q13.1 in a region that has been associated with diabetes and obesity. Although there has been a partial characterization of the 3' end of the human PTP-1B gene, the complete gene organization has not been described. In order to further characterize the PTP-1B gene, we have cloned and determined the genomic organization for both the human and mouse PTP-1B genes including the promoter. The human gene spans >74 kb and features a large first intron of >54 kb; the mouse gene likewise contains a large first intron, although the exact size has not been determined. The organization of the human and mouse PTP-1B genes is identical except for an additional exon at the 3' end of the human that is absent in the mouse. The mouse PTP-1B gene maps to the distal arm of mouse chromosome 2 in the region H2-H3. This region is associated with a mouse obesity quantitative trait locus (QTL) and is syntenic with human chromosome 20. The promoter region of both the human and mouse genes contain no TATA box but multiple GC-rich sequences that contain a number of consensus SP-1 binding sites. The basal activity of the human PTP-1B promoter was characterized in Hep G2 cells using up to 8 kb of 5' flanking sequence. A 432 bp promoter construct immediately upstream of the ATG was able to confer maximal promoter activity. Within this sequence, there are at least three GC-rich sequences and one CCAAT box, and deletion of any of these elements results in decreased promoter activity. In addition, the promoter in a number of mouse strains contains, 3.5 kb upstream of the start codon, an insertion of an intracisternal a particle (IAP) element that possibly could alter the expression of PTP-1B mRNA in these strains. PMID:11137300

  13. LOCALIZATION OF THE MOUSE THYMIDINE KINASE GENE TO THE DISTAL PORTION OF CHROMOSOME 11

    EPA Science Inventory

    We report the regional mapping of the thymidine kinase (tk-1) gene in the mouse using two complementary analyses: 1) investigation of chromosome aberrations associated with tx-1 gene inactivation in the L5178Y TX+/-3.7.2c cell line and (2) in situ molecular hybridization of a clo...

  14. Mouse. cap alpha. -globin genes and. cap alpha. -globin-like pseudogenes are not syntenic

    SciTech Connect

    Popp, R.A.; Lalley, P.A.; Whitney, J.B.; Anderson, W.F.

    1981-10-01

    A genetic polymorphism for a Bgl I endonuclease site near the ..cap alpha..-globin-like pseudogene ..cap alpha..-4 of C57BL/6 and C3H/HeN mice was used to show that ..cap alpha..-4 was not affected by three independent mutations in which the adult globin genes ..cap alpha..-1 and ..cap alpha..-2 were deleted. These results indicated that ..cap alpha..-4 might not be located adjacent to the adult ..cap alpha..-globin genes on chromosome 11. Restriction endonuclease analysis of DNA of a primary clone of a Chinese hamster-mouse somatic cell hybrid that had lost mouse chromosomes 11 and 18 showed that this clone lacked the adult murine globin genes ..cap alpha..-1 and ..cap alpha..-2 but it did contain the ..cap alpha..-globin-like pseudogenes ..cap alpha..-3 and ..cap alpha..-4. These results indicated that the adult ..cap alpha..-globin genes and ..cap alpha..-globin-like pseudogenes are not located on the same chromosome. Similar analyses of several other Chinese hamster-mouse somatic cell hybrids that had segregated other mouse chromosomes indicated that the ..cap alpha..-globin-like pseudogenes ..cap alpha..-3 and ..cap alpha..-4 are located on mouse chromosomes 15 and 17, respectively. These data explain why ..cap alpha..-3 and ..cap alpha..-4 were not affected by the three independently induced deletion-type mutations that cause ..cap alpha..-thalassemia in the mouse.

  15. Metallothionein function and genetic regulation in yeast

    SciTech Connect

    Ecker, D.J.; Butt, T.R.; Crooke, S.T.

    1986-05-01

    Copper resistance in yeast is mediated by the CUP1 locus which codes for yeast metallothionein (MT). A genetic approach was taken to study yeast MT gene regulation and to test the function of MT in the detoxification of metal ions other than copper. A yeast strain was constructed (cup1/sup ..delta../) in which the MT structural and regulatory sequences were deleted. The deleted gene was then replaced with the following genetically modified forms of MT on high copy episomal plasmid (YE/sup p/ 13): 1) the intact yeast gene with normal structural and regulatory sequences; 2) a constitutively expressed yeast promoter (TDH) running the yeast MT structural gene. Metal resistance in the cup1/sup ..delta../ strain and the cup1/sup ..delta../ strain transformed with the MT plasmid constructions was compared on metal-supplemented agar plates. Both of the high copy MT plasmids conferred in excess of 500-fold greater copper resistance to the cup1/sup ..delta../ strain. Increased cadmium resistance was not observed in any of the strains that had MT under normal regulatory control. However, the strain with constitutively expressed MT was in excess of 1000-fold more resistant to cadmium. Neither of the MT constructions conferred resistance to Hg,Zn,Co,Ni,Ag,Au,Pt,La,U or Sn. MT gene induction measured by the analysis of MT mRNA on northern blots showed that the yeast MT promoter is not induced by Cd, Zn, Au, Hg, Ag, superoxide, hydrogen peroxide, steroid hormones or heat shock.

  16. Comparative analysis of sequence characteristics of imprinted genes in human, mouse, and cattle

    PubMed Central

    Zaitoun, Ismail; Kim, Eui-Soo

    2007-01-01

    Genomic imprinting is an epigenetic mechanism that results in monoallelic expression of genes depending on parent-of-origin of the allele. Although the conservation of genomic imprinting among mammalian species has been widely reported for many genes, there is accumulating evidence that some genes escape this conservation. Most known imprinted genes have been identified in the mouse and human, with few imprinted genes reported in cattle. Comparative analysis of genomic imprinting across mammalian species would provide a powerful tool for elucidating the mechanisms regulating the unique expression of imprinted genes. In this study we analyzed the imprinting of 22 genes in human, mouse, and cattle and found that in only 11 was imprinting conserved across the three species. In addition, we analyzed the occurrence of the sequence elements CpG islands, C + G content, tandem repeats, and retrotransposable elements in imprinted and in nonimprinted (control) cattle genes. We found that imprinted genes have a higher G + C content and more CpG islands and tandem repeats. Short interspersed nuclear elements (SINEs) were notably fewer in number in imprinted cattle genes compared to control genes, which is in agreement with previous reports for human and mouse imprinted regions. Long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs) were found to be significantly underrepresented in imprinted genes compared to control genes, contrary to reports on human and mouse. Of considerable significance was the finding of highly conserved tandem repeats in nine of the genes imprinted in all three species. Electronic supplementary material The online version of this article (doi: 10.1007/s00335-007-9039-z) contains supplementary material, which is available to authorized users. PMID:17653590

  17. Mouse lactoferrin gene: a marker for estrogen and epidermal growth factor.

    PubMed Central

    Teng, C

    1995-01-01

    Lactoferrin mRNA in the 21-day-old mouse uterus can be increased several hundredfold by estrogen. The physiological role of lactoferrin in mouse uterus is unclear; however, it can be a useful marker for the estrogen action in the uterus. The structural organization and the chromosome location of the lactoferrin gene are similar to members of the transferrin gene family. At the 5' flanking region of the lactoferrin gene, we have characterized two modules that respond to estrogen and growth factor stimulation. Each module is composed of either overlapping or multiple transcription factor-binding elements. The well-characterized estrogen and growth factor response modules in the mouse lactoferrin gene could serve as the foundation to understand the intricate molecular mechanisms of estrogen action and its relationship to growth factors. Images Figure 1. Figure 1. PMID:8593866

  18. An animal model for Norrie disease (ND): gene targeting of the mouse ND gene.

    PubMed

    Berger, W; van de Pol, D; Bchner, D; Oerlemans, F; Winkens, H; Hameister, H; Wieringa, B; Hendriks, W; Ropers, H H

    1996-01-01

    In order to elucidate the cellular and molecular processes which are involved in Norrie disease (ND), we have used gene targeting technology to generate ND mutant mice. The murine homologue of the ND gene was cloned and shown to encode a polypeptide that shares 94% of the amino acid sequence with its human counterpart. RNA in situ hybridization revealed expression in retina, brain and the olfactory bulb and epithelium of 2 week old mice. Hemizygous mice carrying a replacement mutation in exon 2 of the ND gene developed retrolental structures in the vitreous body and showed an overall disorganization of the retinal ganglion cell layer. The outer plexiform layer disappears occasionally, resulting in a juxtaposed inner and outer nuclear layer. At the same regions, the outer segments of the photoreceptor cell layer are no longer present. These ocular findings are consistent with observations in ND patients and the generated mouse line provides a faithful model for study of early pathogenic events in this severe X-linked recessive neurological disorder. PMID:8789439

  19. Chromosomal mapping of the structural gene coding for the mouse cell adhesion molecule uvomorulin.

    PubMed Central

    Eistetter, H R; Adolph, S; Ringwald, M; Simon-Chazottes, D; Schuh, R; Guénet, J L; Kemler, R

    1988-01-01

    The gene coding for the mouse cell adhesion molecule uvomorulin has been mapped to chromosome 8. Uvomorulin cDNA clone F5H3 identified restriction fragment length polymorphisms in Southern blots of genomic DNA from mouse species Mus musculus domesticus and Mus spretus. By analyzing the segregation pattern of the gene in 75 offspring from an interspecific backcross a single genetic locus, Um, was defined on chromosome 8. Recombination frequency between Um and the co-segregating loci serum esterase 1 (Es-1) and tyrosine aminotransferase (Tat) places Um about 14 centimorgan (cM) distal to Es-1, and 5 cM proximal to Tat. In situ hybridization of uvomorulin [3H]cDNA to mouse metaphase chromosomes located the Um locus close to the distal end of chromosome 8 (bands C3-E1). Since uvomorulin is evolutionarily highly conserved, its chromosomal assignment adds an important marker to the mouse genetic map. Images PMID:2897121

  20. Sequence divergence and chromosomal rearrangements during the evolution of human pseudoautosomal genes and their mouse homologs

    SciTech Connect

    Ellison, J.; Li, X.; Francke, U.

    1994-09-01

    The pseudoautosomal region (PAR) is an area of sequence identity between the X and Y chromosomes and is important for mediating X-Y pairing during male meiosis. Of the seven genes assigned to the human PAR, none of the mouse homologs have been isolated by a cross-hybridization strategy. Two of these homologs, Csfgmra and II3ra, have been isolated using a functional assay for the gene products. These genes are quite different in sequence from their human homologs, showing only 60-70% sequence similarity. The Csfgmra gene has been found to further differ from its human homolog in being isolated not on the sex chromosomes, but on a mouse autosome (chromosome 19). Using a mouse-hamster somatic cell hybrid mapping panel, we have mapped the II3ra gene to yet another mouse autosome, chromosome 14. Attempts to clone the mouse homolog of the ANT3 locus resulted in the isolation of two related genes, Ant1 and Ant2, but failed to yield the Ant3 gene. Southern blot analysis of the ANT/Ant genes showed the Ant1 and Ant2 sequences to be well-conserved among all of a dozen mammals tested. In contrast, the ANT3 gene only showed hybridization to non-rodent mammals, suggesting it is either greatly divergent or has been deleted in the rodent lineage. Similar experiments with other human pseudoautosomal probes likewise showed a lack of hybridization to rodent sequences. The results show a definite trend of extensive divergence of pseudoautosomal sequences in addition to chromosomal rearrangements involving X;autosome translocations and perhaps gene deletions. Such observations have interesting implications regarding the evolution of this important region of the sex chromosomes.

  1. Structure of 4-hydrophenylpyruvic acid dioxygenase (HPD) gene and its mutation in tyrosinemic mouse strain III

    SciTech Connect

    Awata, H.; Endo, F.; Matsuda, I.

    1994-09-01

    4-Hydroxphenylpyruvic acid dioxygenase (HPD) is an important enzyme in tyrosine catabolism in most organisms. The activity of this enzyme is expressed mainly in the liver and is developmentally regulated in mammals. A genetic deficiency of the enzyme in man and mouse leads to hereditary tyrosinemia type 3. Using human HPD cDNA as a probe, a chromosomal gene related to HPD was isolated from human and mouse gene libraries. The human HPD gene is over 30 kilo-bases long and is split into 14 exons. Analysis of the 5{prime} flanking sequence of the gene suggests that expression of the gene is regulated by hepatocyte-specific and liver-enriched transcription factors, as well as by hormones. These features of the 5{prime} flanking region of the gene are similar to those of other genes which are specifically expressed in hepatocytes and which are developmentally regulated. The gene for mouse HPD has a similar structure and we obtained evidence for a nucleotide substitution which generates a termination codon in exon 7 of the HPD gene in III mice. This mutation associates a partial exon skipping and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Thus, mouse strain III can serve as a genetic model for human tyrosinemia type 3. Ongoing studies are expected to elucidate the disease process involved in hereditary tyrosinemia type 1 and to shed light on mechanisms that mediate developmental regulation of HPD gene expression. In addition, mouse strain III together with recently established models for tyrosinemia type 1 will facilitate studies on hereditary tyrosinemias.

  2. Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes

    PubMed Central

    Soh, Y.Q. Shirleen; Alföldi, Jessica; Pyntikova, Tatyana; Brown, Laura G.; Graves, Tina; Minx, Patrick J.; Fulton, Robert S.; Kremitzki, Colin; Koutseva, Natalia; Mueller, Jacob L.; Rozen, Steve; Hughes, Jennifer F.; Owens, Elaine; Womack, James E.; Murphy, William J.; Cao, Qing; de Jong, Pieter; Warren, Wesley C.; Wilson, Richard K.; Skaletsky, Helen; Page, David C.

    2014-01-01

    Summary We sequenced the MSY (Male-Specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only two percent of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 50 of the MSY's genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs, but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism. PMID:25417157

  3. Gene Expression Analyses of Mouse Aortic Endothelium in Response to Atherogenic Stimuli

    PubMed Central

    Erbilgin, Ayca; Siemers, Nathan; Kayne, Paul; Yang, Wen-pin; Berliner, Judith; Lusis, Aldons J.

    2014-01-01

    Objective Endothelial cells are central to the initiation of atherosclerosis, yet there has been limited success in studying their gene expression in the mouse aorta. To address this, we developed a method for determining the global transcriptional changes that occur in the mouse endothelium in response to atherogenic conditions and applied it to investigate inflammatory stimuli. Approach and Results We characterized a method for the isolation of endothelial cell RNA with high purity directly from mouse aortas and adapted this method to allow for the treatment of aortas ex vivo before RNA collection. Expression array analysis was performed on endothelial cell RNA isolated from control and hyperlipidemic prelesion mouse aortas, and 797 differentially expressed genes were identified. We also examined the effect of additional atherogenic conditions on endothelial gene expression, including ex vivo treatment with inflammatory stimuli, acute hyperlipidemia, and age. Of the 14 most highly differentially expressed genes in endothelium from prelesion aortas, 8 were also perturbed significantly by ?1 atherogenic conditions: 2610019E17Rik, Abca1, H2-Ab1, H2-D1, Pf4, Ppbp, Pvrl2, and Tnnt2. Conclusions We demonstrated that RNA can be isolated from mouse aortic endothelial cells after in vivo and ex vivo treatments of the murine vessel wall. We applied these methods to identify a group of genes, many of which have not been described previously as having a direct role in atherosclerosis, that were highly regulated by atherogenic stimuli and may play a role in early atherogenesis. PMID:23990205

  4. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  5. [The cloning and expression of a novel mouse gene mLPTS and its subcellular localization].

    PubMed

    Liao, Cheng; Zhao, Mu-Jun; Li, Zai-Ping

    2002-10-01

    A novel mouse gene mLPTS was cloned by EST assembling, RT-PCR and DNA sequencing. The gene fragment for mLPTS is 1244 bp in length, encoding a protein of 332 amino acids. The amino acid sequence of mLPTS has 78% homologue with that of LPTS gene, which is a novel liver cancer-related gene identified through positional candidate cloning stratage by our laboratory. The expression of LPTS gene was ubiquitous in normal human tissues, whereas levels appeared to be significantly reduced, or sometime undetectable in HCC cells and neoplastic tissues, and it might be involved in the negative regulation of cell proliferation. The expression of mLPTS gene was found in all mouse tissues analyzed, same with that of LPTS gene in human. There was only one transcript for mLPTS gene in mouse tissues. The phylogenetic tree was constructed through the amino acids sequence analysis and the study of the sequence homologue among different species. Next, mLPTS gene was cloned into green fluorescent protein eukarytic expression vector and then transfected into CHO cell line. The green fluorescent was mostly limited in the nucleolus, showing that the gene products of mLPTS in eukaryocytes were located in the nucleolus. PMID:12561469

  6. A Survey of Imprinted Gene Expression in Mouse Trophoblast Stem Cells

    PubMed Central

    Calabrese, J. Mauro; Starmer, Joshua; Schertzer, Megan D.; Yee, Della; Magnuson, Terry

    2015-01-01

    Several hundred mammalian genes are expressed preferentially from one parental allele as the result of a process called genomic imprinting. Genomic imprinting is prevalent in extra-embryonic tissue, where it plays an essential role during development. Here, we profiled imprinted gene expression via RNA-Seq in a panel of six mouse trophoblast stem lines, which are ex vivo derivatives of a progenitor population that gives rise to the placental tissue of the mouse. We found evidence of imprinted expression for 48 genes, 31 of which had been described previously as imprinted and 17 of which we suggest as candidate imprinted genes. An equal number of maternally and paternally biased genes were detected. On average, candidate imprinted genes were more lowly expressed and had weaker parent-of-origin biases than known imprinted genes. Several known and candidate imprinted genes showed variability in parent-of-origin expression bias between the six trophoblast stem cell lines. Sixteen of the 48 known and candidate imprinted genes were previously or newly annotated noncoding RNAs and six encoded for a total of 60 annotated microRNAs. Pyrosequencing across our panel of trophoblast stem cell lines returned levels of imprinted expression that were concordant with RNA-Seq measurements for all eight genes examined. Our results solidify trophoblast stem cells as a cell culture-based experimental model to study genomic imprinting, and provide a quantitative foundation upon which to delineate mechanisms by which the process is maintained in the mouse. PMID:25711832

  7. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

    PubMed Central

    Miller, R D; Hogg, J; Ozaki, J H; Gell, D; Jackson, S P; Riblet, R

    1995-01-01

    The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs. Images Fig. 3 PMID:7479885

  8. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

    PubMed

    Miller, R D; Hogg, J; Ozaki, J H; Gell, D; Jackson, S P; Riblet, R

    1995-11-01

    The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs. PMID:7479885

  9. Molecular cloning and structural analysis of mouse gene and pseudogenes for proliferating cell nuclear antigen.

    PubMed Central

    Yamaguchi, M; Hayashi, Y; Hirose, F; Matsuoka, S; Moriuchi, T; Shiroishi, T; Moriwaki, K; Matsukage, A

    1991-01-01

    We have isolated clones containing the entire mouse proliferating cell nuclear antigen (PCNA) gene of 3890 bp and flanking sequences using a rat PCNA cDNA as a probe. The mouse gene has 6 exons whose sequences and junction points of exons with introns are extensively homologous to the human gene while sizes and nucleotide sequences of introns are much less conserved than exons. By a transient expression assay of chloramphenicol acetyltransferase, the promoter of this gene is localized within 200 bp upstream of the transcription initiation site. We have also isolated two processed pseudogenes. Homology between the first one (psi PCNA-I) and the exons of the PCNA gene was 76.8% in the region so far sequenced. The second one (psi PCNA-II) consists of a region highly homologous to the entire exons of the PCNA gene, and only 9 out of total 1256 bp are different from the corresponding exon sequence of the gene. The 5'-flanking region of the psi PCNA-II did not function as an active promoter. Surveys in various wild and laboratory mice genomes suggest that the psi PCNA-II was generated through the reverse transcription process of the PCNA mRNA about 5 x 10(5) years ago in the domesticus subspecies of Mus musculus, the house mouse. The psi PCNA-II is tentatively mapped in the chromosome 17 of the C57BL mouse. Images PMID:1674997

  10. Number of CpG islands and genes in human and mouse.

    PubMed Central

    Antequera, F; Bird, A

    1993-01-01

    Estimation of gene number in mammals is difficult due to the high proportion of noncoding DNA within the nucleus. In this study, we provide a direct measurement of the number of genes in human and mouse. We have taken advantage of the fact that many mammalian genes are associated with CpG islands whose distinctive properties allow their physical separation from bulk DNA. Our results suggest that there are approximately 45,000 CpG islands per haploid genome in humans and 37,000 in the mouse. Sequence comparison confirms that about 20% of the human CpG islands are absent from the homologous mouse genes. Analysis of a selection of genes suggests that both human and mouse are losing CpG islands over evolutionary time due to de novo methylation in the germ line followed by CpG loss through mutation. This process appears to be more rapid in rodents. Combining the number of CpG islands with the proportion of island-associated genes, we estimate that the total number of genes per haploid genome is approximately 80,000 in both organisms. Images Fig. 1 PMID:7505451

  11. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

    PubMed Central

    Blatt, C; Eversole-Cire, P; Cohn, V H; Zollman, S; Fournier, R E; Mohandas, L T; Nesbitt, M; Lugo, T; Jones, D T; Reed, R R

    1988-01-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins. Images PMID:2902634

  12. Inferring nonneutral evolution from human-chimp-mouse orthologous gene trios.

    PubMed

    Clark, Andrew G; Glanowski, Stephen; Nielsen, Rasmus; Thomas, Paul D; Kejariwal, Anish; Todd, Melissa A; Tanenbaum, David M; Civello, Daniel; Lu, Fu; Murphy, Brian; Ferriera, Steve; Wang, Gary; Zheng, Xianqgun; White, Thomas J; Sninsky, John J; Adams, Mark D; Cargill, Michele

    2003-12-12

    Even though human and chimpanzee gene sequences are nearly 99% identical, sequence comparisons can nevertheless be highly informative in identifying biologically important changes that have occurred since our ancestral lineages diverged. We analyzed alignments of 7645 chimpanzee gene sequences to their human and mouse orthologs. These three-species sequence alignments allowed us to identify genes undergoing natural selection along the human and chimp lineage by fitting models that include parameters specifying rates of synonymous and nonsynonymous nucleotide substitution. This evolutionary approach revealed an informative set of genes with significantly different patterns of substitution on the human lineage compared with the chimpanzee and mouse lineages. Partitions of genes into inferred biological classes identified accelerated evolution in several functional classes, including olfaction and nuclear transport. In addition to suggesting adaptive physiological differences between chimps and humans, human-accelerated genes are significantly more likely to underlie major known Mendelian disorders. PMID:14671302

  13. Assessment of orthologous splicing isoforms in human and mouse orthologous genes

    PubMed Central

    2010-01-01

    Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts) we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species-specific, suggests that, still maintaining the conventional definition of gene orthology, a new concept of "splicing orthology" can be defined at transcript level. PMID:20920313

  14. The mouse lysosomal membrane protein 1 gene as a candidate for the motorneuron degeneration (mnd) locus

    SciTech Connect

    Bermingham, N.A.; Martin, J.E.; Fisher, E.M.C.

    1996-03-01

    The motorneuron degeneration (mnd) mutation causes one of the few late-onset progressive neurodegenerations in mice; therefore, the mnd mouse is a valuable paradigm for studying neurodegenerative biology. The mnd mutation may also model human neuronal ceroid lipofuscinosis (NCL) or Batten disease. Mnd maps to the centromeric region of mouse chromosome 8, which likely corresponds to portions of human chromosomes 13,8, or 19; we note that the chromosome 13 portion maps close to a region thought to contain the human Type V NCL locus. We have identified candidate genes for the mnd locus from human chromosomes 13, 8, and 19, and we are mapping these genes in the mouse to determine their proximity to the mutated locus and to refine the comparative human-mouse map in this area. A candidate gene from human chromosome 13 is LAMP1, which encodes lysosomal membrane protein 1. We found that Lamp1 in the mouse lies within the region of the mnd mutation. Therefore, we sequenced Lamp1 cDNAs from homozygous mnd mice and unrelated wildtype C57BL/6 mice. We find no differences between the two cDNA species in the regions examined, and expression analysis shows a similar LAMP1 protein distribution in wildtype and mutant mice, suggesting that an abnormal accumulation of material within normal lysosome structures is unlikely to be the pathogenetic mechanism in the mnd mouse. 19 refs., 3 figs.

  15. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    SciTech Connect

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J.; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  16. Significance of metallothioneins in aging brain.

    PubMed

    Sharma, Sushil; Ebadi, Manuchair

    2014-01-01

    Aging is an inevitable biological process, associated with gradual and spontaneous biochemical and physiological changes, and increased susceptibility to diseases. Chronic inflammation and oxidative stress are hallmarks of aging. Metallothioneins (MTs) are low molecular weight, zinc-binding, anti-inflammatory, and antioxidant proteins that provide neuroprotection in the aging brain through zinc-mediated transcriptional regulation of genes involved in cell growth, proliferation, and differentiation. In addition to Zn(2+) homeostasis, antioxidant role of MTs is routed through -SH moieties on cysteine residues. MTs are induced in aging brain as a defensive mechanism to attenuate oxidative and nitrative stress implicated in broadly classified neurodegenerative ?-synucleinopathies. In addition, MTs as free radical scavengers inhibit Charnoly body (CB) formation to provide mitochondrial neuroprotection in the aging brain. In general, MT-1 and MT-2 induce cell growth and differentiation, whereas MT-3 is a growth inhibitory factor, which is reduced in Alzheimer's disease. MTs are down-regulated in homozygous weaver (wv/wv) mice exhibiting progressive neurodegeneration, early aging, morbidity, and mortality. These neurodegenerative changes are attenuated in MTs over-expressing wv/wv mice, suggesting the neuroprotective role of MTs in aging. This report provides recent knowledge regarding the therapeutic potential of MTs in neurodegenerative disorders of aging such as Parkinson's disease and Alzheimer's disease. PMID:24389356

  17. Metallothionein as an Anti-Inflammatory Mediator

    PubMed Central

    Inoue, Ken-ichiro; Takano, Hirohisa; Shimada, Akinori; Satoh, Masahiko

    2009-01-01

    The integration of knowledge concerning the regulation of MT, a highly conserved, low molecular weight, cystein-rich metalloprotein, on its proposed functions is necessary to clarify how MT affects cellular processes. MT expression is induced/enhanced in various tissues by a number of physiological mediators. The cellular accumulation of MT depends on the availability of cellular zinc derived from the diet. MT modulates the binding and exchange/transport of heavy metals such as zinc, cadmium, or copper under physiological conditions and cytoprotection from their toxicities, and the release of gaseous mediators such as hydroxyl radicals or nitric oxide. In addition, MT reportedly affects a number of cellular processes, such as gene expression, apoptosis, proliferation, and differentiation. Given the genetic approach, the apparently healthy status of MT-deficient mice argues against an essential biological role for MT; however, this molecule may be critical in cells/tissues/organs in times of stress, since MT expression is also evoked/enhanced by various stresses. In particular, because metallothionein (MT) is induced by inflammatory stress, its roles in inflammation are implied. Also, MT expression in various organs/tissues can be enhanced by inflammatory stimuli, implicating in inflammatory diseases. In this paper, we review the role of MT of various inflammatory conditions. PMID:19436762

  18. Characterizing Embryonic Gene Expression Patterns in the Mouse Using Nonredundant Sequence-Based Selection

    PubMed Central

    Sousa-Nunes, Rita; Rana, Amer Ahmed; Kettleborough, Ross; Brickman, Joshua M.; Clements, Melanie; Forrest, Alistair; Grimmond, Sean; Avner, Philip; Smith, James C.; Dunwoodie, Sally L.; Beddington, Rosa S.P.

    2003-01-01

    This article investigates the expression patterns of 160 genes that are expressed during early mouse development. The cDNAs were isolated from 7.5 d postcoitum (dpc) endoderm, a region that comprises visceral endoderm (VE), definitive endoderm, and the node–tissues that are required for the initial steps of axial specification and tissue patterning in the mouse. To avoid examining the same gene more than once, and to exclude potentially ubiquitously expressed housekeeping genes, cDNA sequence was derived from 1978 clones of the Endoderm library. These yielded 1440 distinct cDNAs, of which 123 proved to be novel in the mouse. In situ hybridization analysis was carried out on 160 of the cDNAs, and of these, 29 (18%) proved to have restricted expression patterns. PMID:14613977

  19. Aberrant profile of gene expression in cloned mouse embryos derived from donor cumulus nuclei.

    PubMed

    Tong, Guo Qing; Heng, Boon Chin; Tan, Lay Geok; Ng, Soon Chye

    2006-08-01

    Somatic cell nuclear transfer has successfully been used to clone several mammalian species including the mouse, albeit with extremely low efficiency. This study investigated gene expression in cloned mouse embryos derived from cumulus cell donor nuclei, in comparison with in vivo fertilized mouse embryos, at progressive developmental stages. Enucleation was carried out by the conventional puncture method rather than by the piezo-actuated technique, whereas nuclear transfer was achieved by direct cumulus nuclear injection. Embryonic development was monitored from chemically induced activation on day 0 until the blastocyst stage on day 4. Poor developmental competence of cloned embryos was observed, which was confirmed by lower cell counts in cloned blastocysts, compared with the in vivo fertilized controls. Subsequently, real-time polymerase chain reaction was used to analyze and compare embryonic gene expression at the 2-cell, 4-cell, and blastocyst stages, between the experimental and control groups. The results showed reduced expression of the candidate genes in cloned 2-cell stage embryos, as manifested by poor developmental competence, compared with expression in the in vivo fertilized controls. Cloned 4-cell embryos and blastocysts, which had overcome the developmental block at the 2-cell stage, also showed up-regulated and down-regulated expression of several genes, strongly suggesting incomplete nuclear reprogramming. We have therefore demonstrated that aberrant embryonic gene expression is associated with low developmental competence of cloned mouse embryos. To improve the efficiency of somatic cell nuclear transfer, strategies to rectify aberrant gene expression in cloned embryos should be investigated. PMID:16596391

  20. eMouseAtlas informatics: embryo atlas and gene expression database.

    PubMed

    Armit, Chris; Richardson, Lorna; Hill, Bill; Yang, Yiya; Baldock, Richard A

    2015-10-01

    A significant proportion of developmental biology data is presented in the form of images at morphologically diverse stages of development. The curation of these datasets presents different challenges to that of sequence/text-based data. Towards this end, the eMouseAtlas project created a digital atlas of mouse embryo development as a means of understanding developmental anatomy and exploring the relationship between genes and development in a spatial context. Using the morphological staging system pioneered by Karl Theiler, the project has generated 3D models of post-implantation mouse development and used them as a spatial framework for the delineation of anatomical components and for archiving in situ gene expression data in the EMAGE database. This has allowed us to develop a unique online resource for mouse developmental biology. We describe here the underlying structure of the resource, as well as some of the tools that have been developed to allow users to mine the curated image data. These tools include our IIP3D/X3DOM viewer that allows 3D visualisation of anatomy and/or gene expression in the context of a web browser, and the eHistology resource that extends this functionality to allow visualisation of high-resolution cellular level images of histology sections. Furthermore, we review some of the informatics aspects of eMouseAtlas to provide a deeper insight into the use of the atlas and gene expression database. PMID:26296321

  1. High-resolution gene expression atlases for adult and developing mouse brain and spinal cord.

    PubMed

    Henry, Alex M; Hohmann, John G

    2012-10-01

    Knowledge of the structure, genetics, circuits, and physiological properties of the mammalian brain in both normal and pathological states is ever increasing as research labs worldwide probe the various aspects of brain function. Until recently, however, comprehensive cataloging of gene expression across the central nervous system has been lacking. The Allen Institute for Brain Science, as part of its mission to propel neuroscience research, has completed several large gene-mapping projects in mouse, nonhuman primate, and human brain, producing informative online public resources and tools. Here we present the Allen Mouse Brain Atlas, covering ~20,000 genes throughout the adult mouse brain; the Allen Developing Mouse Brain Atlas, detailing expression of approximately 2,000 important developmental genes across seven embryonic and postnatal stages of brain growth; and the Allen Spinal Cord Atlas, revealing expression for ~20,000 genes in the adult and neonatal mouse spinal cords. Integrated data-mining tools, including reference atlases, informatics analyses, and 3-D viewers, are described. For these massive-scale projects, high-throughput industrial techniques were developed to standardize and reliably repeat experimental goals. To verify consistency and accuracy, a detailed analysis of the 1,000 most viewed genes for the adult mouse brain (according to website page views) was performed by comparing our data with peer-reviewed literature and other databases. We show that our data are highly consistent with independent sources and provide a comprehensive compendium of information and tools used by thousands of researchers each month. All data and tools are freely available via the Allen Brain Atlas portal (www.brain-map.org). PMID:22832508

  2. Construction of a mouse model of factor VIII deficiency by gene targeting

    SciTech Connect

    Bi, L.; Lawler, A.; Gearhart, J.

    1994-09-01

    To develop a small animal model of hemophilia A for gene therapy experiments, we set out to construct a mouse model for factor VIII deficiency by gene targeting. First, we screened a mouse liver cDNA library using a human FVIII cDNA probe. We cloned a 2.6 Kb partial mouse factor VIII cDNA which extends from 800 base pairs of the 3{prime} end of exon 14 to the 5{prime} end of exon 26. A mouse genomic library made from strain 129 was then screened to obtain genomic fragments covering the exons desired for homologous recombination. Two genomic clones were obtained, and one covering exon 15 through 22 was used for gene targeting. To make gene targeting constructs, a 5.8 Kb genomic DNA fragment covering exons 15 to 19 of the mouse FVIII gene was subcloned, and the neo expression cassette was inserted into exons 16 and 17 separately by different strategies. These two constructs were named MFVIIIC-16 and MFVIIIC-17. The constructs were linearized and transfected into strain 129 mouse ES cells by electroporation. Factor VIII gene-knockout ES cell lines were selected by G-418 and screened by genomic Southern blots. Eight exon 16 targeted cell lines and five exon 17 targeted cell lines were obtained. Three cell lines from each construct were injected into blastocysts and surgically transferred into foster mothers. Multiple chimeric mice with 70-90% hair color derived from the ES-cell genotype were seen with both constructs. Germ line transmission of the ES-cell genotype has been obtained for the MFVIIIC-16 construct, and multiple hemophilia A carrier females have been identified. Factor VIII-deficient males will be conceived soon.

  3. Increased levels of metallothionein in placenta of smokers.

    PubMed

    Ronco, Ana Maria; Arguello, Graciela; Suazo, Myriam; Llanos, Miguel N

    2005-03-01

    Experiments were designed to evaluate and compare metallothionein (MT), zinc and cadmium levels in human placentas of smoking and non-smoking women. Smoking was assessed by self-reported cigarette consumption and urine cotinine levels before delivery. Smoking pregnant women with urine cotinine levels higher than 130 ng/ml were included in the smoking group. Determination of placental MT was performed by western blot analysis after tissue homogenization and saturation with cadmium chloride (1000 ppm). Metallothionein was analyzed with a monoclonal antibody raised against MT-1 and MT-2 and with a second anti mouse antibody conjugated to alkaline phosphatase. Zinc and cadmium were determined by neutron activation analysis and atomic absorption spectrometry respectively. Smokers showed higher placental MT and cadmium levels, together with decreased newborn birth weights, as compared to non-smokers. The semi-quantitative analysis of western blots by band densitometry indicated that darker bands corresponded to MT present in smokers' samples. This study confirms that cigarette smoking increases cadmium accumulation in placental tissue and suggests that this element has a stimulatory effect on placental MT production. PMID:15664440

  4. Physical mapping of the retinoid X receptor B gene in mouse and human

    SciTech Connect

    Nagata, T.; Kitagawa, K.; Taketo, M.; Weiss, E.H.; Abe, K.; Ando, A.; Yara-Kikuti, Y.; Inoko, H.; Seldin, M.F.; Ozato, K.

    1995-01-11

    Retinoid X receptors (RXRs) are zinc finger-containing nuclear transcription factors. They belong to the nuclear receptor superfamily that contains retinoid receptors, vitamin D receptors, thyroid hormone receptors, and steroid hormone receptors as well as the so-called orphan receptors. We previously mapped all three RXR genes on mouse chromosomes, using a panel of Mus spretus-Mus musculus interspecific backcross mice: namely, the RXRA-gene (Rxra) on Chr 2 near the centromere, the RXRB gene (Rxrb) on Chr 17 in the H2 region, and the RXRG gene (Rxrg) on distal Chr 1. Using cosmid clones that cover the major histocompatibility complex (MHC) region, we determined the precise physical map positions of the gene encoding mouse and human RXRB, respectively. The mouse gene (Rxrb) maps between H2-Ke4 and H2-Ke5: namely, immediately telomeric to H2-Ke4 which encodes a histidine-rich transmembrane protein, and 12 kilobases centromeric to H2-Ke5 which is expressed in lymphoid tissues, Rxrb and H2-Ke4 are transcribed into opposite directions from a CpG-rich promoter of about 250 base pairs. This gene organization is well conserved also in the human genome at the HLA-DP subregion of Chr 6p, underscoring the strong conservation of the gene organization in the MHC region between the two mammals. 54 refs., 4 figs.

  5. Prediction of Human Disease Genes by Human-Mouse Conserved Coexpression Analysis

    PubMed Central

    Grassi, Elena; Damasco, Christian; Silengo, Lorenzo; Oti, Martin; Provero, Paolo; Di Cunto, Ferdinando

    2008-01-01

    Background Even in the post-genomic era, the identification of candidate genes within loci associated with human genetic diseases is a very demanding task, because the critical region may typically contain hundreds of positional candidates. Since genes implicated in similar phenotypes tend to share very similar expression profiles, high throughput gene expression data may represent a very important resource to identify the best candidates for sequencing. However, so far, gene coexpression has not been used very successfully to prioritize positional candidates. Methodology/Principal Findings We show that it is possible to reliably identify disease-relevant relationships among genes from massive microarray datasets by concentrating only on genes sharing similar expression profiles in both human and mouse. Moreover, we show systematically that the integration of human-mouse conserved coexpression with a phenotype similarity map allows the efficient identification of disease genes in large genomic regions. Finally, using this approach on 850 OMIM loci characterized by an unknown molecular basis, we propose high-probability candidates for 81 genetic diseases. Conclusion Our results demonstrate that conserved coexpression, even at the human-mouse phylogenetic distance, represents a very strong criterion to predict disease-relevant relationships among human genes. PMID:18369433

  6. Gene expression and phenotypic characterization of mouse heart after chronic constant or intermittent hypoxia

    PubMed Central

    Fan, Chenhao; Iacobas, Dumitru A.; Zhou, Dan; Chen, Qiaofang; Lai, James K.; Gavrialov, Orit; Haddad, Gabriel G.

    2010-01-01

    Chronic constant hypoxia (CCH), such as in pulmonary diseases or high altitude, and chronic intermittent hypoxia (CIH), such as in sleep apnea, can lead to major changes in the heart. Molecular mechanisms underlying these cardiac alterations are not well understood. We hypothesized that changes in gene expression could help to delineate such mechanisms. The current study used a neonatal mouse model in CCH or CIH combined with cDNA microarrays to determine changes in gene expression in the CCH or CIH mouse heart. Both CCH and CIH induced substantial alterations in gene expression. In addition, a robust right ventricular hypertrophy and cardiac enlargement was found in CCH- but not in CIH-treated mouse heart. On one hand, upregulation in RNA and protein levels of eukaryotic translation initiation factor-2? and -4E (eIF-2? and eIF-4E) was found in CCH, whereas eIF-4E was downregulated in 1- and 2-wk CIH, suggesting that eIF-4E is likely to play an important role in the cardiac hypertrophy observed in CCH-treated mice. On the other hand, the specific downregulation of heart development-related genes (e.g., notch gene homolog-1, MAD homolog-4) and the upregulation of proteolysis genes (e.g., calpain-5) in the CIH heart can explain the lack of hypertrophy in CIH. Interestingly, apoptosis was enhanced in CCH but not CIH, and this was correlated with an upregulation of proapoptotic genes and downregulation of anti-apoptotic genes in CCH. In summary, our results indicate that 1) the pattern of gene response to CCH is different from that of CIH in mouse heart, and 2) the identified expression differences in certain gene groups are helpful in dissecting mechanisms responsible for phenotypes observed. PMID:15928208

  7. Metallothionein rescues hypoxia-inducible factor-1 transcriptional activity in cardiomyocytes under diabetic conditions.

    PubMed

    Feng, Wenke; Wang, Yuehui; Cai, Lu; Kang, Y James

    2007-08-17

    Metallothionein (MT) is effective in the prevention of diabetic cardiomyopathy, and hypoxia-inducible factor-1 (HIF-1) is known to control vascular endothelial growth factor (VEGF) gene expression and regulate angiogenesis in diabetic hearts. We examined whether or not MT affects HIF-1 activity in the heart of diabetic mice and in the cardiac cells cultured in high glucose (HG) media. Diabetes was induced by streptozotocin in a cardiac-specific MT overexpressing transgenic mouse model. The primary cultures of neonatal cardiomyocytes and the embryonic rat cardiac H9c2 cell line were cultured in HG media. HIF-1 and VEGF were determined by immunofluorescent staining and enzyme-linked immunosorbent assay, respectively. The H9c2 cells were transfected with a hypoxia-responsive element-dependent reporter plasmid and the HIF-1 transcriptional activity was measured by luciferase reporter assay. MT overexpression increased HIF-1alpha in diabetic hearts. HG suppressed CoCl(2)-induced VEGF expression in primary cultures of neonatal cardiomyocytes and MT overexpression suppressed the inhibition. The addition of MT into the cultures of H9c2 cells relieved the HG suppression of hypoxia-induced luciferase activity. This study indicates that MT can rescue HIF-1 transcriptional activity in cardiomyocytes under diabetic conditions. PMID:17586470

  8. Integrative analysis of the connectivity and gene expression atlases in the mouse brain.

    PubMed

    Ji, Shuiwang; Fakhry, Ahmed; Deng, Houtao

    2014-01-01

    Brain function is the result of interneuron signal transmission controlled by the fundamental biochemistry of each neuron. The biochemical content of a neuron is in turn determined by spatiotemporal gene expression and regulation encoded into the genomic regulatory networks. It is thus of particular interest to elucidate the relationship between gene expression patterns and connectivity in the brain. However, systematic studies of this relationship in a single mammalian brain are lacking to date. Here, we investigate this relationship in the mouse brain using the Allen Brain Atlas data. We employ computational models for predicting brain connectivity from gene expression data. In addition to giving competitive predictive performance, these models can rank the genes according to their predictive power. We show that gene expression is predictive of connectivity in the mouse brain when the connectivity signals are discretized. When the expression patterns of 4084 genes are used, we obtain a predictive accuracy of 93%. Our results also show that a small number of genes can almost give the full predictive power of using thousands of genes. We can achieve a prediction accuracy of 91% by using only 25 genes. Gene ontology analysis of the highly ranked genes shows that they are enriched for connectivity related processes. PMID:24004696

  9. The Rab protein family: Genetic mapping of six Rab genes in the mouse

    SciTech Connect

    Barbosa, M.D.F.S.; Gutierrez, M.J.; Kingsmore, S.F.

    1995-12-10

    Rab proteins constitute a family of GTP-binding proteins that are located in distinct intracellular compartments and play a role in the regulation of vesicular trafficking. Yeast mutations in Rab gene homologs cause defects in vesicular transport similar to those observed in beige (bg) mice. To investigate Rab genes as candidates for mouse mutations characterized by defects in vesicular trafficking, we utilized an intersubspecific backcross [C57BL/6J-bg{sup J} X (C57BL/6J-bg{sup J} X CAST/Ei)F{sub 1}] segregating for the bg locus. Restriction fragment length polymorphisms (RFLPs) were obtained through Southern hybridization of F{sub 1} and C57BL/6J chromosomal DNA with the coding sequences of Rab genes. These RFLPs and 12 polymorphic microsatellites were used to determine the segregation of the Rab genes in 93 backcross mice. Rab4a, Rab4b, Rab7, Rab10, Rab22, and Rab24 were localized on mouse chromosomes 8, 7, 9, 12, 2, and 13, respectively. Although the results exclude these loci as candidates for bg, they demonstrate a wide dispersion of Rab genes throughout the mouse genome and reveal that Rab4b and Rab24 are possible candidates for the mouse mutations reduced pigmentation (rp) and purkinje cell degeneration (pcd), respectively. 31 refs., 3 figs., 2 tabs.

  10. Potential translational targets revealed by linking mouse grooming behavioral phenotypes to gene expression using public databases

    PubMed Central

    Roth, Andrew; Kyzar, Evan; Cachat, Jonathan; Stewart, Adam Michael; Green, Jeremy; Gaikwad, Siddharth; OLeary, Timothy P.; Tabakoff, Boris; Brown, Richard E.; Kalueff, Allan V.

    2014-01-01

    Rodent self-grooming is an important, evolutionarily conserved behavior, highly sensitive to pharmacological and genetic manipulations. Mice with aberrant grooming phenotypes are currently used to model various human disorders. Therefore, it is critical to understand the biology of grooming behavior, and to assess its translational validity to humans. The present in-silico study used publicly available gene expression and behavioral data obtained from several inbred mouse strains in the open-field, light-dark box, elevated plus- and elevated zero-maze tests. As grooming duration differed between strains, our analysis revealed several candidate genes with significant correlations between gene expression in the brain and grooming duration. The Allen Brain Atlas, STRING, GoMiner and Mouse Genome Informatics databases were used to functionally map and analyze these candidate mouse genes against their human orthologs, assessing the strain ranking of their expression and the regional distribution of expression in the mouse brain. This allowed us to identify an interconnected network of candidate genes (which have expression levels that correlate with grooming behavior), display altered patterns of expression in key brain areas related to grooming, and underlie important functions in the brain. Collectively, our results demonstrate the utility of large-scale, high-throughput data-mining and in-silico modeling for linking genomic and behavioral data, as well as their potential to identify novel neural targets for complex neurobehavioral phenotypes, including grooming. PMID:23123364

  11. A cross-species genetic analysis identifies candidate genes for mouse anxiety and human bipolar disorder

    PubMed Central

    Ashbrook, David G.; Williams, Robert W.; Lu, Lu; Hager, Reinmar

    2015-01-01

    Bipolar disorder (BD) is a significant neuropsychiatric disorder with a lifetime prevalence of ~1%. To identify genetic variants underlying BD genome-wide association studies (GWAS) have been carried out. While many variants of small effect associated with BD have been identified few have yet been confirmed, partly because of the low power of GWAS due to multiple comparisons being made. Complementary mapping studies using murine models have identified genetic variants for behavioral traits linked to BD, often with high power, but these identified regions often contain too many genes for clear identification of candidate genes. In the current study we have aligned human BD GWAS results and mouse linkage studies to help define and evaluate candidate genes linked to BD, seeking to use the power of the mouse mapping with the precision of GWAS. We use quantitative trait mapping for open field test and elevated zero maze data in the largest mammalian model system, the BXD recombinant inbred mouse population, to identify genomic regions associated with these BD-like phenotypes. We then investigate these regions in whole genome data from the Psychiatric Genomics Consortium's bipolar disorder GWAS to identify candidate genes associated with BD. Finally we establish the biological relevance and pathways of these genes in a comprehensive systems genetics analysis. We identify four genes associated with both mouse anxiety and human BD. While TNR is a novel candidate for BD, we can confirm previously suggested associations with CMYA5, MCTP1, and RXRG. A cross-species, systems genetics analysis shows that MCTP1, RXRG, and TNR coexpress with genes linked to psychiatric disorders and identify the striatum as a potential site of action. CMYA5, MCTP1, RXRG, and TNR are associated with mouse anxiety and human BD. We hypothesize that MCTP1, RXRG, and TNR influence intercellular signaling in the striatum. PMID:26190982

  12. Manual Gene Ontology annotation workflow at the Mouse Genome Informatics Database

    PubMed Central

    Drabkin, Harold J.; Blake, Judith A.

    2012-01-01

    The Mouse Genome Database, the Gene Expression Database and the Mouse Tumor Biology database are integrated components of the Mouse Genome Informatics (MGI) resource (http://www.informatics.jax.org). The MGI system presents both a consensus view and an experimental view of the knowledge concerning the genetics and genomics of the laboratory mouse. From genotype to phenotype, this information resource integrates information about genes, sequences, maps, expression analyses, alleles, strains and mutant phenotypes. Comparative mammalian data are also presented particularly in regards to the use of the mouse as a model for the investigation of molecular and genetic components of human diseases. These data are collected from literature curation as well as downloads of large datasets (SwissProt, LocusLink, etc.). MGI is one of the founding members of the Gene Ontology (GO) and uses the GO for functional annotation of genes. Here, we discuss the workflow associated with manual GO annotation at MGI, from literature collection to display of the annotations. Peer-reviewed literature is collected mostly from a set of journals available electronically. Selected articles are entered into a master bibliography and indexed to one of eight areas of interest such as GO or homology or phenotype. Each article is then either indexed to a gene already contained in the database or funneled through a separate nomenclature database to add genes. The master bibliography and associated indexing provide information for various curator-reports such as papers selected for GO that refer to genes with NO GO annotation. Once indexed, curators who have expertise in appropriate disciplines enter pertinent information. MGI makes use of several controlled vocabularies that ensure uniform data encoding, enable robust analysis and support the construction of complex queries. These vocabularies range from pick-lists to structured vocabularies such as the GO. All data associations are supported with statements of evidence as well as access to source publications. PMID:23110975

  13. Characterization of an upstream regulatory sequence and its binding protein in the mouse apolipoprotein E gene.

    PubMed

    Paik, Y K; Reardon, C A; Taylor, J M; Choi, B K

    1995-06-01

    The mouse apolipoprotein (apo) E gene from strain C57BL/6 was isolated from a genomic DNA library and its complete nucleotide sequence, together with 1.3 kilobase of 5' flanking DNA and 300 base pairs of the 3' flanking DNA, was determined. Regulatory sequences in the proximal 5' flanking region of the gene were identified. Using a chloramphenicol acetyltransferase transient assay system, positive and negative cis-acting sequences were mapped within 380 base pairs of the 5' flanking region of the mouse apoE gene. Two nuclear protein binding sites were identified within this region by DNase I footprinting. We have characterized one of these regions, termed mouse apoE regulatory sequence (MARS-2), which spans nucleotides -151 to -133. Gel mobility shift assays using oligonucleotides of the MARS-2 sequence having specific deletions or substitutions as probes or competitors showed that the essential sequence of MARS-2 required for nuclear protein binding consists of 16 nucleotides encompassing -151 to -136. When nuclear extracts from different cells were examined, L cells and mouse liver nuclear protein contained the highest levels of binding protein for the MARS-2 probe. This protein, termed MARS-2 binding protein, was purified from mouse liver nuclear extracts to homogeneity using gel filtration and MARS-2 oligonucleotide-specific column chromatographic procedures. The Mr = 66,000 binding protein showed a gel mobility shift band that was identical to that of crude nuclear extracts. PMID:7599186

  14. Genomic structure and expression analysis of the mouse testis-specific ribbon protein (Trib) gene.

    PubMed

    Arango, Nelson A; Pearson, Elliot J; Donahoe, Patricia K; Teixeira, Jose

    2004-12-01

    During our analyses of genes required for the development and function of the mouse gonads, we identified a novel testis-specific mRNA, transcribed from a gene that we have named testis-specific ribbon protein (Trib). In the mouse, Trib is located on chromosome 15, overlapping with and transcribed in the opposite orientation of the meiosis specific gene Smc1beta. The deduced amino acid sequence of testis ribbon (TRIB) protein is highly conserved between human, mouse, and rat and contains the ribbon motifs found in the largely uncharacterized microtubule ribbon protein ribbon43a (RIB43A). We show by Northern blot analyses and reverse transcription-polymerase chain reaction (RT-PCR) that Trib mRNA is specifically expressed in the adult testis. In situ hybridization indicates that Trib is expressed solely in germ cells during the leptotene-pachytene stages of spermatogenesis. The high level of evolutionary conservation and the cellular and temporal expression suggest that Trib may be required for mouse spermatogenesis and male fertility. Here, we describe the genomic structure and expression profile of mouse Trib and compare its homology with other ribbon proteins. PMID:15563848

  15. Mapping oxytocin receptor gene expression in the mouse brain and mammary gland using an oxytocin receptor-LacZ reporter mouse.

    PubMed

    Gould, B R; Zingg, H H

    2003-01-01

    The hypothalamic nonapeptide oxytocin (OT) has an established role as a circulating hormone but can also act as a neurotransmitter and as a neuromodulator by interacting with its central OT receptor (OTR). To understand the role of the OTR in the mouse brain we investigated the expression of the OTR gene at the cellular level. We targeted the lacZ reporter gene to the OTR gene locus downstream of the endogenous OTR regulatory elements. Using lactating mouse mammary gland as a control for OTR promoter directed specificity of lacZ gene expression, X-gal histochemistry on tissue sections confirmed that gene expression was restricted to the myoepithelial cells. We also identified for the first time in mice the expression of the OTR gene in neighbouring adipocytes. Further, investigation in the mouse brain identified numerous nuclei containing neurons expressing the OTR gene. Whilst some of these regions had been described for rat or sheep, the OTR-LacZ reporter mouse enabled the identification of novel sites of central OTR gene expression. These regions include the accessory olfactory bulb, the medial septal nucleus, the posterolateral cortical amygdala nucleus, the posterior aspect of the basomedial amygdala nucleus, the medial part of the supramammillary nucleus, the dorsotuberomammillary nucleus, the medial and lateral entorhinal cortices, as well as specific dorsal tegmental, vestibular, spinal trigeminal, and solitary tract subnuclei. By mapping the distribution of OTR gene expression, depicted through histochemical detection of beta-galactosidase, we were able to identify single OTR gene expressing neurons and small neuron clusters that would have remained undetected by conventional approaches. These novel sites of OTR gene expression suggest additional functions of the oxytocinergic system in the mouse. These results lay the foundation for future investigation into the neural role of the OTR and provide a useful model for further study of oxytocin functions in the mouse. PMID:14596857

  16. Structure and evolution of mouse interleukin 6 gene.

    PubMed

    Qin, Z; Richter, G; Diamantstein, T; Blankenstein, T

    1989-11-01

    Restriction fragment length polymorphism in the interleukin 6 gene of murine rodents extending phylogenetically from Mus musculus domesticus to the rat has been analyzed. Most species exhibit distinct restriction site patterns. In contrast, limited polymorphism was found in the tumor necrosis factor alpha gene indicating different selective pressure acting on both genes. The gene encoding interleukin 6 was isolated from a genomic library and the exon/intron organization was determined by restriction analysis and limited DNA sequence analysis. It consists of five exons which distribute over about seven kilobases, thus resembling in structure and organization the human counterpart. Furthermore, no restriction fragment length polymorphisms in the interleukin 6 gene of autoimmune strains NZB, NZW, MRL-lpr/lpr and BxSB could be detected for either EcoRI, BamHI or HindIII. PMID:2575221

  17. Expression Profiling of the Solute Carrier Gene Family in the Mouse BrainS⃞

    PubMed Central

    Dahlin, Amber; Royall, Josh; Hohmann, John G.; Wang, Joanne

    2009-01-01

    The solute carrier (Slc) superfamily is a major group of membrane transport proteins present in mammalian cells. Although Slc transporters play essential and diverse roles in the central nervous system, the localization and function of the vast majority of Slc genes in the mammalian brain are largely unknown. Using high-throughput in situ hybridization data generated by the Allen Brain Atlas, we systematically and quantitatively analyzed the spatial and cellular distribution of 307 Slc genes, which represent nearly 90% of presently known mouse Slc genes, in the adult C57BL/6J mouse brain. Our analysis showed that 252 (82%) of the 307 Slc genes are present in the brain, and a large proportion of these genes were detected at low to moderate expression levels. Evaluation of 20 anatomical brain subdivisions demonstrated a comparable level of Slc gene complexity but significant difference in transcript enrichment. The distribution of the expressed Slc genes was diverse, ranging from near-ubiquitous to highly localized. Functional annotation in 20 brain regions, including the blood-brain and blood-cerebral spinal fluid (CSF) barriers, suggests major roles of Slc transporters in supporting brain energy utilization, neurotransmission, nutrient supply, and CSF production. Furthermore, hierarchical cluster analysis revealed intricate Slc expression patterns associated with neuroanatomical organization. Our studies also revealed Slc genes present within defined brain microstructures and described the putative cell types expressing individual Slc genes. These results provide a useful resource for investigators to explore the roles of Slc genes in neurophysiological and pathological processes. PMID:19179540

  18. Comparative study of apoptosis-related gene loci in human, mouse and rat genomes.

    PubMed

    Yin, Yan-Bin; Zhang, Yong; Yu, Peng; Luo, Jing-Chu; Jiang, Ying; Li, Song-Gang

    2005-05-01

    Many genes are involved in mammalian cell apoptosis pathway. These apoptosis genes often contain characteristic functional domains, and can be classified into at least 15 functional groups, according to previous reports. Using an integrated bioinformatics platform for motif or domain search from three public mammalian proteomes (International Protein Index database for human, mouse, and rat), we systematically cataloged all of the proteins involved in mammalian apoptosis pathway. By localizing those proteins onto the genomes, we obtained a gene locus centric apoptosis gene catalog for human, mouse and rat. Further phylogenetic analysis showed that most of the apoptosis related gene loci are conserved among these three mammals. Interestingly, about one-third of apoptosis gene loci form gene clusters on mammal chromosomes, and exist in the three species, which indicated that mammalian apoptosis gene orders are also conserved. In addition, some tandem duplicated gene loci were revealed by comparing gene loci clusters in the three species. All data produced in this work were stored in a relational database and may be viewed at http://pcas.cbi.pku.edu.cn/database/apd.php. PMID:15880263

  19. Human and Mouse Gene Structure: Comparative Analysis and Application to Exon Prediction

    PubMed Central

    Batzoglou, Serafim; Pachter, Lior; Mesirov, Jill P.; Berger, Bonnie; Lander, Eric S.

    2000-01-01

    We describe a novel analytical approach to gene recognition based on cross-species comparison. We first undertook a comparison of orthologous genomic loci from human and mouse, studying the extent of similarity in the number, size and sequence of exons and introns. We then developed an approach for recognizing genes within such orthologous regions by first aligning the regions using an iterative global alignment system and then identifying genes based on conservation of exonic features at aligned positions in both species. The alignment and gene recognition are performed by new programs called GLASS and ROSETTA, respectively. ROSETTA performed well at exact identification of coding exons in 117 orthologous pairs tested. PMID:10899144

  20. Discordant expression of miR-103/7 and pantothenate kinase host genes in mouse.

    PubMed

    Polster, Brenda J; Westaway, Shawn K; Nguyen, Thuy M; Yoon, Moon Y; Hayflick, Susan J

    2010-01-01

    miR-103 and miR-107, microRNAs hosted by pantothenate kinase genes, are proposed to regulate cellular lipid metabolism. microRNA-mediated regulation is complex, potentially affecting expression of the host gene, related enzymes within the same pathway, or apparently distinct targets. Using qRT-PCR, we demonstrate that miR-103 and miR-107 expression does not correlate with expression of host pantothenate kinase genes in mouse tissues. The miR-103/7 family thus provides an intriguing model for dissecting microRNA transcription, processing and coordinated function within host genes. PMID:20729113

  1. Analysis of mammalian gene function through broad based phenotypic screens across a consortium of mouse clinics

    PubMed Central

    Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl MJ; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie

    2015-01-01

    The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems. PMID:26214591

  2. A short interspersed repetitive element found near some mouse structural genes.

    PubMed

    Lueders, K K; Paterson, B M

    1982-12-11

    We have isolated and characterized a family of interspersed repetitive elements which make up about 1% of the mouse genome. The elements represent a group of homologous but non-identical units about 400 bp in length. Individual members of the family show considerable divergence from one another. The spacial relationships between members of the family and a number of other identified mouse sequences including structural genes have been determined; these elements are found on the 5' as well as 3' sides of various genes at distances ranging from less than 1 to 7.5 kilobases (Kb). The sequences are present in the DNA of all species of Mus. Related sequences are present in the rat genome at a repetition frequency similar to that in the mouse genome. A partial sequence of one member of the family is presented. PMID:6296788

  3. Characterization of the genomic structure of the mouse APLP1 gene

    SciTech Connect

    Zhong, Sue; Wu, Kuo; Black, I.B.; Schaar, D.G.

    1996-02-15

    This article reports on the organization of the mouse APLP1 gene, an evolutionarily conserved amyloid precursor-like protein. The amyloid beta protein, important in Alzheimer diseases, is derived from these precursor proteins. By investigating the expression and structure of this murine gene, it is hoped that more will be learned about the function and regulation of the human homologue. 27 refs., 2 figs.

  4. Genomic integration of adenoviral gene transfer vectors following transduction of fertilized mouse oocytes.

    PubMed

    Larochelle, Nancy; Stucka, Rolf; Rieger, Norman; Schermelleh, Lothar; Schiedner, Gudrun; Kochanek, Stefan; Wolf, Eckhard; Lochmller, Hanns

    2011-02-01

    Adenoviral vectors (AdV) are popular tools to deliver foreign genes into a wide range of cells. They have also been used in clinical gene therapy trials. Studies on AdV-mediated gene transfer to mammalian oocytes and transmission through the germ line have been reported controversially. In the present study we investigated whether AdV sequences integrate into the mouse genome by microinjecting AdV into the perivitelline space of fertilized oocytes. We applied a newly developed PCR technique (HiLo-PCR) for identification of chromosomal junctions next to the integrated AdV. We demonstrate that mouse oocytes can be transduced by different recombinant adenoviral vectors (first generation and gutless). In one transgenic mouse line using the first generation adenoviral vector, the genome has integrated into a highly repetitive cluster located on the Y chromosome. While the transgene (GFP) was expressed in early embryos, no expression was detected in adult transgenic mice. The use of gutless AdV resulted in expression of the transgene, albeit the vector was not transmitted to progeny. These results indicate that under optimized conditions fertilized mouse oocytes are transduced by AdV and give rise to transgenic founder animals. Therefore, adequate precautions should be taken in gene therapy protocols of reproductive patients since transduction of oocytes or early embryos and subsequent chromosomal integration cannot be ruled out entirely. PMID:20464633

  5. The mouse thymosin {beta}4 gene: Structure, promoter identification, and chromosome localization

    SciTech Connect

    Li, Xun; Zimmerman, A.; Yin, H.L.

    1996-03-05

    Thymosin {beta}4 (T{beta}4) is an actin monomer sequestering protein that may have a critical role in modulating the dynamics of actin polymerization and depolymerization in nonmuscle cells. Its regulatory role is consistent with the many examples of transcriptional regulation of T{beta}4 and of tissue-specific expression. Furthermore, lymphocytes have a unique T{beta}4 transcript relative to the ubiquitous transcript found in many other tissues and cells. To determine how T{beta}4 gene expression is regulated and how the alternative transcripts are derived, we cloned the mouse T{beta}4 gene. We established that there is a single mouse T{beta}4 gene and found that the lymphoid-specific transcript is generated by extending the ubiquitous exon 1 with an alternate downstream splice site. The transcription start site is defined by primer extension analysis, and the 5{prime}-flanking region has many of the characteristics of a promoter. It is pyrimidine-rich and contains typical promoter elements, including a GC box, an initiator site, and consensus transcription factor binding sites. The mouse T{beta}4 gene locus (Ptmb4) is located by interspecific backcross mapping to the distal region of the mouse X chromosome, linked to Btk and Gja6. 38 refs., 4 figs., 1 tab.

  6. Isolation, characterization, and chromosomal localization of mouse and human COUP-TF I and II genes

    SciTech Connect

    Qiu, Y.; Krishnan, V.; Zeng, Z.

    1995-09-01

    Chicken ovalbumin upstream promoter transcription factors (COUP-TFs) are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologues have been cloned in many species, from Drosophila to human. The protein sequences of COUP-TFs are highly homologous across species, suggesting functional conservation. Two COUP-TF genes have been cloned from human, and their genomic organizations have been characterized. To determine whether the genomic organization is conserved between human and mouse, we isolated two mouse COUP-TF genes (I and II) and characterized their genomic structures. Both genes have relatively simple structures that are similar to those of their human counterparts. In addition, we mapped mouse COUP-TF I to the distal region of chromosome 13 and COUP-TF II to the central region of chromosome 7. Furthermore, we mapped human COUP-TF I to 5q14 of chromosome 5 and COUP-TF II to 15q26 of chromosome 15. The results demonstrate that COUP-TF genes are located in chromosomal regions that are syntenic between mouse and human. 25 refs., 5 figs.

  7. Cationic star copolymers based on ?-cyclodextrins for efficient gene delivery to mouse embryonic stem cell colonies.

    PubMed

    Loh, Xian Jun; Wu, Yun-Long

    2015-07-11

    A cationic star copolymer with a ?-cyclodextrin core was developed for nonviral gene transfer to mouse embryonic stem cells (mESCs). The copolymer comprises poly(2-dimethyl aminoethyl methacrylate) as the cationic component and poly(2-hydroxyethyl methacrylate) as the non-toxic stealth component. These materials have very low toxicity and show highly efficient transfection to mESC colonies. PMID:26040469

  8. Microarray analysis of metallothioneins in human diseases--A review.

    PubMed

    Krizkova, Sona; Kepinska, Marta; Emri, Gabriella; Rodrigo, Miguel Angel Merlos; Tmejova, Katerina; Nerudova, Danuse; Kizek, Rene; Adam, Vojtech

    2016-01-01

    Metallothioneins (MTs), low molecular mass cysteine-rich proteins, which are able to bind up to 20 monovalent and up to 7 divalent heavy metal ions are widely studied due to their functions in detoxification of metals, scavenging free radicals and cells protection against the oxidative stress. It was found that the loss of the protective effects of MT leads to an escalation of pathogenic processes and carcinogenesis. The most extensive area is MTs expression for oncological applications, where the information about gene patterns is helpful for the identification biological function, resistance to drugs and creating the correct chemotherapy. In other medical applications the effect of oxidative stress to cell lines exposed to heavy metals and hydrogen peroxide is studied as well as influence of drugs and cytokines on MTs expression and MTs expression in the adipose tissue. The precise detection of low metallothionein concentrations and its isoforms is necessary to understand the connection between quantity and isoforms of MTs to size, localization and type of cancer. This information is necessary for well-timed therapy and increase the chance to survival. Microarray chips appear as good possibility for finding all information about expression of MTs genes and isoforms not only in cancer, but also in other diseases, especially diabetes, obesity, cardiovascular diseases, ageing, osteoporosis, psychiatric disorders and as the effects of toxic drugs and pollutants, which is discussed in this review. PMID:26454339

  9. Expression of Notch receptors, ligands and target genes during development of the mouse mammary gland

    PubMed Central

    Raafat, Ahmed; Goldhar, Anita S.; Klauzinska, Malgorzata; Xu, Keli; Amirjazil, Idean; McCurdy, David; Lashin, Karim; Salomon, David; Vonderhaar, Barbara K.; Egan, Sean; Callahan, Robert

    2010-01-01

    Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. Using an immunohistochemical approach with antibodies specific for each Notch receptor, we show that Notch proteins are temporally regulated in mammary epithelial cells during normal mammary gland development in the FVB/N mouse. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. These data define expression of the Notch ligand/receptor system throughout development of the mouse mammary gland and help set the stage for genetic analysis of Notch in this context. PMID:21506125

  10. Locations of human and mouse genes encoding the RFX1 and RFX2 transcription factor proteins

    SciTech Connect

    Doyle, J.; Stubbs, L.; Hoffman, S.

    1996-07-01

    RFX transcription factors constitute a highly conserved family of site-specific DNA binding proteins involved in the expression of a variety of cellular and viral genes, including major histocompatibility complex class II genes and genes in human hepatitis B virus. Five members of the RFX gene family have been isolated from human and mouse, and all share a highly characteristic DNA binding domain that is distinct from other known DNA binding motifs. The human RFX1 and RFX2 genes have been assigned by in situ hybridization to chromosome 19p13.1 and 19p13.3, respectively. In this paper, we present data that localize RFX1 and RFX2 precisely within the detailed physical map of human chromosome 19 and genetic data that assign Rfx1 and Rfx2 to homologous regions of mouse chromosomes 8 and 17, respectively. These data define the established relationships between these homologous mouse and human regions in further detail and provide new tools for linking cloned genes to phenotypes in both species. 26 ref., 2 figs.

  11. Chromosome localizations of genes for five cAMP-specific phosphodiesterases in man and mouse

    SciTech Connect

    Milatovich, A.; Francke, U. ); Bolger, G.; Michaeli, T. )

    1994-03-01

    Cyclic nucleotides are important second messengers that mediate a number of cellular responses to external signals. Cyclic nucleotide phosphodiesterases play a role in signal transduction by regulating the cellular concentrations of these messengers. Here, the authors have applied Southern analyses of somatic cell hybrid lines and of recombinant inbred (RI) mouse strains as well as fluorescence chromosomal in situ hybridization (FISH) to chromosomally localize five cAMP-specific nucleotide phosphodiesterase genes in human and mouse. Genes DPDE1, DPDE2, DPDE3, and DPDE4 that share sequence homology with the Drosophila dunce gene were assigned to human chromosomes 19 (DPDE1 and DPDE2), ga12 (DPDE3), and 1p31 (DPDE4) and to mouse chromosomes 8, 9, 13, and 4, respectively. The high-affinity cAMP-specific phosphodiesterase gene (HCP1) was mapped to human chromosome 8q13-q22. Since these genes are potential candidates for involvement in psychiatric or behavioral disorders, knowledge of their chromosomal localizations will facilitate the discovery of their association with disease genes as they are being mapped by linkage studies.

  12. Reference gene selection for real-time RT-PCR in regenerating mouse livers

    SciTech Connect

    Tatsumi, Kohei; Ohashi, Kazuo Taminishi, Sanae; Okano, Teruo; Yoshioka, Akira; Shima, Midori

    2008-09-12

    The liver has an intrinsic ability to undergo active proliferation and recover functional liver mass in response to an injury response. This regenerative process involves a complex yet well orchestrated change in the gene expression profile. To produce accurate and reliable gene expression of target genes during various stages of liver regeneration, the determination of internal control housekeeping genes (HKGs) those are uniformly expressed is required. In the present study, the gene expression of 8 commonly used HKGs, including GAPDH, ACTB, HPRT1, GUSB, PPIA, TBP, TFRC, and RPL4, were studied using mouse livers that were quiescent and actively regenerating induced by partial hepatectomy. The amplification of the HKGs was statistically analyzed by two different mathematical algorithms, geNorm and NormFinder. Using this method, PPIA and TBP gene expression found to be relatively stable regardless of the stages of liver regeneration and would be ideal for normalization to target gene expression.

  13. A mouse homeobox containing gene on chromosome 11: sequence and tissue-specific expression.

    PubMed Central

    Meijlink, F; de Laaf, R; Verrijzer, P; Destre, O; Kroezen, V; Hilkens, J; Deschamps, J

    1987-01-01

    We have molecularly cloned a mouse homeobox containing gene by isolating cDNA and genomic clones. The gene is located in a previously described cluster on chromosome 11 (Hart et al. (1985) Cell 43, 9-18) and was identified as the Hox2.3 gene. We present the complete mRNA sequence of this gene and describe similarities to other homeobox containing genes, among which its human homologue, the cl gene. High expression of the Hox2.3 gene was found in kidney, testis, and spinal cord of adult mice, in the spinal cord of 12.5-17.5 day embryos and in differentiating EC cells depending on their treatment. Three different treatments of the pluripotent EC cell line P19, each leading to the induction of a specific differentiation pathway, resulted in all cases in induction of Hox2.3; however, major quantitative differences in this response were observed. Images PMID:2889183

  14. Automatic Summarization of Mouse Gene Information by Clustering and Sentence Extraction from MEDLINE Abstracts

    PubMed Central

    Yang, Jianji; Cohen, Aaron M.; Hersh, William

    2007-01-01

    Tools to automatically summarize gene information from the literature have the potential to help genomics researchers better interpret gene expression data and investigate biological pathways. The task of finding information on sets of genes is common for genomic researchers, and PubMed is still the first choice because the most recent and original information can only be found in the unstructured, free text biomedical literature. However, finding information on a set of genes by manually searching and scanning the literature is a time-consuming and daunting task for scientists. We built and evaluated a query-based automatic summarizer of information on mouse genes studied in microarray experiments. The system clusters a set of genes by MeSH, GO and free text features and presents summaries for each gene by ranked sentences extracted from MEDLINE abstracts. Evaluation showed that the system seems to provide meaningful clusters and informative sentences are ranked higher by the algorithm. PMID:18693953

  15. Identification of a Copper-Binding Metallothionein in Pathogenic Mycobacteria

    PubMed Central

    Gold, Ben; Deng, Haiteng; Bryk, Ruslana; Vargas, Diana; Eliezer, David; Roberts, Julia; Jiang, Xiuju; Nathan, Carl

    2009-01-01

    A screen of a genomic library from Mycobacterium tuberculosis (Mtb) identified a small, unannotated open reading frame (MT0196) that encodes a 4.9-kDa, cysteine-rich protein. Despite extensive nucleotide divergence, the amino acid sequence is highly conserved among mycobacteria that are pathogenic in vertebrate hosts. We synthesized the protein and found that it preferentially bound up to 6 Cu(I) ions in a solvent-shielded core. Copper, cadmium and compounds that generate nitric oxide or superoxide induced the genes expression in Mtb up to a thousand-fold. The native protein bound copper within Mtb and partially protected Mtb from copper toxicity. We propose that the product of the MT0196 gene be named mycobacterial metallothionien (MymT). To our knowledge, MymT is the first metallothionein of a Gram-positive bacterium with a demonstrated function. PMID:18724363

  16. Voltage-gated potassium channel genes are clustered in paralogous regions of the mouse genome

    SciTech Connect

    Lock, L.F.; Gilbert, D.J.; Jenkins, N.A.; Copeland, N.G. ); Street, V.A.; Migeon, M.B.; Tempel, B.L. Univ. of Washington School of Medicine, Seattle, WA )

    1994-04-01

    Cloning of the Drosophila Shaker gene established that a neurological phenotype including locomotor dysfunction can be caused by a mutation in a voltage-gated potassium (K) channel gene. Shaker sequences have been used to isolate a large family of related K channel genes from both flies and mammals. Toward elucidating the evolutionary relationship between loci and the potential causal connection that K channels may have to mammalian genetic disorders, the authors report here the genetic mapping of 12-16 different murine, voltage-gated K channel genes. They find that multiple genes, in some cases from distantly related K channel subfamilies, occur in clusters in the mouse genome. These mapping results suggest that the K channel gene subfamilies arose through ancient localized gene duplication events, followed by chromosomal duplications and rearrangements as well as further gene duplication. They also note that several neurologic disorders of both mouse and human are associated with the chromosomal regions containing K channel genes. 78 refs., 5 figs., 1 tab.

  17. Human chromosome 21 gene expression atlas in the mouse.

    PubMed

    Reymond, Alexandre; Marigo, Valeria; Yaylaoglu, Murat B; Leoni, Antonio; Ucla, Catherine; Scamuffa, Nathalie; Caccioppoli, Cristina; Dermitzakis, Emmanouil T; Lyle, Robert; Banfi, Sandro; Eichele, Gregor; Antonarakis, Stylianos E; Ballabio, Andrea

    2002-12-01

    Genome-wide expression analyses have a crucial role in functional genomics. High resolution methods, such as RNA in situ hybridization provide an accurate description of the spatiotemporal distribution of transcripts as well as a three-dimensional 'in vivo' gene expression overview. We set out to analyse systematically the expression patterns of genes from an entire chromosome. We chose human chromosome 21 because of the medical relevance of trisomy 21 (Down's syndrome). Here we show the expression analysis of all identifiable murine orthologues of human chromosome 21 genes (161 out of 178 confirmed human genes) by RNA in situ hybridization on whole mounts and tissue sections, and by polymerase chain reaction with reverse transcription on adult tissues. We observed patterned expression in several tissues including those affected in trisomy 21 phenotypes (that is, central nervous system, heart, gastrointestinal tract, and limbs). Furthermore, statistical analysis suggests the presence of some regions of the chromosome with genes showing either lack of expression or, to a lesser extent, co-expression in specific tissues. This high resolution expression 'atlas' of an entire human chromosome is an important step towards the understanding of gene function and of the pathogenetic mechanisms in Down's syndrome. PMID:12466854

  18. Time course of gene expression during mouse skeletal muscle hypertrophy.

    PubMed

    Chaillou, Thomas; Lee, Jonah D; England, Jonathan H; Esser, Karyn A; McCarthy, John J

    2013-10-01

    The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ≥2-fold increase or ≥50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy. PMID:23869057

  19. Time course of gene expression during mouse skeletal muscle hypertrophy

    PubMed Central

    Lee, Jonah D.; England, Jonathan H.; Esser, Karyn A.; McCarthy, John J.

    2013-01-01

    The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ?2-fold increase or ?50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy. PMID:23869057

  20. Identification and characterization of the genes encoding human and mouse osteoactivin.

    PubMed

    Owen, T A; Smock, S L; Prakash, S; Pinder, L; Brees, D; Krull, D; Castleberry, T A; Clancy, Y C; Marks, S C; Safadi, F F; Popoff, S N

    2003-01-01

    Osteoactivin (OA) is more highly expressed in the bones of osteopetrotic mutant rats (op/op) than in those of their normal littermates and is the homologue of human nmb, a cDNA more highly expressed in melanoma-derived cell lines of low metastatic potential, and of mouse DC-HIL, which has been implicated in endothelial cell adhesion. The human OA gene is found on chromosome 7p15.1 and consists of 11 exons spanning 28.3 kb. Murine OA is encoded by a highly similar gene of 11 exons spanning 20.2 kb on mouse chromosome 6. Human OA uses the same transcriptional initiation site in both bone and kidney as was reported for melanoma cells. OA is expressed in primary human and mouse osteoblast cultures at all stages of differentiation, with increased levels observed concurrently with the expression of osteoblast phenotype markers. OA is also expressed in a wide variety of human and mouse tissues as determined by RT-PCR analysis. Immunohistochemical investigation of OA expression in late mouse embryonic development showed very high, cell-specific expression in the nervous system, basal layer of the skin, germinal cells of hair follicles, and in the forming nephrons of the kidney. Continuing investigation of the cell-specific expression of OA in bone as well as in other tissues will lead to a better understanding of its function in the development of these cell types. PMID:14696968

  1. Characterization of the mouse gene for the heavy metal-responsive transcription factor MTF-1

    PubMed Central

    der Maur, Adrian Auf; Belser, Tanja; Wang, Ying; Gnes, Cagatay; Lichtlen, Peter; Georgiev, Oleg; Schaffner, Walter

    2000-01-01

    MTF-1 is a zinc finger transcription factor that mediates the cellular response to heavy metal stress; its targeted disruption in the mouse leads to liver decay and embryonic lethality at day E14. Recently, we have sequenced the entire MTF-1 gene in the compact genome of the pufferfish Fugu rubripes. Here we have defined the promoter sequences of human and mouse MTF-1 and the genomic structure of the mouse MTF-1 locus. The transcription unit of MTF-1 spans 42 kb (compared to 8.5 kb in Fugu) and is located downstream of the gene for a phosphatase (INPP5P) in mouse, human, and fish. In all of these species, the MTF promoter region has the features of a CpG island. In both mouse and human, the 5? untranslated region harbors conserved short reading frames of unknown function. RNA mapping experiments revealed that in these two species, MTF-1 mRNA is transcribed from a cluster of multiple initiation sites from a TATA-less promoter without metal-responsive elements. Transcription from endogenous and transfected MTF-1 promoters was not affected by heavy metal load or other stressors, in support of the notion that MTF-1 activity is regulated at the posttranscriptional level. Tissue Northern blots normalized for poly A+ RNA indicate that MTF-1 is expressed at similar levels in all tissues, except in the testes, that contain more than 10-fold higher mRNA levels. PMID:11005378

  2. Trio gene is required for mouse learning ability.

    PubMed

    Zong, Wen; Liu, Shuoyang; Wang, Xiaotong; Zhang, Jian; Zhang, Tingting; Liu, Ziyi; Wang, Dongdong; Zhang, Aizhen; Zhu, Minsheng; Gao, Jiangang

    2015-05-22

    Trio is a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Trio regulates cytoskeleton dynamics and actin remodeling and is involved in cell migration and axonal guidance in neuronal development. The null allele of the Trio gene led to embryonic lethality, and Trio null embryos displayed aberrant organization in several regions of the brain at E18.5, including hippocampus. Nestin-Trio-/- mice, in which the Trio gene was deleted specifically in the neuronal system by the Nestin-Cre system, displayed severe phenotypes, including low survival rate, ataxia and multiple developmental defects of the cerebellum. All Nestin-Trio-/- mice died before reaching adulthood, which hinders research on Trio gene function in adult mice. Thus, we generated EMX1-Trio-/- mice by crossing Trio-floxed mice with EMX1-Cre mice in which Cre is expressed in the brain cortex and hippocampus. EMX1-Trio-/- mice can survive to adulthood. Trio gene deletion results in smaller brains, an abnormal hippocampus and disordered granule cells in the dentate gyrus (DG) and cornu ammonis (CA). Behavior tests showed that Trio deletion interfered with the hippocampal-dependent spatial learning in the mice, suggesting that Trio plays critical roles in the learning ability of adult mice. We conclude that the Trio gene regulates the neuronal development of the hippocampus and that it affects the intelligence of adult mice. PMID:25727174

  3. Metallothioneins and cell death by anticancer drugs.

    PubMed

    Lazo, J S; Pitt, B R

    1995-01-01

    Both pharmacologic and genetic methods are now available to manipulate intracellular levels of the protein thiol metallothionein. These approaches have begun to reveal the protective roles metallothioneins (MTs) have against oxygen, nitrogen, and carbon-centered free radicals, as well as the contributory role of MT to resistance to a broad range of electrophilic therapeutic agents, including antineoplastic drugs. We suggest MTs are enlisted to act as primative antioxidant defense mechanisms in mammalian cells and, thus, may have widespread importance in the biology of cell death. PMID:7598510

  4. Differential Expression of Genes within the Cochlea as Defined by a Custom Mouse Inner Ear Microarray

    PubMed Central

    Morris, Ken A.; Snir, Einat; Pompeia, Celine; Koroleva, Irina V.; Kachar, Bechara; Hayashizaki, Yoshihide; Carninci, Piero; Soares, M. Bento

    2005-01-01

    Microarray analyses have contributed greatly to the rapid understanding of functional genomics through the identification of gene networks as well as gene discovery. To facilitate functional genomics of the inner ear, we have developed a mouse inner-ear-pertinent custom microarray chip (CMA-IE1). Nonredundant cDNA clones were obtained from two cDNA library resources: the RIKEN subtracted inner ear set and the NIH organ of Corti library. At least 2000 cDNAs unique to the inner ear were present on the chip. Comparisons were performed to examine the relative expression levels of these unique cDNAs within the organ of Corti, lateral wall, and spiral ganglion. Total RNA samples were obtained from the three cochlear-dissected fractions from adult CF-1 mice. The total RNA was linearly amplified, and a dendrimer-based system was utilized to enhance the hybridization signal. Differentially expressed genes were verified by comparison to known gene expression patterns in the cochlea or by correlation with genes and gene families deduced to be present in the three tissue types. Approximately 2225% of the genes on the array had significant levels of expression. A number of differentially expressed genes were detected in each tissue fraction. These included genes with known functional roles, hypothetical genes, and various unknown or uncharacterized genes. Four of the differentially expressed genes found in the organ of Corti are linked to deafness loci. None of these are hypothetical or unknown genes. PMID:15735932

  5. Effect of microgravity on gene expression in mouse brain

    PubMed Central

    Iacobas, Dumitru A.; Iacobas, Sanda; Nicchia, Grazia Paola; Desaphy, Jean Francois; Camerino, Diana Conte; Svelto, Maria; Spray, David C.

    2009-01-01

    Changes in gravitational force such as that experienced by astronauts during space flight induce a redistribution of fluids from the caudad to the cephalad portion of the body together with an elimination of normal head-to-foot hydrostatic pressure gradients. To assess brain gene profile changes associated with microgravity and fluid shift, a large-scale analysis of mRNA expression levels was performed in the brains of 2-week control and hindlimb-unloaded (HU) mice using cDNA microarrays. Although to different extents, all functional categories displayed significantly regulated genes indicating that considerable transcriptomic alterations are induced by HU. Interestingly, the TIC class (transport of small molecules and ions into the cells) had the highest percentage of up-regulated genes, while the most down-regulated genes were those of the JAE class (cell junction, adhesion, extracellular matrix). TIC genes comprised 16% of those whose expression was altered, including sodium channel, nonvoltage-gated 1 beta (Scnn1b), glutamate receptor (Grin1), voltage-dependent anion channel 1 (Vdac1), calcium channel beta 3 subunit (Cacnb3) and others. The analysis performed by Gene-MAPP revealed several altered protein classes and functional pathways such as blood coagulation and immune response, learning and memory, ion channels and cell junction. In particular, data indicate that HU causes an alteration in hemostasis which resolves in a shift toward a more hyper-coagulative state with an increased risk of venous thrombosis. Furthermore, HU treatment seems to impact on key steps of synaptic plasticity and learning processes. PMID:18704384

  6. Comparison of human chromosome 6p25 with mouse chromosome 13 reveals a greatly expanded ov-serpin gene repertoire in the mouse.

    PubMed

    Kaiserman, Dion; Knaggs, Susan; Scarff, Katrina L; Gillard, Anneliese; Mirza, Ghazala; Cadman, Matthew; McKeone, Richard; Denny, Paul; Cooley, Jessica; Benarafa, Charaf; Remold-O'Donnell, Eileen; Ragoussis, Jiannis; Bird, Phillip I

    2002-03-01

    Ov-serpins are intracellular proteinase inhibitors implicated in the regulation of tumor progression, inflammation, and cell death. The 13 human ov-serpin genes are clustered at 6p25 (3 genes) and 18q21 (10 genes), and share common structures. We show here that a 1-Mb region on mouse chromosome 13 contains at least 15 ov-serpin genes compared with the three ov-serpin genes within 0.35 Mb at human 6p25 (SERPINB1 (MNEI), SERPINB6 (PI-6), SER-PINB9 (PI-9)). The mouse serpins have characteristics of functional inhibitors and fall into three groups on the basis of similarity to MNEI, PI-6, or PI-9. The genes map between the mouse orthologs of the Werner helicase interacting protein and NAD(P)H menadioine oxidoreductase 2 genes, in a region that contains the markers D13Mit136 and D13Mit116. They have the seven-exon structure typical of human 6p25 ov-serpin genes, with identical intron phasing. Most show restricted patterns of expression, with common sites of synthesis being the placenta and immune tissue. Compared with human, this larger mouse serpin repertoire probably reflects the need to regulate a larger proteinase repertoire arising from differing evolutionary pressures on the reproductive and immune systems. PMID:11863365

  7. Gene expression profiles in the fetal mouse brain after etoposide (VP-16) administration.

    PubMed

    Nam, Chunja; Yamauchi, Hirofumi; He, Xi Jun; Woo, Gye-Hyeong; Ahn, Byeongwoo; Nam, Sang-Yoon; Doi, Kunio; Nakayama, Hiroyuki

    2013-01-01

    The aim of this study was to analyze the response of gene expression caused by etoposide (VP-16) in the fetal mouse brain. Four miligrams/kilogram of VP-16 was intraperitoneally injected into pregnant mice on day 12 of gestation (GD 12). Gene expression profiling of the VP-16-treated fetal mouse brain by DNA microarray was performed. The expression changes of the target genes of p53 were also examined by real-time RT-PCR. VP-16 induced S-phase accumulation, G2/M arrest, and eventually apoptosis of neuroepithelial cells in the fetal brain. DNA microarray analysis revealed that 8 of cell cycle control- and apoptosis-related genes were upregulated and that 5 of DNA damage, repair, replication, and transcription genes were also upregulated in the fetal telencephalons at 4 h after VP-16 treatment (HAT). The results of real-time RT-PCR demonstrated that the expression of topoisomerase IIα was increased at 4 and 8 HAT. The expression of pro-apoptotic factors such as puma, noxa, bax, and cyclin G was also increased from 4 to 12 HAT. These results suggest that VP-16 induces DNA damage, DNA repair, cell cycle alternation, and apoptosis in the fetal mouse brain. In addition, VP-16-induced apoptosis is mediated through the mitochondrial pathway in a p53-related manner. The present study will provide a better understanding of the mechanisms of VP-16-induced fetal brain injury. PMID:23615303

  8. A novel downstream regulatory element of the mouse H-2Kb class I major histocompatibility gene.

    PubMed Central

    Krlov, J; Jansa, P; Forejt, J

    1992-01-01

    The H-2Kb gene equipped with a minimal promoter (5' deletion up to -61) was fully expressed in transfected fibroblasts, but inactive in transfected embryonal carcinoma cells. A strong transcriptional regulatory element (H2DRE) was identified when a fragment spanning the second exon and second intron was used to activate transient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in mouse Ltk- or NIH3T3 fibroblasts. Its activity was twice that of a construct where the CAT gene was driven by the H-2Kb 5' enhancer region (H2TF1/KBF1 site) and comparable to that of pRSVCAT construct carrying the strong Rous sarcoma virus LTR enhancer. In accord with regulated transcriptional activity of the intact H-2Kb gene, the H2DRE did not activate the CAT expression in P19 mouse embryonal carcinoma cells. The H2DRE did not function as a typical enhancer since its activity was strongly position dependent. Consistent with its anticipated role in transcription regulation, H2DRE displayed more than five target sites for specifically interacting nuclear factors, two of them being present in H-2 positive fibroblasts, but not in H-2 negative teratocarcinoma cells. None of them was cross-competed by sequences of the 5' enhancer. The results of deletion experiments show that H2DRE is the only regulatory region that can activate transcription from the 5' enhancerless H-2Kb gene in mouse L fibroblasts. Images PMID:1425592

  9. Modeling Chromosomes in Mouse to Explore the Function of Genes, Genomic Disorders, and Chromosomal Organization

    PubMed Central

    Brault, Vronique; Pereira, Patricia; Duchon, Arnaud; Hrault, Yann

    2006-01-01

    One of the challenges of genomic research after the completion of the human genome project is to assign a function to all the genes and to understand their interactions and organizations. Among the various techniques, the emergence of chromosome engineering tools with the aim to manipulate large genomic regions in the mouse model offers a powerful way to accelerate the discovery of gene functions and provides more mouse models to study normal and pathological developmental processes associated with aneuploidy. The combination of gene targeting in ES cells, recombinase technology, and other techniques makes it possible to generate new chromosomes carrying specific and defined deletions, duplications, inversions, and translocations that are accelerating functional analysis. This review presents the current status of chromosome engineering techniques and discusses the different applications as well as the implication of these new techniques in future research to better understand the function of chromosomal organization and structures. PMID:16839184

  10. A high resolution spatiotemporal atlas of gene expression of the developing mouse brain

    PubMed Central

    Thompson, Carol L.; Ng, Lydia; Menon, Vilas; Martinez, Salvador; Lee, Chang-Kyu; Glattfelder, Katie; Sunkin, Susan M.; Henry, Alex; Lau, Christopher; Dang, Chinh; Garcia-Lopez, Raquel; Martinez-Ferre, Almudena; Pombero, Ana; Rubenstein, John L.R.; Wakeman, Wayne B.; Hohmann, John; Dee, Nick; Sodt, Andrew J.; Young, Rob; Smith, Kimberly; Nguyen, Thuc-Nghi; Kidney, Jolene; Kuan, Leonard; Jeromin, Andreas; Kaykas, Ajamete; Miller, Jeremy; Page, Damon; Orta, Geri; Bernard, Amy; Riley, Zackery; Smith, Simon; Wohnoutka, Paul; Hawrylycz, Mike; Puelles, Luis; Jones, Allan R.

    2015-01-01

    SUMMARY To provide a temporal framework for the genoarchitecture of brain development, in situ hybridization data were generated for embryonic and postnatal mouse brain at 7 developmental stages for ~2100 genes, processed with an automated informatics pipeline and manually annotated. This resource comprises 434,946 images, 7 reference atlases, an ontogenetic ontology, and tools to explore co-expression of genes across neurodevelopment. Gene sets coinciding with developmental phenomena were identified. A temporal shift in the principles governing the molecular organization of the brain was detected, with transient neuromeric, plate-based organization of the brain present at E11.5 and E13.5. Finally, these data provided a transcription factor code that discriminates brain structures and identifies the developmental age of a tissue, providing a foundation for eventual genetic manipulation or tracking of specific brain structures over development. The resource is available as the Allen Developing Mouse Brain Atlas (developingmouse.brain-map.org). PMID:24952961

  11. A high-resolution spatiotemporal atlas of gene expression of the developing mouse brain.

    PubMed

    Thompson, Carol L; Ng, Lydia; Menon, Vilas; Martinez, Salvador; Lee, Chang-Kyu; Glattfelder, Katie; Sunkin, Susan M; Henry, Alex; Lau, Christopher; Dang, Chinh; Garcia-Lopez, Raquel; Martinez-Ferre, Almudena; Pombero, Ana; Rubenstein, John L R; Wakeman, Wayne B; Hohmann, John; Dee, Nick; Sodt, Andrew J; Young, Rob; Smith, Kimberly; Nguyen, Thuc-Nghi; Kidney, Jolene; Kuan, Leonard; Jeromin, Andreas; Kaykas, Ajamete; Miller, Jeremy; Page, Damon; Orta, Geri; Bernard, Amy; Riley, Zackery; Smith, Simon; Wohnoutka, Paul; Hawrylycz, Michael J; Puelles, Luis; Jones, Allan R

    2014-07-16

    To provide a temporal framework for the genoarchitecture of brain development, we generated in situ hybridization data for embryonic and postnatal mouse brain at seven developmental stages for ?2,100 genes, which were processed with an automated informatics pipeline and manually annotated. This resource comprises 434,946 images, seven reference atlases, an ontogenetic ontology, and tools to explore coexpression of genes across neurodevelopment. Gene sets coinciding with developmental phenomena were identified. A temporal shift in the principles governing the molecular organization of the brain was detected, with transient neuromeric, plate-based organization of the brain present at E11.5 and E13.5. Finally, these data provided a transcription factor code that discriminates brain structures and identifies the developmental age of a tissue, providing a foundation for eventual genetic manipulation or tracking of specific brain structures over development. The resource is available as the Allen Developing Mouse Brain Atlas (http://developingmouse.brain-map.org). PMID:24952961

  12. Chromosomal localization of a new mouse lens opacity gene (lop18)

    SciTech Connect

    Chang, Bo; Hawes, N.L.; Smith, R.S.

    1996-08-15

    Examination of mouse strains with a slit lamp and indirect ophthalmoscopy revealed that strain CBA/CaGnLe has a white cataract obvious at weaning age. It soon progresses to a large white nuclear cataract with mild cortical changes. Crosses with C57BL/GJ showed that this is inherited as a single recessive fully penetrant gene, which we have designated lop18 (lens opacity 18). Linkage analysis using visible marker T (brachyury), histocompatibility marker H2, and microsatellite markers D17MU21, D17MU28, D17MU38, and D17MU46 shows that the 1op18 gene is located, {approximately}16 cM from the centromere on mouse Chromosome 17. It is a likely candidate mutation for the {alpha}-crystallin (Cryal) gene. 14 refs., 1 fig., 1 tab.

  13. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system

    PubMed Central

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-01-01

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM+ mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab′ fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production. PMID:26956543

  14. Molecular cloning, mapping and characterization of a novel mouse RING finger gene, Mrf1.

    PubMed

    Chang, Ruying; Xu, Xiaoyuan; Li, Ming D

    2002-05-29

    With a combined approach of database search, heterologous polymerase chain reaction (PCR), reverse transcription-PCR, rapid amplification of complementary DNA ends and genomic library screening, we have successfully cloned a mouse RING finger gene, mouse RING finger 1 (Mrf1). The Mrf1 gene has two exons of 63 and 2665 bp, respectively, and one intron of over 13 kb. An open reading frame was identified exclusively in exon 2, which encodes a putative protein of the RING-B box-coiled coil or the tripartite motif type of 403 amino acids. Mrf1 is moderately expressed in the spleen, brain and heart as a single 3.0 kb product and very highly expressed in the testis as two transcripts of 3.0 and 1.5 kb, respectively. The Mrf1 gene was mapped to mouse chromosome 3, between markers D3Mit70 and D3Mit277. Western blotting analysis indicated that an expected protein of approximately 44 kD was detected in the brain extracts of mouse, rat and human. The possible functions of Mrf1 are discussed in the contexts of protein-protein interactions, oncogenesis and ubiquitination. PMID:12095697

  15. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system.

    PubMed

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-01-01

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM(+) mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab' fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production. PMID:26956543

  16. Mapping of the ARIX homeodomain gene to mouse chromosome 7 and human chromosome 11q13

    SciTech Connect

    Johnson, K.R.; Smith, L.; Rhodes, J.

    1996-05-01

    The recently described homeodomain protein ARIX is expressed specifically in noradreneric cell types of the sympathetic nervous system, brain, and adrenal medulla. ARIX interacts with regulatory elements of the genes encoding the noradrenergic biosynthetic enzymes tyrosine hydroxylase and dopamine {beta}-hydroxylase, suggesting a role for ARIX in expression of the noradrenergic phenotype. In the study described here, the mouse and human ARIX genes are mapped. Using segregation analysis of two panels of mouse backcross DNA, mouse Arix was positioned approximately 50 cM distal to the centromere of chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 11q13.3-q13.4. These map locations extend and further define regions of conserved synteny between mouse and human genomes and identify a new candidate gene for inherited developmental disorders linked to human 11q13.

  17. An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse

    PubMed Central

    2013-01-01

    Background We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). Results We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly, abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. Conclusions Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines. PMID:24025402

  18. Dysregulation of gene expression in mouse trisomy 16, an animal model of Down syndrome.

    PubMed Central

    Holtzman, D M; Bayney, R M; Li, Y W; Khosrovi, H; Berger, C N; Epstein, C J; Mobley, W C

    1992-01-01

    In humans, trisomy 21 results in a specific phenotype known as Down syndrome (DS). The mechanism by which an extra copy of normal genes leads to the DS phenotype is unknown. Most studies in DS and other aneuploid organisms have shown that gene dose is proportional to gene expression. To date, most genes examined have encoded either metabolic enzymes or constitutively expressed products. In the trisomy 16 mouse, an animal model of DS, we found marked dysregulation of two developmentally regulated genes, App and Prn-p. Dysregulation varied from tissue to tissue and during development in the same tissue. We conclude that abnormal phenotypes seen in aneuploid conditions may result in part from disordered expression of developmentally regulated genes. Images PMID:1371464

  19. Two genes substitute for the mouse Y chromosome for spermatogenesis and reproduction.

    PubMed

    Yamauchi, Yasuhiro; Riel, Jonathan M; Ruthig, Victor A; Ortega, Eglė A; Mitchell, Michael J; Ward, Monika A

    2016-01-29

    The mammalian Y chromosome is considered a symbol of maleness, as it encodes a gene driving male sex determination, Sry, as well as a battery of other genes important for male reproduction. We previously demonstrated in the mouse that successful assisted reproduction can be achieved when the Y gene contribution is limited to only two genes, Sry and spermatogonial proliferation factor Eif2s3y. Here, we replaced Sry by transgenic activation of its downstream target Sox9, and Eif2s3y, by transgenic overexpression of its X chromosome-encoded homolog Eif2s3x. The resulting males with no Y chromosome genes produced haploid male gametes and sired offspring after assisted reproduction. Our findings support the existence of functional redundancy between the Y chromosome genes and their homologs encoded on other chromosomes. PMID:26823431

  20. Birth defects caused by mutations in human GLI3 and mouse Gli3 genes.

    PubMed

    Naruse, Ichiro; Ueta, Etsuko; Sumino, Yoshiki; Ogawa, Masaya; Ishikiriyama, Satoshi

    2010-03-01

    ABSTRACT GLI3 is the gene responsible for Greig cephalopolysyndactyly syndrome (GCPS), Pallister-Hall syndrome (PHS) and Postaxial polydactyly type-A (PAP-A). Genetic polydactyly mice such as Pdn/Pdn (Polydactyly Nagoya), Xt(H)/Xt(H) (Extra toes) and Xt(J)/Xt(J) (Extra toes Jackson) are the mouse homolog of GCPS, and Gli3(tmlUrtt)/Gli3(tmlUrt) is produced as the mouse homolog of PHS. In the present review, relationships between mutation points of GLI3 and Gli3, and resulting phenotypes in humans and mice are described. It has been confirmed that mutation in the upstream or within the zinc finger domain of the GLI3 gene induces GCPS; that in the post-zinc finger region including the protease cleavage site induces PHS; and that in the downstream of the GLI3 gene induces PAP-A. A mimicking phenomenon was observed in the mouse homolog. Therefore, human GLI3 and mouse Gli3 genes have a common structure, and it is suggested here that mutations in the same functional regions produce similar phenotypes in human and mice. The most important issue might be that GCPS and PHS exhibit an autosomal dominant trait, but mouse homologs, such as Pdn/Pdn, Xt(H)/Xt(H), Xt(J)/Xt(J) and Gli3(tmlUrt)/Gli3(tmlUrt), are autosomal recessive traits in the manifestation of similar phenotypes to human diseases. It is discussed here how the reduced amounts of the GLI3 protein, or truncated mutant GLI3 protein, disrupt development of the limbs, head and face. PMID:20201963

  1. Metallothionein protection of cadmium toxicity

    SciTech Connect

    Klaassen, Curtis D. Liu, Jie; Diwan, Bhalchandra A.

    2009-08-01

    The discovery of the cadmium (Cd)-binding protein from horse kidney in 1957 marked the birth of research on this low-molecular weight, cysteine-rich protein called metallothionein (MT) in Cd toxicology. MT plays minimal roles in the gastrointestinal absorption of Cd, but MT plays important roles in Cd retention in tissues and dramatically decreases biliary excretion of Cd. Cd-bound to MT is responsible for Cd accumulation in tissues and the long biological half-life of Cd in the body. Induction of MT protects against acute Cd-induced lethality, as well as acute toxicity to the liver and lung. Intracellular MT also plays important roles in ameliorating Cd toxicity following prolonged exposures, particularly chronic Cd-induced nephrotoxicity, osteotoxicity, and toxicity to the lung, liver, and immune system. There is an association between human and rodent Cd exposure and prostate cancers, especially in the portions where MT is poorly expressed. MT expression in Cd-induced tumors varies depending on the type and the stage of tumor development. For instance, high levels of MT are detected in Cd-induced sarcomas at the injection site, whereas the sarcoma metastases are devoid of MT. The use of MT-transgenic and MT-null mice has greatly helped define the role of MT in Cd toxicology, with the MT-null mice being hypersensitive and MT-transgenic mice resistant to Cd toxicity. Thus, MT is critical for protecting human health from Cd toxicity. There are large individual variations in MT expression, which might in turn predispose some people to Cd toxicity.

  2. The intracisternal A particle derived solo LTR promoter of the rat oncomodulin gene is not present in the mouse gene.

    PubMed

    Banville, D; Rotaru, M; Boie, Y

    1992-01-01

    The rat gene encoding oncomodulin, a small calcium-binding protein related to parvalbumin, is under the control of a solo long terminal repeat (LTR) derived from an endogenous intracisternal A-particle (IAP). This gene was the first example of a mammalian gene regulated in normal cells by a promoter of retroviral origin (see also article by D. Robins and L. Samuelson in this volume). We show here that the oncomodulin LTR is a member of a small subset of sequence related solo LTR elements present in the rat genome and that a full length IAP genome containing LTRs of this type is no longer present in the rat genome. We have assayed the transcriptional activity of the oncomodulin LTR coupled to the human growth hormone gene as a reporter. Transfections in both Hela cells and 293 cells indicate the oncomodulin LTR promoter is sufficient to efficiently initiate transcription. In 293 cells (human embryo kidney cells transformed with human adenovirus type 5 DNA), the oncomodulin LTR is a strong promoter, capable of bidirectional transcription. Finally, we have determined the structure and the sequence of the mouse oncomodulin gene. Our results suggest that the integration of the IAP particle genome within the rat oncomodulin gene occurred after the rat and the mouse became distinct species. PMID:1468649

  3. Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

    PubMed Central

    Wang, Lingyan; Jiang, Han; Brigande, John V.

    2012-01-01

    The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6. The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol. PMID:22781586

  4. Identification of a conserved set of upregulated genes in mouse skeletal muscle hypertrophy and regrowth

    PubMed Central

    Chaillou, Thomas; Jackson, Janna R.; England, Jonathan H.; Kirby, Tyler J.; Richards-White, Jena; Esser, Karyn A.; Dupont-Versteegden, Esther E.

    2014-01-01

    The purpose of this study was to compare the gene expression profile of mouse skeletal muscle undergoing two forms of growth (hypertrophy and regrowth) with the goal of identifying a conserved set of differentially expressed genes. Expression profiling by microarray was performed on the plantaris muscle subjected to 1, 3, 5, 7, 10, and 14 days of hypertrophy or regrowth following 2 wk of hind-limb suspension. We identified 97 differentially expressed genes (?2-fold increase or ?50% decrease compared with control muscle) that were conserved during the two forms of muscle growth. The vast majority (?90%) of the differentially expressed genes was upregulated and occurred at a single time point (64 out of 86 genes), which most often was on the first day of the time course. Microarray analysis from the conserved upregulated genes showed a set of genes related to contractile apparatus and stress response at day 1, including three genes involved in mechanotransduction and four genes encoding heat shock proteins. Our analysis further identified three cell cycle-related genes at day and several genes associated with extracellular matrix (ECM) at both days 3 and 10. In conclusion, we have identified a core set of genes commonly upregulated in two forms of muscle growth that could play a role in the maintenance of sarcomere stability, ECM remodeling, cell proliferation, fast-to-slow fiber type transition, and the regulation of skeletal muscle growth. These findings suggest conserved regulatory mechanisms involved in the adaptation of skeletal muscle to increased mechanical loading. PMID:25554798

  5. Inferring mouse gene functions from genomic-scale data using a combined functional network/classification strategy

    PubMed Central

    Kim, Wan Kyu; Krumpelman, Chase; Marcotte, Edward M

    2008-01-01

    The complete set of mouse genes, as with the set of human genes, is still largely uncharacterized, with many pieces of experimental evidence accumulating regarding the activities and expression of the genes, but the majority of genes as yet still of unknown function. Within the context of the MouseFunc competition, we developed and applied two distinct large-scale data mining approaches to infer the functions (Gene Ontology annotations) of mouse genes from experimental observations from available functional genomics, proteomics, comparative genomics, and phenotypic data. The two strategies the first using classifiers to map features to annotations, the second propagating annotations from characterized genes to uncharacterized genes along edges in a network constructed from the features offer alternative and possibly complementary approaches to providing functional annotations. Here, we re-implement and evaluate these approaches and their combination for their ability to predict the proper functional annotations of genes in the MouseFunc data set. We show that, when controlling for the same set of input features, the network approach generally outperformed a nave Bayesian classifier approach, while their combination offers some improvement over either independently. We make our observations of predictive performance on the MouseFunc competition hold-out set, as well as on a ten-fold cross-validation of the MouseFunc data. Across all 1,339 annotated genes in the MouseFunc test set, the median predictive power was quite strong (median area under a receiver operating characteristic plot of 0.865 and average precision of 0.195), indicating that a mining-based strategy with existing data is a promising path towards discovering mammalian gene functions. As one product of this work, a high-confidence subset of the functional mouse gene network was produced spanning >70% of mouse genes with >1.6 million associations that is predictive of mouse (and therefore often human) gene function and functional associations. The network should be generally useful for mammalian gene functional analyses, such as for predicting interactions, inferring functional connections between genes and pathways, and prioritizing candidate genes. The network and all predictions are available on the worldwide web. PMID:18613949

  6. A gene expression resource generated by genome-wide lacZ profiling in the mouse.

    PubMed

    Tuck, Elizabeth; Estabel, Jeanne; Oellrich, Anika; Maguire, Anna Karin; Adissu, Hibret A; Souter, Luke; Siragher, Emma; Lillistone, Charlotte; Green, Angela L; Wardle-Jones, Hannah; Carragher, Damian M; Karp, Natasha A; Smedley, Damian; Adams, Niels C; Bussell, James N; Adams, David J; Ramrez-Solis, Ramiro; Steel, Karen P; Galli, Antonella; White, Jacqueline K

    2015-11-01

    Knowledge of the expression profile of a gene is a critical piece of information required to build an understanding of the normal and essential functions of that gene and any role it may play in the development or progression of disease. High-throughput, large-scale efforts are on-going internationally to characterise reporter-tagged knockout mouse lines. As part of that effort, we report an open access adult mouse expression resource, in which the expression profile of 424 genes has been assessed in up to 47 different organs, tissues and sub-structures using a lacZ reporter gene. Many specific and informative expression patterns were noted. Expression was most commonly observed in the testis and brain and was most restricted in white adipose tissue and mammary gland. Over half of the assessed genes presented with an absent or localised expression pattern (categorised as 0-10 positive structures). A link between complexity of expression profile and viability of homozygous null animals was observed; inactivation of genes expressed in ?21 structures was more likely to result in reduced viability by postnatal day 14 compared with more restricted expression profiles. For validation purposes, this mouse expression resource was compared with Bgee, a federated composite of RNA-based expression data sets. Strong agreement was observed, indicating a high degree of specificity in our data. Furthermore, there were 1207 observations of expression of a particular gene in an anatomical structure where Bgee had no data, indicating a large amount of novelty in our data set. Examples of expression data corroborating and extending genotype-phenotype associations and supporting disease gene candidacy are presented to demonstrate the potential of this powerful resource. PMID:26398943

  7. A gene expression resource generated by genome-wide lacZ profiling in the mouse

    PubMed Central

    Tuck, Elizabeth; Estabel, Jeanne; Oellrich, Anika; Maguire, Anna Karin; Adissu, Hibret A.; Souter, Luke; Siragher, Emma; Lillistone, Charlotte; Green, Angela L.; Wardle-Jones, Hannah; Carragher, Damian M.; Karp, Natasha A.; Smedley, Damian; Adams, Niels C.; Bussell, James N.; Adams, David J.; Ramírez-Solis, Ramiro; Steel, Karen P.; Galli, Antonella; White, Jacqueline K.

    2015-01-01

    ABSTRACT Knowledge of the expression profile of a gene is a critical piece of information required to build an understanding of the normal and essential functions of that gene and any role it may play in the development or progression of disease. High-throughput, large-scale efforts are on-going internationally to characterise reporter-tagged knockout mouse lines. As part of that effort, we report an open access adult mouse expression resource, in which the expression profile of 424 genes has been assessed in up to 47 different organs, tissues and sub-structures using a lacZ reporter gene. Many specific and informative expression patterns were noted. Expression was most commonly observed in the testis and brain and was most restricted in white adipose tissue and mammary gland. Over half of the assessed genes presented with an absent or localised expression pattern (categorised as 0-10 positive structures). A link between complexity of expression profile and viability of homozygous null animals was observed; inactivation of genes expressed in ≥21 structures was more likely to result in reduced viability by postnatal day 14 compared with more restricted expression profiles. For validation purposes, this mouse expression resource was compared with Bgee, a federated composite of RNA-based expression data sets. Strong agreement was observed, indicating a high degree of specificity in our data. Furthermore, there were 1207 observations of expression of a particular gene in an anatomical structure where Bgee had no data, indicating a large amount of novelty in our data set. Examples of expression data corroborating and extending genotype-phenotype associations and supporting disease gene candidacy are presented to demonstrate the potential of this powerful resource. PMID:26398943

  8. Positional Cloning of the Mouse Circadian Clock Gene

    PubMed Central

    King, David P.; Zhao, Yaliang; Sangoram, Ashvin M.; Wilsbacher, Lisa D.; Tanaka, Minoru; Antoch, Marina P.; Steeves, Thomas D. L.; Vitaterna, Martha Hotz; Kornhauser, Jon M.; Lowrey, Phillip L.; Turek, Fred W.; Takahashi, Joseph S.

    2013-01-01

    Summary We used positional cloning to identify the circadian Clock gene in mice. Clock is a large transcription unit with 24 exons spanning ~100,000 bp of DNA from which transcript classes of 7.5 and ~10 kb arise. Clock encodes a novel member of the bHLHPAS family of transcription factors. In the Clock mutant allele, an A?T nucleotide transversion in a splice donor site causes exon skipping and deletion of 51 amino acids in the CLOCK protein. Clock is a unique gene with known circadian function and with features predicting DNA binding, protein dimerization, and activation domains. CLOCK represents the second example of a PAS domaincontaining clock protein (besides Drosophila PERIOD), which suggests that this motif may define an evolutionarily conserved feature of the circadian clock mechanism. PMID:9160755

  9. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

    PubMed Central

    Kirov, Julia V.; Adkisson, Michael; Nava, A. J.; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K.; Lloyd, K. C. Kent; de Jong, Pieter; West, David B.

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  10. Analysis of spatial-temporal gene expression patterns reveals dynamics and regionalization in developing mouse brain

    PubMed Central

    Chou, Shen-Ju; Wang, Chindi; Sintupisut, Nardnisa; Niou, Zhen-Xian; Lin, Chih-Hsu; Li, Ker-Chau; Yeang, Chen-Hsiang

    2016-01-01

    Allen Brain Atlas (ABA) provides a valuable resource of spatial/temporal gene expressions in mammalian brains. Despite rich information extracted from this database, current analyses suffer from several limitations. First, most studies are either gene-centric or region-centric, thus are inadequate to capture the superposition of multiple spatial-temporal patterns. Second, standard tools of expression analysis such as matrix factorization can capture those patterns but do not explicitly incorporate spatial dependency. To overcome those limitations, we proposed a computational method to detect recurrent patterns in the spatial-temporal gene expression data of developing mouse brains. We demonstrated that regional distinction in brain development could be revealed by localized gene expression patterns. The patterns expressed in the forebrain, medullary and pontomedullary, and basal ganglia are enriched with genes involved in forebrain development, locomotory behavior, and dopamine metabolism respectively. In addition, the timing of global gene expression patterns reflects the general trends of molecular events in mouse brain development. Furthermore, we validated functional implications of the inferred patterns by showing genes sharing similar spatial-temporal expression patterns with Lhx2 exhibited differential expression in the embryonic forebrains of Lhx2 mutant mice. These analysis outcomes confirm the utility of recurrent expression patterns in studying brain development. PMID:26786896

  11. PiggyBac Mediated Multiplex Gene Transfer in Mouse Embryonic Stem Cell

    PubMed Central

    Lu, Xibin; Huang, Wei

    2014-01-01

    PiggyBac system has been shown to have a high efficiency to mediate gene transfer. However, there are no reports on its efficiency to mediate multiplex transgenes in mouse embryonic stem cells. Here we first established an immortalized feeder cell line by introducing four antibiotic resistance genes simultaneously into the original SNL 76/7 feeder cell line utilizing the PiggyBac system. This is the feeder cell line with the most diverse types of antibiotic resistance genes reported so far, which will enable researchers to perform simultaneous multiplex gene transfer or gene targeting experiments in ES cells. With such feeder cell line, we were able to quantitatively characterize the transposition efficiency of PiggyBac system in mouse ES cells using five transposons carrying different inducible fluorescence proteins and antibiotic resistance genes, and the efficiency ranged from about 2% for one transposon to 0.5% for five transposons. The highly efficient multiplex gene transfer mediated by PiggyBac will no doubt provide researchers with more choices in biomedical research and development. PMID:25517991

  12. Gene structure and chromosomal localization of the mouse homologue of rat OX40 protein.

    PubMed

    Birkeland, M L; Copeland, N G; Gilbert, D J; Jenkins, N A; Barclay, A N

    1995-04-01

    The OX40 protein is expressed only on activated rat CD4+ T blasts and is a member of a superfamily of cell surface molecules which includes CD40, CD30, CD95 (Fas), CD27, 4-1BB antigens and the receptors for tumor necrosis factor (TNF) and nerve growth factor (NGF). The proteins of this group are related to each other by having three to six repeats of a cysteine-rich sequence in their extracellular domains. Members of this family of receptors have also been shown to bind to ligands which are structurally related to TNF. The mouse homologue of the rat OX40 protein was cloned at the cDNA and genomic levels. The gene structure shows that there are several intron/exon borders shared between OX40 and CD27, CD40, TNF receptor type I, CD95 and 4-1BB genes. This group of genes is less closely related structurally to the gene structure of the NGF receptor. The gene encoding murine OX40 has been placed on mouse chromosome 4, in an area which contains the genes for TNF receptor type II and 4-1BB, and is syntenic with a region of human chromosome 1 which contains human TNF receptor type II, OX40, and CD30 genes. PMID:7737295

  13. Analysis of spatial-temporal gene expression patterns reveals dynamics and regionalization in developing mouse brain.

    PubMed

    Chou, Shen-Ju; Wang, Chindi; Sintupisut, Nardnisa; Niou, Zhen-Xian; Lin, Chih-Hsu; Li, Ker-Chau; Yeang, Chen-Hsiang

    2016-01-01

    Allen Brain Atlas (ABA) provides a valuable resource of spatial/temporal gene expressions in mammalian brains. Despite rich information extracted from this database, current analyses suffer from several limitations. First, most studies are either gene-centric or region-centric, thus are inadequate to capture the superposition of multiple spatial-temporal patterns. Second, standard tools of expression analysis such as matrix factorization can capture those patterns but do not explicitly incorporate spatial dependency. To overcome those limitations, we proposed a computational method to detect recurrent patterns in the spatial-temporal gene expression data of developing mouse brains. We demonstrated that regional distinction in brain development could be revealed by localized gene expression patterns. The patterns expressed in the forebrain, medullary and pontomedullary, and basal ganglia are enriched with genes involved in forebrain development, locomotory behavior, and dopamine metabolism respectively. In addition, the timing of global gene expression patterns reflects the general trends of molecular events in mouse brain development. Furthermore, we validated functional implications of the inferred patterns by showing genes sharing similar spatial-temporal expression patterns with Lhx2 exhibited differential expression in the embryonic forebrains of Lhx2 mutant mice. These analysis outcomes confirm the utility of recurrent expression patterns in studying brain development. PMID:26786896

  14. Regulation of expression of TL genes of the mouse Mhc.

    PubMed

    Dolby, N; Mehta, V; Cook, R G

    1990-01-01

    The expression of thymus leukemia (TL) antigens and genes in thymocytes and activated T cells was examined by immunoprecipitation, flow cytometric, northern, and nuclear run-off transcription analyses. Cell surface forms of TL were detectable by immunoprecipitation on activated peripheral T cells from Tla haplotypes except Tla(b), in agreement with expression observed on thymocytes. Approximately 40%-50% of concanavalin A (Con A) or anti-CD3-activated T cells were TL+, with expression detected on both the CD4 and CD8 subsets by dual-color analysis. Activated T cells expressed detectable levels of TL mRNA 48 h after stimulation, but no TL transcripts were detectable in unstimulated splenocytes. However, TL mRNA expression in mature activated T cells did not precisely mimic thymocyte expression: the level of expression was considerably lower in activated T cells, and in most haplotypes the transcripts produced in activated T cells appeared to represent a subset of the transcripts produced in thymocytes. By run-off transcription assays in isolated nuclei, TL gene expression was detected in activated but not resting T cells indicating that lack of expression of TL in resting T cells is not due to message instability. These data demonstrate that TL genes are inducible and transcriptionally regulated. PMID:2125577

  15. Targeted disruption of the mouse Gz-alpha gene: a role for Gz in platelet function?

    PubMed

    Kelleher, K L; Matthaei, K I; Hendry, I A

    2001-03-01

    Gz is one of nine G proteins identified in platelets and its role in these cells is unknown. Our laboratory has generated a mouse deficient in the Gz-alpha gene in the hope of determining its in vivo function. Bleeding times from the tail tip of Gzalpha deficient mice was significantly longer than wild type mice. Platelet aggregation and ATP secretion did not differ between wild type and Gzalpha deficient mice. When mice were presented with a thromboembolism challenge no differences were observed in the survival or mortality of wild type or Gzalpha deficient mice, however a strain difference was observed. Ignoring the genetic background of a mutant mouse might lead to a misinterpretation of results and thus it is absolutely critical to take the genetic background into account when assessing any aspect of a mutant mouse. PMID:11307826

  16. Mapping of the Sca1 and pcd genes on mouse chromosome 13 provides evidence that they are different genes

    SciTech Connect

    Servadio, A.; McCall, A.; Zoghbi, H.; Eicher, E.M.

    1995-10-10

    It is well established that large chromosomal segments have remained intact during the evolution of different mammalian species. Thus, mapping information for a gene in mammalian species facilitates mapping the same gene in another mammalian species. In addition, phenotypically similar diseases that map to linkage conserved regions in two species may be caused by mutations in the same gene. Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited human disorder characterized by progressive ataxia, dysarthria, and dysmetria. SCA1 maps to the short arm of human chromosome (Chr) 6 in the 6p23-p22 region. SCA1 is caused by the expansion of an unstable CAG repeat located within the coding region of a novel protein, ataxin-1, Purkinje cell degeneration (pcd) is a recessively inherited mouse disorder characterized by a moderate ataxia, usually noted by 3-4 weeks of age. Progressive degeneration of Purkinje cells is the underlying pathogenesis in this disorder. The pcd gene was assigned to mouse Chr 13 because it showed linkage to extra toes (Xt) and pearl (pe). Some doubt about this assignment existed, however, because the calculated genetic distance between pcd and Xt was 32 cM and that between pcd and pe was 18 cM. If pcd is located in Chr 13, its placement relative to Xt and pe suggests that it would be located in the region that shares linkage homology with the region that shares linkage homology with the region of human Chr 6 that contains SCA1. Here, we present data that confirm the assignment of pcd to Chr 13, map the mouse Sca1 gene to Chr 13, and eliminate Sca1 as a candidate gene for pcd. 11 refs., 1 tab.

  17. Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

    PubMed Central

    Matsui, Asuka; Yoshida, Aya C.; Kubota, Mayumi; Ogawa, Masaharu; Shimogori, Tomomi

    2011-01-01

    In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses. PMID:21860382

  18. Cloning and sequencing of the mouse Gli2 gene: Localization to the Dominant hemimelia critical region

    SciTech Connect

    Hughes, D.C.; Allen, J.; Prosser, J.

    1997-01-15

    The GLI family of zinc finger genes has been implicated in both neoplastic and developmental disorders. We have cloned and sequenced the mouse homolog of the zinc finger gene Gli2 and demonstrated significant similarity to the human GLI3 gene. We have also localized Gli2 to mouse chromosome 1, in the vicinity of the morphogenetic mutation Dominant hemimelia (Dh), which is characterized by tibial hemimelia, poly/oligodactyly, and a number of visceral abnormalities, most strikingly absence of the spleen. Using a Gli2-associated microsatellite, we demonstrated no recombination between Dh and Gli2 in a Dh intraspecific backcross. Gli2 is expressed in Dh heterozygotes and homozygotes. However, using a combination of mismatch analysis and direct sequencing, we have failed to identify any mutations in the coding sequence of Gli2 from Dh. We have also demonstrated that it is unlikely that there are any Gli genes in the mouse genome in addition to the previously described Gli, Gli2, and Gli3. 52 refs., 4 figs., 4 tabs.

  19. Influence of sex on gene expression in the mouse lacrimal gland.

    PubMed

    Richards, Stephen M; Jensen, Roderick V; Liu, Meng; Sullivan, Benjamin D; Lombardi, Michael J; Rowley, Patricia; Schirra, Frank; Treister, Nathaniel S; Suzuki, Tomo; Steagall, Rebecca J; Yamagami, Hiroko; Sullivan, David A

    2006-01-01

    Significant, sex-associated differences exist in the physiology and pathophysiology of the lacrimal gland. We hypothesize that many of these differences are due to fundamental variations in gene expression. The purpose of this study was to determine the extent to which sex-related differences in gene expression are present in the lacrimal gland. Lacrimal glands were obtained from adult male and female BALB/c mice (n=5-10mice/sex/experiment), pooled according to sex and processed for the isolation of RNA. Samples were analyzed for differentially expressed mRNAs by using Atlas Mouse cDNA Expression Arrays, cDNA amplification techniques, GEM 1 and 2 gene chips, CodeLink bioarrays and quantitative real-time PCR (qPCR) procedures. Quantitative evaluation of Atlas Array gene expression was performed with an image analysis system developed in our laboratory, whereas gene chip data were analyzed with Rosetta Resolver and GeneSifter.Net software. Statistical significance was determined by using Student's t-test. Our results with CodeLink bioarrays show that sex has a significant influence on the expression of over 490 genes in the mouse lacrimal gland. These genes are involved in a wide range of biological processes, molecular functions and cellular components, including such activities as development, growth, transcription, metabolism, signal transduction, transport, receptor activity and protein and nucleic acid binding. The expression of selected genes was confirmed by the use of GEM gene chips and qPCR. Our findings also demonstrate that certain methodological approaches are less useful in attempting to assess the magnitude of sex-associated differences in the lacrimal gland. These results support our hypothesis that sex-related differences in gene expression play a role in the sexual dimorphism of the lacrimal gland. PMID:15979613

  20. Linkage analysis of the whirler deafness gene on mouse chromosome 4

    SciTech Connect

    Fleming, J.; Rogers, M.J.C.; Steel, K.P. ); Brown, S.D.M. )

    1994-05-01

    The whirler mouse harbors an autosomal recessive mutation on mouse chromosome 4 that causes deafness and vestibular dysfunction in the adult that is manifested as head-bobbing and circling behavior. Although there is no obvious human homologue for this mutation as yet, whirler is a potential mouse model for human autosomal recessive deafness. Many genetic markers for this region of mouse chromosome 4 are now available, and the authors have used these to construct genetic linkage maps in both inter- and intraspecific backcrosses as the first step toward the cloning of the whirler gene. A total of 19 loci were analyzed in these crosses, giving the following gene orders: Interspecific cross, centromere-(D4Mit5, D4Mit38)-D4Mit6-(Lv,Tzn,D4Mit44)-wi-Hxb-(D4Mit25, D4Nds9)-(D4Mit7, D4Ler2)-b-D4Mit45-(D4Wsm1, D4Mit27b)-(D4Rck65, D4Mit15), and intraspecific cross, centromere-(Mup-1, wi, Hxb)-b-D4Wsm1. This analysis has positioned the wi locus in the interval between the genes for [delta]-aminolevulinate dehydratase (Lv) and hexabrachion (Hxb). The human homologues of these genes, ALAD and HXB, both lie on human chromosome 9q32-q34. They therefore predict that a human homologue of the wi gene, involved in autosomal recessive deafness, lies in this region of conserved homology on 9q32-q34. 36 refs., 2 figs., 4 tabs.

  1. Positions of pluripotency genes and hepatocyte-specific genes in the nucleus before and after mouse ES cell differentiation.

    PubMed

    Udagawa, K; Ohyama, T

    2014-01-01

    Spatial positioning of genes in the cell nucleus plays an important role in the regulation of genomic functions. Evidence for changes in gene positioning associated with transcriptional activity has been reported. However, our understanding of this phenomenon is still quite limited. We examined how pluripotency genes and hepatocyte-specific genes behave during the differentiation of mouse embryonic stem (ES) cells into hepatocytes, by targeting the loci of the Klf4, Nanog, Oct4, Sox2, Cyp7α1, Pck1, Tat, and Tdo2 genes, and using three-dimensional fluorescence in situ hybridization analyses. We found that each gene has a distinctly inherent localization profile in the ES cell nucleus. During differentiation, the Klf4, Nanog, Oct4, Cyp7α1, Pck1, and Tat loci shifted toward the nuclear center, while the Sox2 and Tdo2 loci shifted toward the periphery. The Klf4, Nanog, Oct4, and Tdo2 seem to prefer the outer regions, rather than the inner regions, when they are active. We also found that the radial positioning of the focused genes in the hepatocyte cell nucleus was highly correlated with the local GC content and the gene density of the surrounding region, but not with gene activity. PMID:24737423

  2. Molecular cloning and chromosomal mapping of the mouse cyclin-dependent kinase 5 gene

    SciTech Connect

    Ohshima, Toshio; Nagle, J.W.; Brady, R.O.; Kozak, C.A.

    1995-08-10

    Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neurons. In vitro, Cdk5 purified from the nervous tissue phosphorylates both high-molecular-weight neurofilament and microtubule-associated tau. The mouse gene encoding Cdk5 (Cdk5) was found to be 5 kb in length and divided into 12 exons. All of the exon-intron junctions matched the expected consensus sequence with the exception of the splice junction for intron 9, which has AT and AC dinucleotides instead of the usual GT and AG bordering sequence. In the 5{prime}-flanking region of mouse Cdk5, several putative promoter elements were present, including AP1, Sp1, PuF, and TATA motifs. A metal regulatory element was also identified at position -207 to -201. Nucleotide sequence analysis of mouse Cdk5 showed high identity to the homologues of other vertebrate species, indicating that this kinase is highly conserved during evolution. Mouse Cdk5 was mapped to the centromeric region of mouse chromosome 5. 20 refs., 2 figs., 1 tab.

  3. Dose-Related Estrogen Effects on Gene Expression in Fetal Mouse Prostate Mesenchymal Cells

    PubMed Central

    Taylor, Julia A.; Richter, Catherine A.; Suzuki, Atsuko; Watanabe, Hajime; Iguchi, Taisen; Coser, Kathryn R.; Shioda, Toshihiro; vom Saal, Frederick S.

    2012-01-01

    Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα) and androgen receptors and convert stimuli from circulating estrogens and androgens into paracrine signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2) in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM) for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM) induce genes involved in morphological tissue development and sterol biosynthesis but suppress genes involved in growth factor signaling. Genes involved in cell adhesion were enriched among both up-regulated and down-regulated genes. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM) were enriched in the glycolytic pathway. At the highest dose (100 nM), E2 induced genes enriched for cell adhesion, steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF- β signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas secreted growth factors and cytokines might play significant roles when estrogen level is high. PMID:23144751

  4. Gene expression in salivary glands: effects of diet and mouse chromosome 17 locus regulating macronutrient intake

    PubMed Central

    Simon, Jacob; DiCarlo, Lisa M; Kruger, Claudia; Johnson, William D; Kappen, Claudia; Richards, Brenda K

    2015-01-01

    Dcpp2, Prrt1, and Has1 are plausible candidate genes for the Mnic1 (macronutrient intake-carbohydrate) locus on mouse chromosome 17, based on their map positions and sequence variants, documented expression in salivary glands, and the important role of saliva in oral food processing and taste. We investigated the effects of genotype and diet on gene expression in salivary glands (parotid, submandibular, sublingual) of carbohydrate-preferring, C57BL6J.CAST/EiJ-17.1 subcongenic mice compared to fat-preferring wild-type C57BL/6J. To achieve accurate normalization of real-time quantitative RT-PCR data, we evaluated multiple reference genes to identify the most stably expressed control genes in salivary gland tissues, and then used geometric averaging to produce a reliable normalization factor. Gene expression was measured in mice fed different diets: (1) rodent chow, (2) macronutrient selection diets, (3) high-fat diet, and (4) low-fat diet. In addition, we measured salivary hyaluronan concentrations. All three genes showed strain differences in expression, in at least one major salivary gland, and diet effects were observed in two glands. Dcpp2 expression was limited primarily to sublingual gland, and strongly decreased in B6.CAST-17.1 subcongenic mice compared to wild-type B6, regardless of diet. In contrast, both genotype and diet affected Prrt1 and Has1 expression, in a gland-specific manner, for example, Prrt1 expression in the parotid gland alone was strongly reduced in both mouse strains when fed macronutrient selection diet compared to chow. Notably, we discovered an association between diet composition and salivary hyaluronan content. These results demonstrate robust effects of genetic background and diet composition on candidate gene expression in mouse salivary glands. PMID:25713331

  5. Mouse model systems to study sex chromosome genes and behavior: relevance to humans

    PubMed Central

    Cox, Kimberly H.; Bonthuis, Paul J.; Rissman, Emilie F.

    2014-01-01

    Sex chromosome genes directly influence sex differences in behavior. The discovery of the Sry gene on the Y chromosome (Gubbay et al., 1990; Koopman et al., 1990) substantiated the sex chromosome mechanistic link to sex differences. Moreover, the pronounced connection between X chromosome gene mutations and mental illness produces a strong sex bias in these diseases. Yet, the dominant explanation for sex differences continues to be the gonadal hormones. Here we review progress made on behavioral differences in mouse models that uncouple sex chromosome complement from gonadal sex. We conclude that many social and cognitive behaviors are modified by sex chromosome complement, and discuss the implications for human research. Future directions need to include identification of the genes involved and interactions with these genes and gonadal hormones. PMID:24388960

  6. The mouse formin (Fmn) gene: Genomic structure, novel exons, and genetic mapping

    SciTech Connect

    Wang, C.C.; Chan, D.C.; Leder, P.

    1997-02-01

    Mutations in the mouse formin (Fmn) gene, formerly known as the limb deformity (ld) gene, give rise to recessively inherited limb deformities and renal malformations or aplasia. The Fmn gene encodes many differentially processed transcripts that are expressed in both adult and embryonic tissues. To study the genomic organization of the Fmn locus, we have used Fmn probes to isolate and characterize genomic clones spanning 500 kb. Our analysis of these clones shows that the Fmn gene is composed of at least 24 exons and spans 400 kb. We have identified two novel exons that are expressed in the developing embryonic limb bud as well as adult tissues such as brain and kidney. We have also used a microsatellite polymorphism from within the Fmn gene to map it genetically to a 2.2-cM interval between D2Mit58 and D2Mit103. 36 refs., 6 figs., 1 tab.

  7. Lethal thalassemia after insertional disruption of the mouse major adult beta-globin gene.

    PubMed

    Shehee, W R; Oliver, P; Smithies, O

    1993-04-15

    Thalassemias are hereditary anemias caused by mutations that disturb the normal 1:1 balance of alpha- and beta-globin chains that form hemoglobin. We have disrupted the major adult beta-globin gene (b1) in mouse embryonic stem cells by using homologous recombination to insert selectable sequences into the gene. Mice homozygous for this insertional disruption of the b1 gene (Hbbth-2/Hbbth-2) are severely anemic and die perinatally. In contrast, approximately 60% of mice homozygous for deletion of the same gene (Hbbth-1/Hbbth-1) survive to adulthood and are much less anemic [Skow, L. C., Burkhart, B. A., Johnson, F. M., Popp, R. A., Goldberg, S. Z., Anderson, W. F., Barnett, L. B. & Lewis, S. E. (1983) Cell 34, 1043-1052]. These different phenotypes have implications for the control of beta-globin gene expression. PMID:8475058

  8. Structure of gene mts1, transcribed in metastatic mouse tumor cells.

    PubMed

    Tulchinsky, E M; Grigorian, M S; Ebralidze, A K; Milshina, N I; Lukanidin, E M

    1990-03-15

    Different oncogenes are implicated in the genesis of tumors. However, little is known so far about the genes which are activated at the latest stages of tumor progression. While studying two genetically related mouse lines, highly metastatic CSML-100 and nearly nonmetastatic CSML-0, we have cloned the cDNA of the gene, mts1, which is specifically expressed in different metastatic cells. The gene contains an open reading frame of 101 amino acids and shows homology with a family of Ca2(+)-binding proteins. Here, we present data on the structure of a 17-kb genomic clone of mts1 with surrounding sequences. The gene contains two introns and three exons. The mts1 upstream region has been cloned in a plasmid containing the cat gene. The results of transient expression of the mts1-cat plasmid in NIH3T3 cells indicate the presence of a transcription regulator of mts1. PMID:2332170

  9. A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts

    NASA Astrophysics Data System (ADS)

    Pandian, Ganesh N.; Nakano, Yusuke; Sato, Shinsuke; Morinaga, Hironobu; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi

    2012-07-01

    Cellular reprogramming involves profound alterations in genome-wide gene expression that is precisely controlled by a hypothetical epigenetic code. Small molecules have been shown to artificially induce epigenetic modifications in a sequence independent manner. Recently, we showed that specific DNA binding hairpin pyrrole-imidazole polyamides (PIPs) could be conjugated with chromatin modifying histone deacetylase inhibitors like SAHA to epigenetically activate certain pluripotent genes in mouse fibroblasts. In our steadfast progress to improve the efficiency of SAHA-PIPs, we identified a novel compound termed, ? that could dramatically induce the endogenous expression of Oct-3/4 and Nanog. Genome-wide gene analysis suggests that in just 24 h and at nM concentration, ? induced multiple pluripotency-associated genes including Rex1 and Cdh1 by more than ten-fold. ? treated MEFs also rapidly overcame the rate-limiting step of epithelial transition in cellular reprogramming by switching ``'' the complex transcriptional gene network.

  10. Gene order is conserved within the human chromosome 21 linkage group on mouse chromosome 10

    SciTech Connect

    Irving, N.G.; Cabin, D.E.; Swanson, D.A.; Reeves, R.H. )

    1994-05-01

    One hundred progeny from each of two intersubspecific mouse backcrosses were used to construct a comparative genetic map of a region of mouse chromosome 10 (MMU10) that is homologous to the distal tip of the long arm of human chromosome 21 (HSA21). The analysis included five genes and three simple sequence repeat markers, two of which flanked the HSA21-homologous cluster on either side. Analysis of 200 backcross progeny detected at least one crossover between each pair of adjacent genes and demonstrated that the proximal to distal orientation of the cluster was reversed between human and mouse. The order was determined to be Fyn-1-D10Mit20-S100b-Col6a1-Itgb2-Pfk1/D10Mit7-D10Mit11. Comparative mapping supports the order of corresponding markers on HSA21 determined using pulsed-field gel electrophoresis and radiation hybrid line data. However, sequence tagged site content mapping of human yeast artificial chromosomes (YACs) yielded conflicting data on the relative positions of human COL6A1 and S100B on HSA21. This discrepancy was resolved here by demonstrating that several key YACs used in the human contig analysis were mistyped for S100B. The murine map reported here provides a scaffold for construction of physical maps and yeast artificial chromosome contigs that will be useful in the development of mouse models for the study of Down syndrome. 28 refs., 4 figs., 2 tabs.

  11. Lactoferrin-iCre: a new mouse line to study uterine epithelial gene function.

    PubMed

    Daikoku, Takiko; Ogawa, Yuya; Terakawa, Jumpei; Ogawa, Akiyo; DeFalco, Tony; Dey, Sudhansu K

    2014-07-01

    Transgenic animal models are valuable for studying gene function in various tissue compartments. Mice with conditional deletion of genes in the uterus using the Cre-loxP system serve as powerful tools to study uterine biology. The uterus is comprised of 3 major tissue types: myometrium, stroma, and epithelium. Proliferation and differentiation in each uterine cell type are differentially regulated by ovarian hormones, resulting in spatiotemporal control of gene expression. Therefore, examining gene function in each uterine tissue type will provide more meaningful information regarding uterine biology during pregnancy and disease states. Although currently available Cre mouse lines have been very useful in exploring functions of specific genes in uterine biology, overlapping expression of these Cre lines in more than 1 tissue type and in other reproductive organs sometimes makes interpretation of results difficult. In this article, we report the generation of a new iCre knock-in mouse line, in which iCre is expressed from endogenous lactoferrin (Ltf) promoter. Ltf-iCre mice primarily direct recombination in the uterine epithelium in adult females and in immature females after estrogen treatment. These mice will allow for specific interrogation of gene function in the mature uterine epithelium, providing a helpful tool to uncover important aspects of uterine biology. PMID:24823394

  12. Systems Biology-Based Identification of Mycobacterium tuberculosis Persistence Genes in Mouse Lungs

    PubMed Central

    Dutta, Noton K.; Bandyopadhyay, Nirmalya; Veeramani, Balaji; Lamichhane, Gyanu; Karakousis, Petros C.; Bader, Joel S.

    2014-01-01

    ABSTRACT Identifying Mycobacterium tuberculosis persistence genes is important for developing novel drugs to shorten the duration of tuberculosis (TB) treatment. We developed computational algorithms that predict M.tuberculosis genes required for long-term survival in mouse lungs. As the input, we used high-throughput M.tuberculosis mutant library screen data, mycobacterial global transcriptional profiles in mice and macrophages, and functional interaction networks. We selected 57 unique, genetically defined mutants (18 previously tested and 39 untested) to assess the predictive power of this approach in the murine model of TB infection. We observed a 6-fold enrichment in the predicted set of M.tuberculosis genes required for persistence in mouse lungs relative to randomly selected mutant pools. Our results also allowed us to reclassify several genes as required for M.tuberculosis persistence in vivo. Finally, the new results implicated additional high-priority candidate genes for testing. Experimental validation of computational predictions demonstrates the power of this systems biology approach for elucidating M.tuberculosis persistence genes. PMID:24549847

  13. Molecular cloning and functional characterization of the mouse mafB gene.

    PubMed

    Huang, K; Serria, M S; Nakabayashi, H; Nishi, S; Sakai, M

    2000-01-25

    The Maf family of the transcription factors plays a pivotal role in controlling development and cellular differentiation. To clarify the molecular mechanisms controlling mafB expression, a genomic clone of the mouse mafB gene was isolated and analyzed. RNase protection analysis determined the transcription initiation site at 389 bp upstream from the translation initiation site. The 3' end of the gene is located at 946 bp downstream from the termination codon. The gene lacks intron structure. Sequence analysis showed a TATA-like sequence (5'-GATAAAA-3') and an inverted CCAAT-box (5'-ATTGG-3') in the promoter region. Upstream of these sequences, there are several potential regulatory elements, including two GC-boxes (5'-GGGCGG-3'), and a palindromic sequence (5'-GTCAGCTGAC-3') which contains two Maf recognition elements (MARE, 5'-GCTGAC-3') and an E-box (5'-CAGCTG-3'). Transient transfection analysis with the 5'-flanking region of the mafB gene demonstrated that these elements are important for mafB gene expression. In addition, cotransfection analysis indicated that the MyoD activates the mouse mafB promoter and the gene is positively auto-regulated by its own product. PMID:10721736

  14. Expression Profile of DNA Damage Signaling Genes in Proton Exposed Mouse Brain

    NASA Astrophysics Data System (ADS)

    Ramesh, Govindarajan; Wu, Honglu

    Exposure of living systems to radiation results in a wide assortment of lesions, the most signif-icant of is damage to genomic DNA which induce several cellular functions such as cell cycle arrest, repair, apoptosis etc. The radiation induced DNA damage investigation is one of the im-portant area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes particularly, damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR array in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2Gy proton exposed mouse brain tissues as compared with control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed brain tissue undergo severe DNA damage which in turn destabilize the chromatin stability.

  15. CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation.

    PubMed

    Shinmyo, Yohei; Tanaka, Satoshi; Tsunoda, Shinichi; Hosomichi, Kazuyoshi; Tajima, Atsushi; Kawasaki, Hiroshi

    2016-01-01

    The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo. PMID:26857612

  16. Lactoferrin-iCre: A New Mouse Line to Study Uterine Epithelial Gene Function

    PubMed Central

    Terakawa, Jumpei; Ogawa, Akiyo; DeFalco, Tony; Dey, Sudhansu K.

    2014-01-01

    Transgenic animal models are valuable for studying gene function in various tissue compartments. Mice with conditional deletion of genes in the uterus using the Cre-loxP system serve as powerful tools to study uterine biology. The uterus is comprised of 3 major tissue types: myometrium, stroma, and epithelium. Proliferation and differentiation in each uterine cell type are differentially regulated by ovarian hormones, resulting in spatiotemporal control of gene expression. Therefore, examining gene function in each uterine tissue type will provide more meaningful information regarding uterine biology during pregnancy and disease states. Although currently available Cre mouse lines have been very useful in exploring functions of specific genes in uterine biology, overlapping expression of these Cre lines in more than 1 tissue type and in other reproductive organs sometimes makes interpretation of results difficult. In this article, we report the generation of a new iCre knock-in mouse line, in which iCre is expressed from endogenous lactoferrin (Ltf) promoter. Ltf-iCre mice primarily direct recombination in the uterine epithelium in adult females and in immature females after estrogen treatment. These mice will allow for specific interrogation of gene function in the mature uterine epithelium, providing a helpful tool to uncover important aspects of uterine biology. PMID:24823394

  17. Cloning and chromosomal localization of a paralog and a mouse homolog of the human transaldolase gene.

    PubMed

    Kusuda, J; Hirai, M; Toyoda, A; Tanuma, R; Nomura-Kitabayashi, A; Hashimoto, K

    1998-03-16

    A sequence homologous to the transaldolase gene (TALDO) was identified in a polymorphic cosmid DNA mapped on human chromosome 11p15 by exon trapping with pSPL3. Analysis of lambda clones contiguous to the cosmid clone showed that the related gene (TALDOR) consists of 8 exons spanning approximately 19kb from the translation start site to the polyadenylation signal. The exon sequence of TALDOR was almost identical with that of TALDO localized on 1p33-34. 1, but its exons corresponding to exons 4 and 5 of TALDO were found to be split by 4 introns in TALDOR. To examine the evolutionary conservation of two genes for transaldolase, we have isolated the cDNA for its mouse homolog and determined the nucleotide sequence covering the complete coding region. Fluorescence in situ hybridization using the cDNA as a probe showed that the mouse transaldolase gene (Taldo) is localized on chromosome 7 F3-F4 as a single copy gene. This chromosomal region is known to be syntenic to human chromosome 11p15 rather than to 1p33-p34.1, suggesting that TALDOR is the ancestral form. The existence of TALDOR implies a duplication of the mammalian transaldolase gene after divergence of rodent and primate. PMID:9524206

  18. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model*

    PubMed Central

    Tong, Guo-qing; Heng, Boon-chin; Ng, Soon-chye

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 mol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes. PMID:17657853

  19. Activation of Type III Interferon Genes by Pathogenic Bacteria in Infected Epithelial Cells and Mouse Placenta

    PubMed Central

    Bierne, Hélène; Tailleux, Ludovic; Subtil, Agathe; Lebreton, Alice; Paliwal, Anupam; Gicquel, Brigitte; Staeheli, Peter; Lecuit, Marc; Cossart, Pascale

    2012-01-01

    Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-λ) genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-λ genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-λ genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-λ genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-λ. We also found that IFN-λ genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-λ2/λ3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-λ2. Together, these results suggest that IFN-λ may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues. PMID:22720036

  20. CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

    PubMed Central

    Shinmyo, Yohei; Tanaka, Satoshi; Tsunoda, Shinichi; Hosomichi, Kazuyoshi; Tajima, Atsushi; Kawasaki, Hiroshi

    2016-01-01

    The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo. PMID:26857612

  1. Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

    PubMed Central

    Wert, Katherine J.; Skeie, Jessica M.; Davis, Richard J.; Tsang, Stephen H.; Mahajan, Vinit B.

    2012-01-01

    The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue. PMID:23207897

  2. Meta-Analysis of Differentiating Mouse Embryonic Stem Cell Gene Expression Kinetics Reveals Early Change of a Small Gene Set

    PubMed Central

    Glover, Clive H; Marin, Michael; Eaves, Connie J; Helgason, Cheryl D; Piret, James M; Bryan, Jennifer

    2006-01-01

    Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types. PMID:17121458

  3. Gene expression signature of cerebellar hypoplasia in a mouse model of Down syndrome during postnatal development

    PubMed Central

    Laffaire, Julien; Rivals, Isabelle; Dauphinot, Luce; Pasteau, Fabien; Wehrle, Rosine; Larrat, Benoit; Vitalis, Tania; Moldrich, Randal X; Rossier, Jean; Sinkus, Ralph; Herault, Yann; Dusart, Isabelle; Potier, Marie-Claude

    2009-01-01

    Background Down syndrome is a chromosomal disorder caused by the presence of three copies of chromosome 21. The mechanisms by which this aneuploidy produces the complex and variable phenotype observed in people with Down syndrome are still under discussion. Recent studies have demonstrated an increased transcript level of the three-copy genes with some dosage compensation or amplification for a subset of them. The impact of this gene dosage effect on the whole transcriptome is still debated and longitudinal studies assessing the variability among samples, tissues and developmental stages are needed. Results We thus designed a large scale gene expression study in mice (the Ts1Cje Down syndrome mouse model) in which we could measure the effects of trisomy 21 on a large number of samples (74 in total) in a tissue that is affected in Down syndrome (the cerebellum) and where we could quantify the defect during postnatal development in order to correlate gene expression changes to the phenotype observed. Statistical analysis of microarray data revealed a major gene dosage effect: for the three-copy genes as well as for a 2 Mb segment from mouse chromosome 12 that we show for the first time as being deleted in the Ts1Cje mice. This gene dosage effect impacts moderately on the expression of euploid genes (2.4 to 7.5% differentially expressed). Only 13 genes were significantly dysregulated in Ts1Cje mice at all four postnatal development stages studied from birth to 10 days after birth, and among them are 6 three-copy genes. The decrease in granule cell proliferation demonstrated in newborn Ts1Cje cerebellum was correlated with a major gene dosage effect on the transcriptome in dissected cerebellar external granule cell layer. Conclusion High throughput gene expression analysis in the cerebellum of a large number of samples of Ts1Cje and euploid mice has revealed a prevailing gene dosage effect on triplicated genes. Moreover using an enriched cell population that is thought responsible for the cerebellar hypoplasia in Down syndrome, a global destabilization of gene expression was not detected. Altogether these results strongly suggest that the three-copy genes are directly responsible for the phenotype present in cerebellum. We provide here a short list of candidate genes. PMID:19331679

  4. Identification of sexually dimorphic genes in the neonatal mouse cortex and hippocampus.

    PubMed

    Armoskus, Chris; Moreira, Debbie; Bollinger, Kayla; Jimenez, Oliva; Taniguchi, Saori; Tsai, Houng-Wei

    2014-05-01

    The cerebral cortex and hippocampus are important for the control of cognitive functions and social behaviors, many of which are sexually dimorphic and tightly regulated by gonadal steroid hormones via activation of their respective nuclear receptors. As different levels of sex steroid hormones are present between the sexes during early development and their receptors act as transcription factors to regulate gene expression, we hypothesize that sexually dimorphic gene expression in the developing mouse cortex and hippocampus might result in sex differences in brain structures and neural circuits governing distinct behaviors between the sexes as adults. To test our hypothesis, we used gene expression microarrays to identify 90 candidate genes differentially expressed in the neonatal cortex/hippocampus between male and female mice, including 55 male-biased and 35 female-biased genes. Among these genes, sexually dimorphic expression of eight sex chromosome genes was confirmed by reverse transcription with quantitative PCR (RT-qPCR), including three located on the X chromosome (Xist, Eif2s3x, and Kdm6a), three on the Y chromosome (Ddx3y, Eif2s3y, and Kdm5d), and two in the pseudoautosomal region of the X and Y chromosomes (Erdr1 and Mid1). In addition, five autosomal genes (Cd151, Dab2, Klk8, Meg3, and Prkdc) were also validated for their sexually dimorphic expression in the neonatal mouse cortex/hippocampus. Gene Ontology annotation analysis suggests that many of these sexually dimorphic genes are involved in histone modifications, cell proliferation/death, androgen/estrogen signaling pathways, and synaptic organization, and these biological processes have been implicated in differential neural development, cognitive function, and neurological diseases between the sexes. PMID:24661915

  5. Identification of Sexually Dimorphic Genes in the Neonatal Mouse Cortex and Hippocampus

    PubMed Central

    Armoskus, Chris; Moreira, Debbie; Bollinger, Kayla; Jimenez, Oliva; Taniguchi, Saori; Tsai, Houng-Wei

    2014-01-01

    The cerebral cortex and hippocampus are important for the control of cognitive functions and social behaviors, many of which are sexually dimorphic and tightly regulated by gonadal steroid hormones via activation of their respective nuclear receptors. As different levels of sex steroid hormones are present between the sexes during early development and their receptors act as transcription factors to regulate gene expression, we hypothesize that sexually dimorphic gene expression in the developing mouse cortex and hippocampus might result in sex differences in brain structures and neural circuits governing distinct behaviors between the sexes as adults. To test our hypothesis, we used gene expression microarrays to identify 90 candidate genes differentially expressed in the neonatal cortex/hippocampus between male and female mice, including 55 male-biased and 35 female-biased genes. Among these genes, sexually dimorphic expression of eight sex chromosome genes was confirmed by reverse transcription with quantitative PCR (RT-qPCR), including three located on the X chromosome (Xist, Eif2s3x, and Kdm6a), three on the Y chromosome (Ddx3y, Eif2s3y, and Kdm5d), and two in the pseudoautosomal region of the X and Y chromosomes (Erdr1 and Mid1). In addition, five autosomal genes (Cd151, Dab2, Klk8, Meg3, and Prkdc) were also validated for their sexually dimorphic expression in the neonatal mouse cortex/hippocampus. Gene Ontology annotation analysis suggests that many of these sexually dimorphic genes are involved in histone modifications, cell proliferation/death, androgen/estrogen signaling pathways, and synaptic organization, and these biological processes have been implicated in differential neural development, cognitive function, and neurological diseases between the sexes. PMID:24661915

  6. From zebrafish heart jogging genes to mouse and human orthologs: using Gene Ontology to investigate mammalian heart development.

    PubMed Central

    Lovering, Ruth C

    2014-01-01

    For the majority of organs in developing vertebrate embryos, left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer’s vesicle in the zebrafish. In the zebrafish, laterality cues from the Kuppfer’s vesicle determine asymmetry in the developing heart, the direction of ‘heart jogging’ and the direction of ‘heart looping’.  ‘Heart jogging’ is the term given to the process by which the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward ‘jog’. Heart jogging is not considered to occur in mammals, although a leftward shift of the developing mouse caudal heart does occur prior to looping, which may be analogous to zebrafish heart jogging. Previous studies have characterized 30 genes involved in zebrafish heart jogging, the majority of which have well defined orthologs in mouse and human and many of these orthologs have been associated with early mammalian heart development.    We undertook manual curation of a specific set of genes associated with heart development and we describe the use of Gene Ontology term enrichment analyses to examine the cellular processes associated with heart jogging.  We found that the human, mouse and zebrafish ‘heart jogging orthologs’ are involved in similar organ developmental processes across the three species, such as heart, kidney and nervous system development, as well as more specific cellular processes such as cilium development and function. The results of these analyses are consistent with a role for cilia in the determination of left-right asymmetry of many internal organs, in addition to their known role in zebrafish heart jogging.    This study highlights the importance of model organisms in the study of human heart development, and emphasises both the conservation and divergence of developmental processes across vertebrates, as well as the limitations of this approach. PMID:24627794

  7. From zebrafish heart jogging genes to mouse and human orthologs: using Gene Ontology to investigate mammalian heart development.

    PubMed

    Khodiyar, Varsha K; Howe, Doug; Talmud, Philippa J; Breckenridge, Ross; Lovering, Ruth C

    2013-01-01

    For the majority of organs in developing vertebrate embryos, left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer's vesicle in the zebrafish. In the zebrafish, laterality cues from the Kuppfer's vesicle determine asymmetry in the developing heart, the direction of 'heart jogging' and the direction of 'heart looping'.  'Heart jogging' is the term given to the process by which the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward 'jog'. Heart jogging is not considered to occur in mammals, although a leftward shift of the developing mouse caudal heart does occur prior to looping, which may be analogous to zebrafish heart jogging. Previous studies have characterized 30 genes involved in zebrafish heart jogging, the majority of which have well defined orthologs in mouse and human and many of these orthologs have been associated with early mammalian heart development.    We undertook manual curation of a specific set of genes associated with heart development and we describe the use of Gene Ontology term enrichment analyses to examine the cellular processes associated with heart jogging.  We found that the human, mouse and zebrafish 'heart jogging orthologs' are involved in similar organ developmental processes across the three species, such as heart, kidney and nervous system development, as well as more specific cellular processes such as cilium development and function. The results of these analyses are consistent with a role for cilia in the determination of left-right asymmetry of many internal organs, in addition to their known role in zebrafish heart jogging.    This study highlights the importance of model organisms in the study of human heart development, and emphasises both the conservation and divergence of developmental processes across vertebrates, as well as the limitations of this approach. PMID:24627794

  8. Endocrine Parameters and Phenotypes of the Growth Hormone Receptor Gene Disrupted (GHR−/−) Mouse

    PubMed Central

    List, Edward O.; Sackmann-Sala, Lucila; Berryman, Darlene E.; Funk, Kevin; Kelder, Bruce; Gosney, Elahu S.; Okada, Shigeru; Ding, Juan; Cruz-Topete, Diana

    2011-01-01

    Disruption of the GH receptor (GHR) gene eliminates GH-induced intracellular signaling and, thus, its biological actions. Therefore, the GHR gene disrupted mouse (GHR−/−) has been and is a valuable tool for helping to define various parameters of GH physiology. Since its creation in 1995, this mouse strain has been used by our laboratory and others for numerous studies ranging from growth to aging. Some of the most notable discoveries are their extreme insulin sensitivity in the presence of obesity. Also, the animals have an extended lifespan, which has generated a large number of investigations into the roles of GH and IGF-I in the aging process. This review summarizes the many results derived from the GHR−/− mice. We have attempted to present the findings in the context of current knowledge regarding GH action and, where applicable, to discuss how these mice compare to GH insensitivity syndrome in humans. PMID:21123740

  9. Isolation of mouse osteocytes using cell fractionation for gene expression analysis.

    PubMed

    Halleux, Christine; Kramer, Ina; Allard, Cyril; Kneissel, Michaela

    2012-01-01

    Osteocytes are the terminally differentiated cells of the osteoblastic lineage embedded within the mineralized bone matrix. T: hey have been identified as key players in mechanotransduction and in mineral and phosphate homeostasis. In addition, they appear to have a role in mediating bone formation, since they secrete the bone formation inhibitor sclerostin. In contrast to osteoblasts and osteoclasts, which reside on the bone surface, it has been difficult to isolate and analyze cellular and molecular properties of osteocytes due to their specific location inside the "hard" mineralized bone compartment. This chapter describes a method to isolate osteocytes from newborn mouse calvaria and adult mouse long bone, followed by immediate total RNA extraction allowing to selectively study osteocytic versus osteoblastic gene expression by quantitative real-time polymerase chain reaction (qPCR). The osteocyte-enriched cell fraction isolated by this method can further be purified by FACS and selectively expresses osteocytic marker genes, such as Dmp1 and Sost. PMID:22130922

  10. The BioMart interface to the eMouseAtlas gene expression database EMAGE.

    PubMed

    Stevenson, Peter; Richardson, Lorna; Venkataraman, Shanmugasundaram; Yang, Yiya; Baldock, Richard

    2011-01-01

    Here, we describe the BioMart interface to the eMouseAtlas gene expression database EMAGE. EMAGE is a spatiotemporal database of in situ gene expression patterns in the developing mouse embryo. BioMart provides a generic web query interface and programmable access using web services. The BioMart interface extends access to EMAGE via a powerful method of structuring complex queries and one with which users may already be familiar with from other BioMart implementations. The interface is structured into several data sets providing the user with comprehensive query access to the EMAGE data. The federated nature of BioMart allows scope for integration and cross querying of EMAGE with other similar BioMarts. PMID:21930504

  11. Analyses of Allele-Specific Gene Expression in Highly Divergent Mouse Crosses Identifies Pervasive Allelic Imbalance

    PubMed Central

    Crowley, James J; Zhabotynsky, Vasyl; Sun, Wei; Huang, Shunping; Pakatci, Isa Kemal; Kim, Yunjung; Wang, Jeremy R; Morgan, Andrew P; Calaway, John D; Aylor, David L; Yun, Zaining; Bell, Timothy A; Buus, Ryan J; Calaway, Mark E; Didion, John P; Gooch, Terry J; Hansen, Stephanie D; Robinson, Nashiya N; Shaw, Ginger D; Spence, Jason S; Quackenbush, Corey R; Barrick, Cordelia J; Nonneman, Randal J.; Kim, Kyungsu; Xenakis, James; Xie, Yuying; Valdar, William; Lenarcic, Alan B; Wang, Wei; Welsh, Catherine E; Fu, Chen-Ping; Zhang, Zhaojun; Holt, James; Guo, Zhishan; Threadgill, David W; Tarantino, Lisa M; Miller, Darla R; Zou, Fei; McMillan, Leonard; Sullivan, Patrick F; de Villena, Fernando Pardo-Manuel

    2015-01-01

    Complex human traits are influenced by variation in regulatory DNA through mechanisms that are not fully understood. Since regulatory elements are conserved between humans and mice, a thorough annotation of cis regulatory variants in mice could aid in this process. Here we provide a detailed portrait of mouse gene expression across multiple tissues in a three-way diallel. Greater than 80% of mouse genes have cis regulatory variation. These effects influence complex traits and usually extend to the human ortholog. Further, we estimate that at least one in every thousand SNPs creates a cis regulatory effect. We also observe two types of parent-of-origin effects, including classical imprinting and a novel, global allelic imbalance in favor of the paternal allele. We conclude that, as with humans, pervasive regulatory variation influences complex genetic traits in mice and provide a new resource toward understanding the genetic control of transcription in mammals. PMID:25730764

  12. In vivo gene editing in dystrophic mouse muscle and muscle stem cells.

    PubMed

    Tabebordbar, Mohammadsharif; Zhu, Kexian; Cheng, Jason K W; Chew, Wei Leong; Widrick, Jeffrey J; Yan, Winston X; Maesner, Claire; Wu, Elizabeth Y; Xiao, Ru; Ran, F Ann; Cong, Le; Zhang, Feng; Vandenberghe, Luk H; Church, George M; Wagers, Amy J

    2016-01-22

    Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle. PMID:26721686

  13. A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

    SciTech Connect

    Laig-Webster, M.; Lim, M.E.; Chehab, F.F.

    1994-09-01

    The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.

  14. Polydactyly Nagoya, Pdn: A new mutant gene in the mouse.

    PubMed

    Hayasaka, I; Nakatsuka, T; Fujii, T; Naruse, I; Oda, S

    1980-10-01

    A new hereditary polydactyly (gene symbol Pdn) was found in the course of breeding JCL : ICR mice. The genetic analysis indicated that the polydactyly was an autosomal dominant trait. The homozygotes died within two days after birth. The homozygous fetuses or newborn had 1-3 extra-digits both in te fore- and hindlimbs on the preaxial side. They occasionally showed exencephaly, cleft palate, open eyelid, short tibia and fibula or deformed sternum. The heterozygotes had one extra-digit preaxial side. They occasionally showed exencephaly, heterozygotes had one extra-digit preaxially in the hindlimb and an enlarged first digit on the forelimb which often showed bifurcated distal phalanx. A tab on the postaxial side of the forelimb was found in all homozygotes and in some heterozygotes. PMID:7202526

  15. Identification and characterization of mouse otic sensory lineage genes

    PubMed Central

    Hartman, Byron H.; Durruthy-Durruthy, Robert; Laske, Roman D.; Losorelli, Steven; Heller, Stefan

    2015-01-01

    Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations) from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5) as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting cells. PMID:25852475

  16. DISC1 mouse models as a tool to decipher gene-environment interactions in psychiatric disorders

    PubMed Central

    Cash-Padgett, Tyler; Jaaro-Peled, Hanna

    2013-01-01

    DISC1 was discovered in a Scottish pedigree in which a chromosomal translocation that breaks this gene segregates with psychiatric disorders, mainly depression and schizophrenia. Linkage and association studies in diverse populations support DISC1 as a susceptibility gene to a variety of neuropsychiatric disorders. Many Disc1 mouse models have been generated to study its neuronal functions. These mouse models display variable phenotypes, some of them relevant to schizophrenia, others to depression. The Disc1 mouse models are popular genetic models for studying gene-environment interactions in schizophrenia. Five different Disc1 models have been combined with environmental factors. The environmental stressors employed can be classified as either early immune activation or later social paradigms. These studies cover major time points along the neurodevelopmental trajectory: prenatal, early postnatal, adolescence, and adulthood. Various combinations of molecular, anatomical and behavioral methods have been used to assess the outcomes. Additionally, three of the studies sought to rescue the resulting abnormalities. Here we provide background on the environmental paradigms used, summarize the results of these studies combining Disc1 mouse models with environmental stressors and discuss what we can learn and how to proceed. A major question is how the genetic and environmental factors determine which psychiatric disorder will be clinically manifested. To address this we can take advantage of the many Disc1 models available and expose them to the same environmental stressor. The complementary experiment would be to expose the same model to different environmental stressors. DISC1 is an ideal gene for this approach, since in the Scottish pedigree the same chromosomal translocation results in different psychiatric conditions. PMID:24027503

  17. The Hox-2 Homeo Box Gene Complex on Mouse Chromosome 11 Is Closely Linked to Re

    PubMed Central

    Hart, C. P.; Dalton, D. K.; Nichols, L.; Hunihan, L.; Roderick, T. H.; Langley, S. H.; Taylor, B. A.; Ruddle, F. H.

    1988-01-01

    Restriction fragment length polymorphisms have been identified between inbred strains of mice for the homeo box gene complex Hox-2. These genetic markers were used to follow the segregation of different Hox-2 alleles among recombinant inbred strains of mice and among the progeny of a three point genetic cross. The results place the Hoax-2 locus approximately 1 cM from the rex (Re) locus on mouse chromosome 11. PMID:2896141

  18. The Mouse Solitary Odorant Receptor Gene Promoters as Models for the Study of Odorant Receptor Gene Choice

    PubMed Central

    Degl'Innocenti, Andrea

    2016-01-01

    Background In vertebrates, several anatomical regions located within the nasal cavity mediate olfaction. Among these, the main olfactory epithelium detects most conventional odorants. Olfactory sensory neurons, provided with cilia exposed to the air, detect volatile chemicals via an extremely large family of seven-transmembrane chemoreceptors named odorant receptors. Their genes are expressed in a monogenic and monoallelic fashion: a single allele of a single odorant receptor gene is transcribed in a given mature neuron, through a still uncharacterized molecular mechanism known as odorant receptor gene choice. Aim Odorant receptor genes are typically arranged in genomic clusters, but a few are isolated (we call them solitary) from the others within a region broader than 1 Mb upstream and downstream with respect to their transcript's coordinates. The study of clustered genes is problematic, because of redundancy and ambiguities in their regulatory elements: we propose to use the solitary genes as simplified models to understand odorant receptor gene choice. Procedures Here we define number and identity of the solitary genes in the mouse genome (C57BL/6J), and assess the conservation of the solitary status in some mammalian orthologs. Furthermore, we locate their putative promoters, predict their homeodomain binding sites (commonly present in the promoters of odorant receptor genes) and compare candidate promoter sequences with those of wild-caught mice. We also provide expression data from histological sections. Results In the mouse genome there are eight intact solitary genes: Olfr19 (M12), Olfr49, Olfr266, Olfr267, Olfr370, Olfr371, Olfr466, Olfr1402; five are conserved as solitary in rat. These genes are all expressed in the main olfactory epithelium of three-day-old mice. The C57BL/6J candidate promoter of Olfr370 has considerably varied compared to its wild-type counterpart. Within the putative promoter for Olfr266 a homeodomain binding site is predicted. As a whole, our findings favor Olfr266 as a model gene to investigate odorant receptor gene choice. PMID:26794459

  19. Organization of the mouse 5-HT3 receptor gene and functional expression of two splice variants.

    PubMed

    Werner, P; Kawashima, E; Reid, J; Hussy, N; Lundström, K; Buell, G; Humbert, Y; Jones, K A

    1994-10-01

    The structure of the mouse 5-HT3 receptor gene, 5-HT3R-A, is most similar to nicotinic acetylcholine receptor (nAChR) genes, in particular to the gene encoding the neuronal nAChR subunit alpha 7. These genes share among other things the location of three adjacent introns, suggesting that 5-HT3R-A and nAChR genes arose from a common precursor gene. The alternative use of two adjacent splice acceptor sites in intron 8 creates, in addition to the original 5-HT3R-A cDNA (5-HT3R-AL), a shorter isoform (5-HT3R-AS) which lacks six codons in the segment that translates into the major intracellular domain. This splice consensus sequence is not found in human genomic DNA. In mouse, we demonstrate by RNAse protection assay that 5-HT3R-AS mRNA is approximately 5 times more abundant than 5-HT3R-AL mRNA in both neuroblastoma cell lines and neuronal tissues. We used the Semliki Forest virus expression system for electrophysiological characterization of 5-HT3R-AS and 5-HT3R-AL in mammalian cells. No differences in electrophysiological characteristics, such as voltage dependence, desensitization kinetics, or unitary conductance were found between homomeric 5-HT3R-AS and 5-HT3R-AL receptors. Their properties are very similar to those of 5-HT3 receptors in mouse neuroblastoma cell lines. PMID:7854052

  20. A High-Resolution Anatomical Framework of the Neonatal Mouse Brain for Managing Gene Expression Data

    PubMed Central

    Lee, Erh-Fang; Boline, Jyl; Toga, Arthur W.

    2007-01-01

    This study aims to provide a high-resolution atlas and use it as an anatomical framework to localize the gene expression data for mouse brain on postnatal day 0 (P0). A color Nissl-stained volume with a resolution of 13.3??50??13.3??3 was constructed and co-registered to a standard anatomical space defined by an averaged geometry of C57BL/6J P0 mouse brains. A 145 anatomical structures were delineated based on the histological images. Anatomical relationships of delineated structures were established based on the hierarchical relations defined in the atlas of adult mouse brain (MacKenzie-Graham et al., 2004) so the P0 atlas can be related to the database associated with the adult atlas. The co-registered multimodal atlas as well as the original anatomical delineations is available for download at http://www.loni.ucla.edu/Atlases/. The region-specific anatomical framework based on the neonatal atlas allows for the analysis of gene activity within a high-resolution anatomical space at an early developmental stage. We demonstrated the potential application of this framework by incorporating gene expression data generated using in situ hybridization to the atlas space. By normalizing the gene expression patterns revealed by different images, experimental results from separate studies can be compared and summarized in an anatomical context. Co-displaying multiple registered datasets in the atlas space allows for 3D reconstruction of the co-expression patterns of the different genes in the atlas space, hence providing better insight into the relationship between the differentiated distribution pattern of gene products and specific anatomical systems. PMID:18974801

  1. Identification of a mouse gene required for binding of Rauscher MuLV envelope gp70.

    PubMed

    Hilkens, J; Colombatti, A; Strand, M; Nichols, E; Ruddle, F H; Hilgers, J

    1979-01-01

    Mouse chromosome segregating somatic cell hybrids were established between a mouse thymic leukemai cell line (GRSL) and Chinese hamster E36 cells. The GRSL cells specifically bound purified Rauscher leukemia virus gp70 while the E36 cells exhibited no binding. The hybrids selectively bound Ruascher gp70 depending on the presence of a mouse cellular gene for the ecotropic murine luekemia gp70 receptor. A syntenic relationship was observed between the DIP-3 chromosome marker (on chromosome 5) and the gp70 receptor in primary clones and subclones of these hybrids; this was confirmed by chromosome analysis. The involvement of H-2 in the binding of Rauscher MuLV gp70 could be ruled out, because discordancies of the receptor presence and H-2 absence as well as of the receptor absence and H-2 presence type could be observed. Our results indicate that the Rec-1 (replication ecotropic MuLV) gene of Gazdar et al. (4) may well be the receptor gene for the ecotropic murine leukemia virus. PMID:432756

  2. The Construction of Transgenic and Gene Knockout/Knockin Mouse Models of Human Disease

    PubMed Central

    Doyle, Alfred; McGarry, Michael P.; Lee, Nancy A.; Lee, James J.

    2012-01-01

    The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research, including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individuals gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care. PMID:21800101

  3. Endocrine genes

    SciTech Connect

    Lau, Y.F.

    1988-01-01

    This book contains 13 chapters. Some of the titles are: Gene Transfer and Expression of Mammalian Cell Receptors; Mapping Endocrine Genes with Sorted Human Chromosomes; Structure, Function, Hormonal Regulation of Steroidogenic Enzyme Genes; Molecular Analysis of Steroid Hormone Action Using the Human Metallothionein Genes as a Model.

  4. A Comparative Study of Mouse Hepatic and Intestinal Gene Expression Profiles under PPAR? Knockout by Gene Set Enrichment Analysis

    PubMed Central

    He, Kan; Wang, Qishan; Yang, Yumei; Wang, Minghui; Pan, Yuchun

    2011-01-01

    Gene expression profiling of PPAR? has been used in several studies, but fewer studies went further to identify the tissue-specific pathways or genes involved in PPAR? activation in genome-wide. Here, we employed and applied gene set enrichment analysis to two microarray datasets both PPAR? related respectively in mouse liver and intestine. We suggested that the regulatory mechanism of PPAR? activation by WY14643 in mouse small intestine is more complicated than in liver due to more involved pathways. Several pathways were cancer-related such as pancreatic cancer and small cell lung cancer, which indicated that PPAR? may have an important role in prevention of cancer development. 12 PPAR? dependent pathways and 4 PPAR? independent pathways were identified highly common in both liver and intestine of mice. Most of them were metabolism related, such as fatty acid metabolism, tryptophan metabolism, pyruvate metabolism with regard to PPAR? regulation but gluconeogenesis and propanoate metabolism independent of PPAR? regulation. Keratan sulfate biosynthesis, the pathway of regulation of actin cytoskeleton, the pathways associated with prostate cancer and small cell lung cancer were not identified as hepatic PPAR? independent but as WY14643 dependent ones in intestinal study. We also provided some novel hepatic tissue-specific marker genes. PMID:21811494

  5. The Immunoglobulin-like Gene spe-45 Acts during Fertilization in Caenorhabditis elegans like the Mouse Izumo1 Gene.

    PubMed

    Nishimura, Hitoshi; Tajima, Tatsuya; Comstra, Heather Skye; Gleason, Elizabeth J; L'Hernault, Steven W

    2015-12-21

    The Caenorhabditis elegans spe-9 class genes, which show specific or predominant expression in the male germline, are indispensable for fertilization [1, 2]. However, due to the rapid evolution of genes involved in reproduction, we do not currently know if there are spe-9 class genes in mammals that play similar roles during fertilization to those found in C.elegans. In mice, the Izumo1 gene encodes a sperm-specific transmembrane (TM) protein with a single immunoglobulin (Ig)-like domain that is absolutely required for gamete fusion [3, 4]. In this study, we hypothesized that C.elegans has a new member of the spe-9 class genes coding for an IZUMO1-like protein. We screened C.elegans microarray data [5, 6] to identify male germline-enriched genes that encode membrane proteins with Ig-like domains. Adeletion (tm3715) in one such gene (F28D1.8) caused hermaphrodites to show a male germline-dependent self-sterility, so we have named it spe-45. Mutant spe-45 worms seemed to normally undergo spermatogenesis (spermatid production by meiosis) and spermiogenesis (spermatid activation into actively motile spermatozoa). spe-45 mutant spermatozoa, however, could not complete gamete fusion, which is a characteristic of all spe-9 class mutants [1, 2]. Moreover, spe-45 self-sterile worms were rescued by a transgene expressing chimeric SPE-45 protein in which its Ig-like domain was replaced by the Ig-like domain from mouse IZUMO1. Hence, C.elegans SPE-45 and mouse IZUMO1 appear to have retained a common function(s) that is required during fertilization. PMID:26671669

  6. Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model

    PubMed Central

    Bard-Chapeau, Emilie A.; Nguyen, Anh-Tuan; Rust, Alistair G.; Sayadi, Ahmed; Lee, Philip; Chua, Belinda Q; New, Lee-Sun; de Jong, Johann; Ward, Jerrold M.; Chin, Christopher KY.; Chew, Valerie; Toh, Han Chong; Abastado, Jean-Pierre; Benoukraf, Touati; Soong, Richie; Bard, Frederic A.; Dupuy, Adam J.; Johnson, Randy L.; Radda, George K.; Chan, Eric CY.; Wessels, Lodewyk FA.; Adams, David J.

    2014-01-01

    The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC we performed a near saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers, and a bewildering number (2860) of candidate later stage drivers, that were enriched for genes mutated, deregulated, or that function in signaling pathways important for human HCC, with a striking 1199 genes linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC. PMID:24316982

  7. Genetic and Molecular Basis of QTL of Diabetes in Mouse: Genes and Polymorphisms

    PubMed Central

    Gao, Peng; Jiao, Yan; Xiong, Qing; Wang, Cong-Yi; Gerling, Ivan; Gu, Weikuan

    2008-01-01

    A systematic study has been conducted of all available reports in PubMed and OMIM (Online Mendelian Inheritance in Man) to examine the genetic and molecular basis of quantitative genetic loci (QTL) of diabetes with the main focus on genes and polymorphisms. The major question is, What can the QTL tell us? Specifically, we want to know whether those genome regions differ from other regions in terms of genes relevant to diabetes. Which genes are within those QTL regions, and, among them, which genes have already been linked to diabetes? whether more polymorphisms have been associated with diabetes in the QTL regions than in the non-QTL regions. Our search revealed a total of 9038 genes from 26 type 1 diabetes QTL, which cover 667,096,006 bp of the mouse genomic sequence. On one hand, a large number of candidate genes are in each of these QTL; on the other hand, we found that some obvious candidate genes of QTL have not yet been investigated. Thus, the comprehensive search of candidate genes for known QTL may provide unexpected benefit for identifying QTL genes for diabetes. PMID:19471607

  8. Stage-specific reference genes significant for quantitative PCR during mouse retinal development.

    PubMed

    Adachi, Hiroko; Tominaga, Hiroyuki; Maruyama, Yuko; Yoneda, Kazuhito; Maruyama, Kazuichi; Yoshii, Kengo; Kinoshita, Shigeru; Nakano, Masakazu; Tashiro, Kei

    2015-08-01

    Developing mouse retina has been serving as an ideal model for investigating the molecular mechanism of neural development and angiogenesis, because several significant events associated with these physiological phenomena are drastically occurring in conjunction with retinal development. However, as many genes are influencing on each other to establish mature retina within 21 days from E10 to P12, we must carefully design the experiments, such as in the case of quantitating the amount of altered gene expression toward the establishment of retina by quantitative PCR. As we have seen considerable variations of quantitative results in different developmental stages of retina depending on the reference genes used for compensation, we here attempted to determine a reliable reference gene to accurately quantitate the target genes in each stage. According to the results of in silico prediction and comparison with a database of SAGE, we found that the most stable gene from early to late stages was Sdha, whereas one of the most popular housekeeping genes, Actb, was the one that could mislead the quantitative results even in the adult stage. Consequently, we pointed out the importance of selecting an appropriate reference gene, especially to quantitate the amount of gene expression in the developmental stages of a certain tissue. PMID:26059597

  9. High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

    PubMed Central

    Brommage, Robert; Liu, Jeff; Hansen, Gwenn M; Kirkpatrick, Laura L; Potter, David G; Sands, Arthur T; Zambrowicz, Brian; Powell, David R; Vogel, Peter

    2014-01-01

    Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets. PMID:26273529

  10. High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes.

    PubMed

    Brommage, Robert; Liu, Jeff; Hansen, Gwenn M; Kirkpatrick, Laura L; Potter, David G; Sands, Arthur T; Zambrowicz, Brian; Powell, David R; Vogel, Peter

    2014-01-01

    Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets. PMID:26273529

  11. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    SciTech Connect

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. )

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  12. Adeno-associated virus-mediated gene transfer targeting normal and traumatized mouse utricle.

    PubMed

    Wang, G-P; Guo, J-Y; Peng, Z; Liu, Y-Y; Xie, J; Gong, S-S

    2014-11-01

    Balance dysfunction is closely associated with loss of vestibular hair cells (HCs). Gene therapy shows promise when used to protect or regenerate vestibular HCs to preserve or restore adequate vestibular function. Adeno-associated virus (AAV) vectors allow long-term gene expression in the absence of toxicity. To noninvasively define an AAV serotype exhibiting favorable tropism toward the vestibular sensory epithelium, we characterized the transgene expression potential of AAV vectors (serotypes 1, 2, 5, 6 and 8) inoculated into adult mouse utricle via canalostomy. We found that AAV8 was the most effective AAV vector in utricular gene transfer. Swim tests and measurements of auditory brainstem response revealed minimal loss of vestibular function and hearing after canalostomy. In the normal utricle after AAV8 infusion, transduction efficiency peaked at 7 days, and was maintained thereafter, in vestibular HCs, and at 3 days in supporting cells (SCs). In the streptomycin-lesioned utricle, the SC transduction efficiency peaked at 7 days and decreased at 30 days. In conclusion, AAV8-mediated gene transfer via canalostomy facilitates efficient and safe transduction in mouse vestibular sensory epithelium, and may in the future become clinically relevant for human vestibular gene therapy. PMID:25119376

  13. Cloning and characterization of promoter of the mouse mafB gene.

    PubMed

    Huang, K

    1999-11-01

    The MafB transcription factor plays a pivotal role in controlling the development and differentiation. The author reports the isolation and analysis of genomic clone of the mouse mafB gene. The gene lacks intron structure, at least, within its coding and 5'-untranslated sequences that are similar to the chicken mafB gene. RNA protection analysis determined one transcription initiation site of the gene at 389-bp upstream from the translation initiation site. Sequence analysis showed that the 5'-flanking region upstream to the ATG codon did not contain a conventional TATA box. A TATA-like sequence (5'-GATAAAA-3') and an inverted CCAAT-box (5'-ATTGG-3') were found to be located at nucleotide -31 and -86, referring to the transcription initiation site, respectively. Upstream to these sequences, there were several potential regulatory elements, including two GC-boxes (5'-GGGCGG-3': from -148 to -143; and from -123 to -118), and a palindromic sequence (5'-GTCAGCTGAC-3': from -164 to -155) which contained two halves-MARE (Maf recognition element), 5'-GCTGAC-3', and an E-box (5'-CAGCTG-3'). Promoter activity of the 5'-flanking region was analyzed by reporter transfection assay, which suggested that these segments were an important transcriptional activator. It was also suggested that MyoD transactivated the mouse mafB promoter and this gene was positively autoregulated by its product, MafB. PMID:10642889

  14. Toward a Systems Biology of Mouse Inner Ear Organogenesis: Gene Expression Pathways, Patterns and Network Analysis

    PubMed Central

    Sajan, Samin A.; Warchol, Mark E.; Lovett, Michael

    2007-01-01

    We describe the most comprehensive study to date on gene expression during mouse inner ear (IE) organogenesis. Samples were microdissected from mouse embryos at E9E15 in half-day intervals, a period that spans all of IE organogenesis. These included separate dissections of all discernible IE substructures such as the cochlea, utricle, and saccule. All samples were analyzed on high density expression microarrays under strict statistical filters. Extensive confirmatory tests were performed, including RNA in situ hybridizations. More than 5000 genes significantly varied in expression according to developmental stage, tissue, or both and defined 28 distinct expression patterns. For example, upregulation of 315 genes provided a clear-cut signature of early events in IE specification. Additional, clear-cut, gene expression signatures marked specific structures such as the cochlea, utricle, or saccule throughout late IE development. Pathway analysis identified 53 signaling cascades enriched within the 28 patterns. Many novel pathways, not previously implicated in IE development, including ?-adrenergic, amyloid, estrogen receptor, circadian rhythm, and immune system pathways, were identified. Finally, we identified positional candidate genes in 54 uncloned nonsyndromic human deafness intervals. This detailed analysis provides many new insights into the spatial and temporal genetic specification of this complex organ system. PMID:17660535

  15. Structure and expression of mouse mitochondrial voltage dependent anion channel genes

    SciTech Connect

    Craigen, W.J.; Lovell, R.S.; Sampson, M.J.

    1994-09-01

    Voltage dependent anion channels (VDACs) are small abundant proteins of the outer mitochondrial membrane that interact with the adenine nucleotide translocater and bind glycerol kinase and hexokinase. Kinase binding is developmentally regulated, tissue specific, and increased in various tumor cell lines. VDACs are also components of the peripheral benzodiazepine receptor and GABA{sub A} receptor. Two human VDAC cDNAs have previously been reported, and expression of these isoforms appears ubiquitous. Genomic Southern analysis suggests the presence of other as yet uncharacterised VDAC genes. To study VDAC function in a mammal more amenable to experimental manipulation, we have isolated three mouse VDAC genes by cDNA cloning from a mouse brain cDNA library. DNA sequencing of the cDNAs shows that they share 65-75% amino acid identity. Northern analysis indicates that MVDAC1 is expressed most highly in kidney, heart, and brain. Using an MVDAC3 3{prime} untranslated exon as a probe, three distinct transcripts can be detected. The gene structure for MVDAC3 and MVDAC2 has been completed and suggests that the VDAC isoforms did not arise by gene duplication and divergence. The intron/exon boundaries are not conserved between MVDAC1 and MVDAC3, and MVDAC2 appears to be encoded by a single intronless gene.

  16. Proteomics and bioinformatics analysis of mouse hypothalamic neurogenesis with or without EPHX2 gene deletion

    PubMed Central

    Zhong, Lijun; Zhou, Juntuo; Wang, Dawei; Zou, Xiajuan; Lou, Yaxin; Liu, Dan; Yang, Bin; Zhu, Yi; Li, Xiaoxia

    2015-01-01

    The aim of this study was to identify differently expressed proteins in the presence and absence of EPHX2 gene in mouse hypothalamus using proteomics profiling and bioinformatics analysis. This study was performed on 3 wild type (WT) and 3 EPHX2 gene global knockout (KO) mice (EPHX2 -/-). Using the nano- electrospray ionization (ESI)-LC-MS/MS detector, we identified 31 over-expressed proteins in WT mouse hypothalamus compared to the KO counterparts. Gene Ontology (GO) annotation in terms of the protein-protein interaction network indicated that cellular metabolic process, protein metabolic process, signaling transduction and protein post-translation biological processes involved in EPHX2 -/- regulatory network. In addition, signaling pathway enrichment analysis also highlighted chronic neurodegenerative diseases and some other signaling pathways, such as TGF-beta signaling pathway, T cell receptor signaling pathway, ErbB signaling pathway, Neurotrophin signaling pathway and MAPK signaling pathway, were strongly coupled with EPHX2 gene knockout. Further studies into the molecular functions of EPHX2 gene in hypothalamus will help to provide new perspective in neurogenesis. PMID:26722453

  17. Isolation and characterization of two mouse Pi-class glutathione S-transferase genes.

    PubMed Central

    Bammler, T K; Smith, C A; Wolf, C R

    1994-01-01

    Pi-class glutathione S-transferases (GSTs) play an important role in the detoxification of chemical toxins and mutagens and are implicated in neoplastic development and drug resistance. In all species characterized to date, only one functional Pi-class GST gene has been described. In this report we have identified two actively transcribed murine Pi-class GST genes, Gst p-1 and Gst p-2. The coding regions of Gst p-1 and the mouse Pi-class GST cDNA (GST-II) reported by Hatayama, Satoh and Satoh (1990) (Nucleic Acids Res. 18, 4606) are identical, whereas Gst p-2 encodes a protein that has not been described previously. The two genes are approximately 3 kb long and contain seven exons interrupted by six introns. In addition to a TATA box and a sequence motif matching the phorbol-ester-responsive element, the promoters of Gst p-1 and Gst p-2 exhibit one and two G+C boxes (GGGCGG) respectively. The cDNAs of the two genes were isolated from total liver RNA using reverse PCR. The peptide sequence deduced from the cDNAs share 97% identity and differ in six amino acids. Both genes are transcribed at significantly higher levels in male mouse liver than in female, and Gst p-1 mRNA is more abundant in both sexes than Gst p-2. Images Figure 4 Figure 5 PMID:8135745

  18. Analysis of imprinted gene expression and implantation in haploid androgenetic mouse embryos.

    PubMed

    Hu, M; Zhao, Z; TuanMu, L-C; Wei, H; Gao, F; Li, L; Ying, J; Zhang, S

    2015-02-01

    The successful development of mammalian embryos requires both parental genomes. Nuclear transfer techniques have been adapted to generate uniparental embryos, which possess two sets of paternal or maternal genomes. These embryos fail to develop to term because of abnormal imprinted gene expression, which is not regulated by Mendelian inheritance. Uniparental embryos provide us with an important model to investigate imprinted gene function and ontogenesis. To evaluate the pre- and post-developmental ability of haploid androgenetic mouse embryos, and to analyse the expression of imprinted genes Igf2r, Asb4 and Mest in haploid androgenetic/gynogenetic blastocysts, we produced the haploid mouse embryos using the enucleation technique, examined their development at 6.5 dpc and quantified gene expression by quantitative real-time PCR. The results demonstrated that the developmental potential of haploid embryos was severely impaired and revealed that the haploid androgenones could induce the deciduas reaction, but failed to retain a live foetus at 6.5 dpc. Expression of imprinted genes Igf2r and Asb4 was unregulated in haploid androgenetic/gynogenetic blastocysts. PMID:24387305

  19. Cloning and tissue-specific expression of the gene for mouse C-reactive protein.

    PubMed

    Ku, N O; Mortensen, R F

    1993-10-15

    C-reactive protein is a serum acute-phase reactant that increases several thousand-fold in concentration during inflammation in most mammals. However, mouse C-reactive protein is considered to be a minor acute-phase reactant, since its blood level increases only from approx. 0.1 to 1-2 micrograms/ml. A mouse genomic clone of approximately 5 kb was obtained to determine the molecular basis for the regulation of the expression of mouse C-reactive protein. Several cis-acting elements in the 5' flanking region that potentially regulate transcription were identified: two glucocorticoid-responsive elements, two CCAAT-enhancer-binding protein C (C/EBP) consensus elements that are required for the interleukin-1 responsiveness of some acute-phase reactant genes, an interleukin-6-responsive element, two hepatocyte nuclear factor-1 (HNF-1) elements and a single heat-shock element. Transfection of the hepatoma cell line Hep 3B.2 with a pCAT expression vector containing the 5' flanking sequence from -1083 to -3 bp from the transcriptional start site, and truncations of this sequence, localized elements that control the tissue-specific expression of mouse C-reactive protein to the two HNF-1 elements and a C/EBP, interleukin-1-responsive element located between -220 and -153, and -90 and -50 bp from the transcriptional start site. A constitutive nuclear protein from mouse-liver hepatocytes specifically binds to the HNF-1 elements. These findings explain the tissue-specific expression of the gene, as well as its limited expression during the acute-phase response. PMID:7916620

  20. Localization of complement factor H gene expression and protein distribution in the mouse outer retina

    PubMed Central

    Smit-McBride, Zeljka; Oltjen, Sharon L.; Radu, Roxana A.; Estep, Jason; Nguyen, Anthony T.; Gong, Qizhi

    2015-01-01

    Purpose To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. Methods Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. Results Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results. Conclusions Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC. PMID:25684976

  1. Modeling disease mutations by gene targeting in one-cell mouse embryos

    PubMed Central

    Meyer, Melanie; Ortiz, Oskar; Hrabé de Angelis, Martin; Wurst, Wolfgang; Kühn, Ralf

    2012-01-01

    Gene targeting by zinc-finger nucleases in one-cell embryos provides an expedite mutagenesis approach in mice, rats, and rabbits. This technology has been recently used to create knockout and knockin mutants through the deletion or insertion of nucleotides. Here we apply zinc-finger nucleases in one-cell mouse embryos to generate disease-related mutants harboring single nucleotide or codon replacements. Using a gene-targeting vector or a synthetic oligodesoxynucleotide as template for homologous recombination, we introduced missense and silent mutations into the Rab38 gene, encoding a small GTPase that regulates intracellular vesicle trafficking. These results demonstrate the feasibility of seamless gene editing in one-cell embryos to create genetic disease models and establish synthetic oligodesoxynucleotides as a simplified mutagenesis tool. PMID:22660928

  2. Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

    PubMed Central

    Bennis, Anna; Gorgels, Theo G. M. F.; ten Brink, Jacoline B.; van der Spek, Peter J.; Bossers, Koen; Heine, Vivi M.; Bergen, Arthur A.

    2015-01-01

    Background The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE. Methods We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE. Results We defined sets of mouse (64), human (171) and mouse–human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like “organ development” and “disorders related to neurological tissue”. However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch’s membrane, immune-regulation and outer blood retina barrier. Conclusion These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular degeneration. PMID:26517551

  3. Cloning and characterization of the mouse glucokinase gene locus and identification of distal liver-specific DNase I hypersensitive sites

    SciTech Connect

    Postic, C.; Niswender, K.D.; Shelton, K.D.; Pettepher, C.C.; Granner, D.K.; Magnuson, M.A.

    1995-10-10

    We cloned and characterized an 83-kb fragment of mouse genomic DNA containing the entire glucokinase (GK) gene. The 11 exons of the gene span a total distance of 49 kb, with exons 1{beta} and 1L being separated by 35 kb. A total of 25,266 bp of DNA sequence information was determined: from {approximately}-9.2 to {approximately}+15 kb (24,195 bp), relative to the hepatocyte transcription start site, and from -335 to -736 bp (1071 bp), relative to the transcription start site in {beta} cells. These sequences revealed that mouse GK is >94% identical to rat and human GK. Mouse hepatic GK mRNA is regulated by fasting and refeeding, as also occurs in the rat. Alignment of the upstream and downstream promoter regions of the mouse, rat, and human genes revealed several evolutionarily conserved regions that may contribute to transcriptional regulation. However, fusion gene studies in transgenic mice indicate that the conserved regions near the transcription start site in hepatocytes are themselves not sufficient for position-independent expression in liver. Analysis of the chromatin structure of a 48-kb region of the mouse gene using DNase I revealed eight liver-specific hypersensitive sites whose locations ranged from 0.1 to 36 kb upstream of the liver transcription start site. The availability of a single, contiguous DNA fragment containing the entire mouse GK gene should allow further studies of cell-specific expression of GK to be performed. 46 refs., 8 figs.

  4. Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

    PubMed Central

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Ltge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10?6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells. PMID:22194948

  5. Early Maternal Alcohol Consumption Alters Hippocampal DNA Methylation, Gene Expression and Volume in a Mouse Model

    PubMed Central

    Marjonen, Heidi; Sierra, Alejandra; Nyman, Anna; Rogojin, Vladimir; Gröhn, Olli; Linden, Anni-Maija; Hautaniemi, Sampsa; Kaminen-Ahola, Nina

    2015-01-01

    The adverse effects of alcohol consumption during pregnancy are known, but the molecular events that lead to the phenotypic characteristics are unclear. To unravel the molecular mechanisms, we have used a mouse model of gestational ethanol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first 8 days of gestation (GD 0.5-8.5). Early neurulation takes place by the end of this period, which is equivalent to the developmental stage early in the fourth week post-fertilization in human. During this exposure period, dynamic epigenetic reprogramming takes place and the embryo is vulnerable to the effects of environmental factors. Thus, we hypothesize that early ethanol exposure disrupts the epigenetic reprogramming of the embryo, which leads to alterations in gene regulation and life-long changes in brain structure and function. Genome-wide analysis of gene expression in the mouse hippocampus revealed altered expression of 23 genes and three miRNAs in ethanol-exposed, adolescent offspring at postnatal day (P) 28. We confirmed this result by using two other tissues, where three candidate genes are known to express actively. Interestingly, we found a similar trend of upregulated gene expression in bone marrow and main olfactory epithelium. In addition, we observed altered DNA methylation in the CpG islands upstream of the candidate genes in the hippocampus. Our MRI study revealed asymmetry of brain structures in ethanol-exposed adult offspring (P60): we detected ethanol-induced enlargement of the left hippocampus and decreased volume of the left olfactory bulb. Our study indicates that ethanol exposure in early gestation can cause changes in DNA methylation, gene expression, and brain structure of offspring. Furthermore, the results support our hypothesis of early epigenetic origin of alcohol-induced disorders: changes in gene regulation may have already taken place in embryonic stem cells and therefore can be seen in different tissue types later in life. PMID:25970770

  6. BMD Regulation on Mouse Distal Chromosome 1, Candidate Genes, and Response to Ovariectomy or Dietary Fat

    PubMed Central

    Beamer, Wesley G; Shultz, Kathryn L; Coombs, Harold F; DeMambro, Victoria E; Reinholdt, Laura G; Ackert-Bicknell, Cheryl L; Canalis, Ernesto; Rosen, Clifford J; Donahue, Leah Rae

    2011-01-01

    The distal end of mouse chromosome 1 (Chr 1) harbors quantitative trait loci (QTLs) that regulate bone mineral density (BMD) and share conserved synteny with human chromosome 1q. The objective of this article was to map this mouse distal Chr 1 region and identify gene(s) responsible for BMD regulation in females. We used X-ray densitometry [ie, dual-energy X-ray Absorptiometry (DXA), microcomputed tomography (CT), and peripheral quantitative computed tomography (pQCT)] to phenotype a set of nested congenic strains constructed from C57BL/6BmJ (B6/Bm) and C3H/HeJ (C3H) mice to map the region associated with the BMD QTL. The critical region has been reduced to an interval of 0.152 Mb that contributes to increased BMD when C3H alleles are present. Histomorphometry and osteoblast cultures indicated that increased osteoblast activity was associated with increased BMD in mouse strains with C3H alleles in this critical region. This region contains two genes, Aim2, which binds with cytoplasmic dsDNA and results in apoptosis, and AC084073.22, a predicted gene of unknown function. Ovariectomy induced bone loss in the B6/Bm progenitor and the three congenic strains regardless of the alleles present in the critical BMD region. High dietary fat treatment (thought to suppress distal Chr 1 QTL for BMD in mice) did not induce bone loss in the congenics carrying C3H alleles in the critical 0.152 Mb carrying the AIM2 and AC084073.22 genes. Gene expression studies in whole bone of key congenics showed differential expression of AC084073.22 for strains carrying B6/Bm versus C3H alleles but not for Aim2. In conclusion, our data suggest that osteoblasts are the cellular target of gene action and that AC084073.22 is the best candidate for female BMD regulation in the distal region of mouse Chr 1. 2011 American Society for Bone and Mineral Research. PMID:20687154

  7. Detection of retinoblastoma gene deletions in spontaneous and radiation-induced mouse lung adenocarcinomas by polymerase chain reaction

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E. )

    1994-03-01

    A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma gene using histological sections from radiation-induced and spontaneous tumors as the DNA source. Six mouse Rb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments relative to control PCR products on a Southern blot indicated a deletion of that portion of the mouse Rb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death. Spontaneous tumors as well as those from irradiated mice (5.69 Gy [sup 60]Co [gamma] rays or 0.6 Gy JANUS neutrons, which have been found to have approximately equal radiobiological effectiveness) were analyzed for mouse Rb deletions. Tumors in 6 neutron-irradiated mice had no mouse Rb deletions. However, 1 of 6 tumors from [gamma]-irradiated mice (17%) and 6 of 18 spontaneous tumors from unirradiated mice (33%) showed a deletion in one or both mouse Rb alleles. All deletions detected were in the 5[prime] region of the mouse Rb gene. 36 refs., 2 figs., 2 tabs.

  8. Integrative gene regulatory network analysis reveals light-induced regional gene expression phase shift programs in the mouse suprachiasmatic nucleus.

    PubMed

    Zhu, Haisun; Vadigepalli, Rajanikanth; Rafferty, Rachel; Gonye, Gregory E; Weaver, David R; Schwaber, James S

    2012-01-01

    We use the multigenic pattern of gene expression across suprachiasmatic nuclei (SCN) regions and time to understand the dynamics within the SCN in response to a circadian phase-resetting light pulse. Global gene expression studies of the SCN indicate that circadian functions like phase resetting are complex multigenic processes. While the molecular dynamics of phase resetting are not well understood, it is clear they involve a "functional gene expression program", e.g., the coordinated behavior of functionally related genes in space and time. In the present study we selected a set of 89 of these functionally related genes in order to further understand this multigenic program. By use of high-throughput qPCR we studied 52 small samples taken by anatomically precise laser capture from within the core and shell SCN regions, and taken at time points with and without phase resetting light exposure. The results show striking regional differences in light response to be present in the mouse SCN. By using network-based analyses, we are able to establish a highly specific multigenic correlation between genes expressed in response to light at night and genes normally activated during the day. The light pulse triggers a complex and highly coordinated network of gene regulation. The largest differences marking neuroanatomical location are in transmitter receptors, and the largest time-dependent differences occur in clock-related genes. Nighttime phase resetting appears to recruit transcriptional regulatory processes normally active in the day. This program, or mechanism, causes the pattern of core region gene expression to transiently shift to become more like that of the shell region. PMID:22662235

  9. Genome-Wide Analyses of Gene Expression during Mouse Endochondral Ossification

    PubMed Central

    James, Claudine G.; Stanton, Lee-Anne; Agoston, Hanga; Ulici, Veronica; Underhill, T. Michael; Beier, Frank

    2010-01-01

    Background Endochondral ossification is a complex process involving a series of events that are initiated by the establishment of a chondrogenic template and culminate in its replacement through the coordinated activity of osteoblasts, osteoclasts and endothelial cells. Comprehensive analyses of in vivo gene expression profiles during these processes are essential to obtain a complete understanding of the regulatory mechanisms involved. Methodology/Principal Findings To address these issues, we completed a microarray screen of three zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs both confirmed reported data and provided novel insights into endochondral ossification. Parallel comparisons of the microdissected tibiae data set with our previously completed micromass culture screen further corroborated the suitability of micromass cultures for modeling gene expression in chondrocyte development. The micromass culture system demonstrated striking similarities to the in vivo microdissected tibiae screen; however, the micromass system was unable to accurately distinguish gene expression differences in the hypertrophic and mineralized zones of the tibia. Conclusions/Significance These studies allow us to better understand gene expression patterns in the growth plate and endochondral bones and provide an important technical resource for comparison of gene expression in diseased or experimentally-manipulated cartilages. Ultimately, this work will help to define the genomic context in which genes are expressed in long bones and to understand physiological and pathological ossification. PMID:20084171

  10. A catalogue of genes in mouse embryonal carcinoma F9 cells identified with expressed sequence tags.

    PubMed

    Nishiguchi, S; Sakuma, R; Nomura, M; Zou, Z; Jearanaisilavong, J; Joh, T; Yasunaga, T; Shimada, K

    1996-04-01

    We used expressed sequence tags (ESTs) to identify genes expressed in mouse embryonal carcinoma F9 cells and prepared 2132 ESTs from undifferentiated F9 cDNA libraries: 1026 were prepared after randomly selecting clones from one of the libraries and the remaining 1106 ESTs were prepared after classifying 2896 clones of the libraries into four classes, according to the levels and patterns of expression. Among the former 1026 ESTs, 797 (78%) matched known genes, 61 (6%) matched database sequences of uncharacterized cDNAs, and 168 (16%) represented novel genes. The ESTs matching known genes were catalogued according to putative structural and cellular functions. As many as 53% were related to transcription and translation, and 19% were related to energy metabolism, including transcripts of mitochondrial DNA. These percentages were significantly higher in F9 cells than in the human heart and brain, and a human liver cell line, HepG2. We found that approximately 7% of the ESTs corresponding to low-abundance mRNAs are either related to retinoic acid-regulated genes or mammalian development- and/or differentiation-related genes. Cataloguing of the genes expressed in the F9 cells paves the way for isolating genes involved in early mammalian development. PMID:8743579

  11. Deafness Gene Expression Patterns in the Mouse Cochlea Found by Microarray Analysis

    PubMed Central

    Yoshimura, Hidekane; Takumi, Yutaka; Nishio, Shin-ya; Suzuki, Nobuyoshi; Iwasa, Yoh-ichiro; Usami, Shin-ichi

    2014-01-01

    Background Tonotopy is one of the most fundamental principles of auditory function. While gradients in various morphological and physiological characteristics of the cochlea have been reported, little information is available on gradient patterns of gene expression. In addition, the audiograms in autosomal dominant non syndromic hearing loss can be distinctive, however, the mechanism that accounts for that has not been clarified. We thought that it is possible that tonotopic gradients of gene expression within the cochlea account for the distinct audiograms. Methodology/Principal Findings We compared expression profiles of genes in the cochlea between the apical, middle, and basal turns of the mouse cochlea by microarray technology and quantitative RT-PCR. Of 24,547 genes, 783 annotated genes expressed more than 2-fold. The most remarkable finding was a gradient of gene expression changes in four genes (Pou4f3, Slc17a8, Tmc1, and Crym) whose mutations cause autosomal dominant deafness. Expression of these genes was greater in the apex than in the base. Interestingly, expression of the Emilin-2 and Tectb genes, which may have crucial roles in the cochlea, was also greater in the apex than in the base. Conclusions/Significance This study provides baseline data of gradient gene expression in the cochlea. Especially for genes whose mutations cause autosomal dominant non syndromic hearing loss (Pou4f3, Slc17a8, Tmc1, and Crym) as well as genes important for cochlear function (Emilin-2 and Tectb), gradual expression changes may help to explain the various pathological conditions. PMID:24676347

  12. Analysis of tumor suppressor gene on human chromosome 9 in mouse x human somatic cell hybrids

    SciTech Connect

    Porterfield, B.W.; Olopade, O.I.; Rowley, J.D.; Diaz, M.O.

    1994-09-01

    Deletions of the short arm of human chromosome 9 (9p) are common in human leukemia and solid tumors. The minimum region of overlap of these deletions, located between the interferon genes and the methylthioadenosine phosphorylase gene, is partially synthenic with a region of mouse chromosome 4 that has tumor suppressor activity. Somatic cell hybrids between tumorigenic, MTAP-deficient, mouse L cells, and MTAP-competent human cells containing either a normal copy of 9p or a 9p with a deletion involving band 9p21 were selected in culture conditions that require MTAP activity for continued growth. Somatic cell hybrids that contained a normal copy of 9p rarely formed tumors in nude mice. Cells from the rare tumors that grew had lost the normal 9p. Hybrid cells that contained a 9p with deletions formed tumors more frequently, and cells from these tumors retained the 9p deletion chromosome. These results provide evidence that a tumor suppressor gene (or genes) is located on human chromosome 9 within the region of deletion.

  13. Survival benefit and phenotypic improvement by hamartin gene therapy in a tuberous sclerosis mouse brain model.

    PubMed

    Prabhakar, Shilpa; Zhang, Xuan; Goto, June; Han, Sangyeul; Lai, Charles; Bronson, Roderick; Sena-Esteves, Miguel; Ramesh, Vijaya; Stemmer-Rachamimov, Anat; Kwiatkowski, David J; Breakefield, Xandra O

    2015-10-01

    We examined the potential benefit of gene therapy in a mouse model of tuberous sclerosis complex (TSC) in which there is embryonic loss of Tsc1 (hamartin) in brain neurons. An adeno-associated virus (AAV) vector (serotype rh8) expressing a tagged form of hamartin was injected into the cerebral ventricles of newborn pups with the genotype Tsc1(cc) (homozygous for a conditional floxed Tsc1 allele) SynI-cre(+), in which Tsc1 is lost selectively in neurons starting at embryonic day 12. Vector-treated Tsc1(cc)SynIcre(+) mice showed a marked improvement in survival from a mean of 22 days in non-injected mice to 52 days in AAV hamartin vector-injected mice, with improved weight gain and motor behavior in the latter. Pathologic studies showed normalization of neuron size and a decrease in markers of mTOR activation in treated as compared to untreated mutant littermates. Hence, we show that gene replacement in the brain is an effective therapeutic approach in this mouse model of TSC1. Our strategy for gene therapy has the advantages that therapy can be achieved from a single application, as compared to repeated treatment with drugs, and that AAV vectors have been found to have minimal to no toxicity in clinical trials for other neurologic conditions. Although there are many additional issues to be addressed, our studies support gene therapy as a useful approach in TSC patients. PMID:26019056

  14. Enhanced transcriptome maps from multiple mouse tissues reveal evolutionary constraint in gene expression.

    PubMed

    Pervouchine, Dmitri D; Djebali, Sarah; Breschi, Alessandra; Davis, Carrie A; Barja, Pablo Prieto; Dobin, Alex; Tanzer, Andrea; Lagarde, Julien; Zaleski, Chris; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Wang, Huaien; Bussotti, Giovanni; Pei, Baikang; Balasubramanian, Suganthi; Monlong, Jean; Harmanci, Arif; Gerstein, Mark; Beer, Michael A; Notredame, Cedric; Guig, Roderic; Gingeras, Thomas R

    2015-01-01

    Mice have been a long-standing model for human biology and disease. Here we characterize, by RNA sequencing, the transcriptional profiles of a large and heterogeneous collection of mouse tissues, augmenting the mouse transcriptome with thousands of novel transcript candidates. Comparison with transcriptome profiles in human cell lines reveals substantial conservation of transcriptional programmes, and uncovers a distinct class of genes with levels of expression that have been constrained early in vertebrate evolution. This core set of genes captures a substantial fraction of the transcriptional output of mammalian cells, and participates in basic functional and structural housekeeping processes common to all cell types. Perturbation of these constrained genes is associated with significant phenotypes including embryonic lethality and cancer. Evolutionary constraint in gene expression levels is not reflected in the conservation of the genomic sequences, but is associated with conserved epigenetic marking, as well as with characteristic post-transcriptional regulatory programme, in which sub-cellular localization and alternative splicing play comparatively large roles. PMID:25582907

  15. Enhanced transcriptome maps from multiple mouse tissues reveal evolutionary constraint in gene expression

    PubMed Central

    Pervouchine, Dmitri D.; Djebali, Sarah; Breschi, Alessandra; Davis, Carrie A.; Barja, Pablo Prieto; Dobin, Alex; Tanzer, Andrea; Lagarde, Julien; Zaleski, Chris; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Wang, Huaien; Bussotti, Giovanni; Pei, Baikang; Balasubramanian, Suganthi; Monlong, Jean; Harmanci, Arif; Gerstein, Mark; Beer, Michael A.; Notredame, Cedric; Guig, Roderic; Gingeras, Thomas R.

    2015-01-01

    Mice have been a long-standing model for human biology and disease. Here we characterize, by RNA sequencing, the transcriptional profiles of a large and heterogeneous collection of mouse tissues, augmenting the mouse transcriptome with thousands of novel transcript candidates. Comparison with transcriptome profiles in human cell lines reveals substantial conservation of transcriptional programmes, and uncovers a distinct class of genes with levels of expression that have been constrained early in vertebrate evolution. This core set of genes captures a substantial fraction of the transcriptional output of mammalian cells, and participates in basic functional and structural housekeeping processes common to all cell types. Perturbation of these constrained genes is associated with significant phenotypes including embryonic lethality and cancer. Evolutionary constraint in gene expression levels is not reflected in the conservation of the genomic sequences, but is associated with conserved epigenetic marking, as well as with characteristic post-transcriptional regulatory programme, in which sub-cellular localization and alternative splicing play comparatively large roles. PMID:25582907

  16. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.

    PubMed

    Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. PMID:26006729

  17. Restricted development of mouse triploid fetuses with disorganized expression of imprinted genes.

    PubMed

    Yamazaki, Wataru; Takahashi, Masashi; Kawahara, Manabu

    2015-12-01

    Eukaryotic species commonly contain a diploid complement of chromosomes. The diploid state appears to be advantageous for mammals because it enables sexual reproduction and facilitates genetic recombination. Nonetheless, the effects of DNA ploidy on mammalian ontogeny have yet to be understood. The present study shows phenotypic features and expression patterns of imprinted genes in tripronucleate diandric and digynic triploid (DAT and DGT) mouse fetuses on embryonic day 10.5 (E10.5). Measurement of crown-rump length revealed that the length of DGT fetuses (1.87 0.13 mm; mean standard error of the mean) was much smaller than that of diploid fetuses (4.81 0.05 mm). However, no significant difference was observed in the crown-rump length between diploid and DAT fetuses (3.86 0.43 mm). In DGT fetuses, the expression level of paternally expressed genes, Igf2, Dlk1, Ndn, and Peg3, remained significantly reduced and that of maternally expressed genes, Igf2r and Grb10, increased. Additionally, in DAT fetuses, the Igf2 mRNA expression level was approximately twice that in diploid fetuses, as expected. These results provide the first demonstration that imprinted genes in mouse triploid fetuses show distinctive expression patterns independent of the number of parental-origin haploid sets. These data suggest that both DNA ploidy and asymmetrical functions of parental genomes separately influence mammalian ontogeny. PMID:25318586

  18. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1994-05-01

    From 1971--1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF{sub 1} mice irradiated with {sup 60}Co {gamma}-rays or JANUS fission-spectrum neutrons. Polymerase chain reaction (PCR) technique was used to detect deletions in the mouse retinoblastoma (mRb) gene. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. Absence of any of these fragments on a Southern blot indicated a deletion of that portion of the mRb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice were analyzed for mRb deletions. In all normal mouse tissues studies all six mRb exon fragments were present on Southern blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, 1 of 6 tumors from {gamma}-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice showed a deletion in one or both mRb alleles. All deletions detected were in the 5{prime} region of the mRb gene.

  19. EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos.

    PubMed

    Auclair, Ghislain; Borgel, Julie; Sanz, Lionel A; Vallet, Judith; Guibert, Sylvain; Dumas, Michael; Cavelier, Patricia; Girardot, Michael; Forn, Thierry; Feil, Robert; Weber, Michael

    2016-02-01

    The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2(-/-) embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development. PMID:26576615

  20. Inactivation of the retinoblastoma gene yields a mouse model of malignant colorectal cancer.

    PubMed

    Parisi, T; Bronson, R T; Lees, J A

    2015-11-26

    The retinoblastoma gene (Rb) is mutated at significant frequency in various human epithelial tumors, including colorectal cancer, and is strongly associated with metastatic disease. However, sole inactivation of Rb in the mouse has so far failed to yield epithelial cancers. Here, we specifically inactivate Rb and/or p53 in the urogenital epithelium and the intestine. We find that the loss of both tumor suppressors is unable to yield tumors in the transitional epithelium lining the bladder, kidneys and ureters. Instead, these mice develop highly metastatic tumors of neuroendocrine, not epithelial, origin within the urogenital tract to give prostate cancer in the males and vaginal tumors in the females. Additionally, we discovered that the sole inactivation of Rb in the intestine was sufficient to induce formation of metastatic colorectal adenocarcinomas. These tumors closely mirror the human disease in regard to the age of onset, histological appearance, invasiveness and metastatic potential. Like most human colorectal carcinomas, our murine Rb-deficient tumors demonstrate genomic instability and they show activation of β-catenin. Deregulation of the Wnt/β-catenin pathway is specific to the intestinal tumors, as genomic instability but not activation of β-catenin was observed in the neuroendocrine tumors. To date, attempts to generate genetically engineered mouse models of colorectal cancer tumors have yielded mostly cancer of the small intestine, which rarely occurs in humans. Our system provides the opportunity to accurately model and study colorectal cancer in the mouse via a single gene mutation. PMID:25745996

  1. NFAT5 regulates transcription of the mouse telomerase reverse transcriptase gene

    SciTech Connect

    Fujiki, Tsukasa; Udono, Miyako; Kotake, Yojiro; Yamashita, Makiko; Shirahata, Sanetaka; Katakura, Yoshinori

    2010-12-10

    We aimed to clarify the transcription-regulation mechanisms of the mouse telomerase reverse transcriptase gene (mTERT). First, we searched for the promoter region required for transcriptional activation of mTERT and identified an enhancer cis-element (named mTERT-EE) located between - 200 and - 179 bp of the mouse TERT gene (mTERT). EMSA results suggested that nuclear factor of activated T cells (NFAT) member proteins bind to mTERT-EE. We then identified NFAT5 as the factor binding to mTERT-EE and found that it activates the transcription of the mTERT core promoter. The results that siRNA directed against NFAT5 significantly reduced mTERT expression and mTERT core promoter activity and that the expressions of NFAT5 and mTERT were well correlated in various mouse tissues except liver suggest that NFAT5 dominantly and directly regulates mTERT expression. To clarify their functionality further, we investigated the effect of hypertonic stress, a known stimulus affecting the expression and transcriptional activity of NFAT5, on mTERT expression. The result indicated that hypertonic stress activates mTERT transcription via the activation and recruitment of NFAT5 to the mTERT promoter. These results provide useful information about the transcription-regulation mechanisms of mTERT.

  2. Using mouse models of autism spectrum disorders to study the neurotoxicology of gene-environment interactions

    PubMed Central

    Schwartzer, Jared J.; Koenig, Claire M.; Berman, Robert F

    2012-01-01

    To better study the role of genetics in autism, mouse models have been developed which mimic the genetics of specific autism spectrum and related disorders. These models have facilitated research on the role genetic susceptibility factors in the pathogenesis of autism in the absence of environmental factors. Inbred mouse strains have been similarly studied to assess the role of environmental agents on neurodevelopment, typically without the complications of genetic heterogeneity of the human population. What has not been as actively pursued, however, is the methodical study of the interaction between these factors (e.g., gene and environmental interactions in neurodevelopment). This review suggests that a genetic predisposition paired with exposure to environmental toxicants play an important role in the etiology of neurodevelopmental disorders including autism, and may contribute to the largely unexplained rise in the number of children diagnosed with autism worldwide. Specifically, descriptions of the major mouse models of autism and toxic mechanisms of prevalent environmental chemicals are provided followed by a discussion of current and future research strategies to evaluate the role of gene and environment interactions in neurodevelopmental disorders. PMID:23010509

  3. Transcriptional Regulation of Mouse PXR Gene: An Interplay of Transregulatory Factors

    PubMed Central

    Kumari, Sangeeta; Mukhopadhyay, Gauranga; Tyagi, Rakesh K.

    2012-01-01

    Pregnane X Receptor (PXR) is an important ligand-activated nuclear receptor functioning as a master regulator of expression of phase I, phase II drug metabolizing enzymes, and members of the drug transporters. PXR is primarily expressed in hepatic tissues and to lesser extent in other non-hepatic tissues both in human and in mice. Although its expression profile is well studied but little is known about the regulatory mechanisms that govern PXR gene expression in these cells. In the present study, we have cloned and characterized over 5 kb (?4963 to +54) region lying upstream of mouse PXR transcription start site. Promoter-reporter assays revealed that the proximal promoter region of up to 1 kb is sufficient to support the expression of PXR in the mouse liver cell lines. It was evident that the 500 bp proximal promoter region contains active binding sites for Ets, Tcf, Ikarose and nuclear factor families of transcription factors. Electrophoretic mobility shift assays demonstrated that the minimal region of 134 bp PXR promoter was able to bind Ets-1 and ?-catenin proteins. This result was further confirmed by chromatin immunoprecipitation analysis. In summary, the present study identified a promoter region of mouse PXR gene and the transregulatory factors responsible for PXR promoter activity. The results presented herein are expected to provide important cues to gain further insight into the regulatory mechanisms of PXR function. PMID:22952895

  4. A short upstream promoter region mediates transcriptional regulation of the mouse doublecortin gene in differentiating neurons

    PubMed Central

    2010-01-01

    Background Doublecortin (Dcx), a MAP (Microtubule-Associated Protein), is transiently expressed in migrating and differentiating neurons and thereby characterizes neuronal precursors and neurogenesis in developing and adult neurogenesis. In addition, reduced Dcx expression during development has been related to appearance of brain pathologies. Here, we attempt to unveil the molecular mechanisms controlling Dcx gene expression by studying its transcriptional regulation during neuronal differentiation. Results To determine and analyze important regulatory sequences of the Dcx promoter, we studied a putative regulatory region upstream from the mouse Dcx coding region (pdcx2kb) and several deletions thereof. These different fragments were used in vitro and in vivo to drive reporter gene expression. We demonstrated, using transient expression experiments, that pdcx2kb is sufficient to control specific reporter gene expression in cerebellar cells and in the developing brain (E14.5). We determined the temporal profile of Dcx promoter activity during neuronal differentiation of mouse embryonic stem cells (mESC) and found that transcriptional activation of the Dcx gene varies along with neuronal differentiation of mESC. Deletion experiments and sequence comparison of Dcx promoters across rodents, human and chicken revealed the importance of a highly conserved sequence in the proximal region of the promoter required for specific and strong expression in neuronal precursors and young neuronal cells. Further analyses revealed the presence in this short sequence of several conserved, putative transcription factor binding sites: LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor) which are effectors of the canonical Wnt pathway; HNF6/OC2 (Hepatocyte Nuclear Factor-6/Oncecut-2) members of the ONECUT family and NF-Y/CAAT (Nuclear Factor-Y). Conclusions Studies of Dcx gene regulatory sequences using native, deleted and mutated constructs suggest that fragments located upstream of the Dcx coding sequence are sufficient to induce specific Dcx expression in vitro: in heterogeneous differentiated neurons from mESC, in primary mouse cerebellar neurons (PND3) and in organotypic slice cultures. Furthermore, a region in the 3'-end region of the Dcx promoter is highly conserved across several species and exerts positive control on Dcx transcriptional activation. Together, these results indicate that the proximal 3'-end region of the mouse Dcx regulatory sequence is essential for Dcx gene expression during differentiation of neuronal precursors. PMID:20509865

  5. Activation of an imprinted Igf 2 gene in mouse somatic cell cultures.

    PubMed Central

    Eversole-Cire, P; Ferguson-Smith, A C; Sasaki, H; Brown, K D; Cattanach, B M; Gonzales, F A; Surani, M A; Jones, P A

    1993-01-01

    The mouse insulin-like growth factor II gene (Igf 2), located on distal chromosome 7, is parentally imprinted such that the paternal allele is expressed while the maternal allele is transcriptionally silent. We derived a cell line from a mouse embryo maternally disomic and paternally deficient for distal chromosome 7 (MatDi7) to determine the stability of gene repression in culture. MatDi7 cells maintained Igf2 in a repressed state even after immortalization, except for one randomly picked clone which spontaneously expressed the gene. Igf 2 was expressed in a cell culture derived from a normal littermate; this expression was growth regulated, with Igf 2 mRNA levels increasing in the stationary phase of growth. Analysis of the methylation status of 28 sites distributed over 10 kb of the gene did not show consistent differences associated with expression level in the normal and MatDi7 cell lines, and the CpG island in the Igf 2 promoter remained unmethylated in all of the cell lines. Only with an oncogenically transformed cell line did the promoter become extensively methylated. We attempted to derepress the imprinted gene in MatDi7 cells by treatments known to alter gene expression. Expression of the Igf 2 allele in MatDi7 cells was increased in a dose-dependent manner by treatment with 5-aza-2'-deoxycytidine or bromodeoxyuridine, agents known to change DNA methylation patterns or chromatin conformation. Treatment of the cells with 1-beta-D-arabinofuranosylcytosine, 2'-deoxycytidine, calcium ionophore, heat shock, cold shock, or sodium butyrate did not result in increases in the levels of Igf 2 expression. It seems likely that the mechanism of the Igf 2 imprint involves subtle changes in the methylation or chromatin conformation of the gene which are affected by 5-aza-2'-deoxycytidine and bromodeoxyuridine. Images PMID:8336727

  6. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

    PubMed Central

    Pasumarthi, Ravi K.; Baridon, Brian; Djan, Esi; Trainor, Amanda; Griffey, Stephen M.; Engelhard, Eric K.; Rapp, Jared; Li, Bowen; de Jong, Pieter J.; Lloyd, K.C. Kent

    2015-01-01

    Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼80% of mutants showed specific staining in one or more tissues, while ∼20% showed no specific staining, ∼13% had staining in only one tissue, and ∼25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known. PMID:25591789

  7. The Role of Metallothionein in Oxidative Stress

    PubMed Central

    Ruttkay-Nedecky, Branislav; Nejdl, Lukas; Gumulec, Jaromir; Zitka, Ondrej; Masarik, Michal; Eckschlager, Tomas; Stiborova, Marie; Adam, Vojtech; Kizek, Rene

    2013-01-01

    Free radicals are chemical particles containing one or more unpaired electrons, which may be part of the molecule. They cause the molecule to become highly reactive. The free radicals are also known to play a dual role in biological systems, as they can be either beneficial or harmful for living systems. It is clear that there are numerous mechanisms participating on the protection of a cell against free radicals. In this review, our attention is paid to metallothioneins (MTs) as small, cysteine-rich and heavy metal-binding proteins, which participate in an array of protective stress responses. The mechanism of the reaction of metallothioneins with oxidants and electrophilic compounds is discussed. Numerous reports indicate that MT protects cells from exposure to oxidants and electrophiles, which react readily with sulfhydryl groups. Moreover, MT plays a key role in regulation of zinc levels and distribution in the intracellular space. The connections between zinc, MT and cancer are highlighted. PMID:23502468

  8. Cardiac gene expression data and in silico analysis provide novel insights into human and mouse taste receptor gene regulation.

    PubMed

    Foster, Simon R; Porrello, Enzo R; Stefani, Maurizio; Smith, Nicola J; Molenaar, Peter; dos Remedios, Cristobal G; Thomas, Walter G; Ramialison, Mirana

    2015-10-01

    G protein-coupled receptors are the principal mediators of the sweet, umami, bitter, and fat taste qualities in mammals. Intriguingly, the taste receptors are also expressed outside of the oral cavity, including in the gut, airways, brain, and heart, where they have additional functions and contribute to disease. However, there is little known about the mechanisms governing the transcriptional regulation of taste receptor genes. Following our recent delineation of taste receptors in the heart, we investigated the genomic loci encoding for taste receptors to gain insight into the regulatory mechanisms that drive their expression in the heart. Gene expression analyses of healthy and diseased human and mouse hearts showed coordinated expression for a subset of chromosomally clustered taste receptors. This chromosomal clustering mirrored the cardiac expression profile, suggesting that a common gene regulatory block may control the taste receptor locus. We identified unique domains with strong regulatory potential in the vicinity of taste receptor genes. We also performed de novo motif enrichment in the proximal promoter regions and found several overrepresented DNA motifs in cardiac taste receptor gene promoters corresponding to ubiquitous and cardiac-specific transcription factor binding sites. Thus, combining cardiac gene expression data with bioinformatic analyses, this study has provided insights into the noncoding regulatory landscape for taste GPCRs. These findings also have broader relevance for the study of taste GPCRs outside of the classical gustatory system, where understanding the mechanisms controlling the expression of these receptors may have implications for future therapeutic development. PMID:25986534

  9. Mapping of the gene for high-density lipoprotein binding protein (Hdlbp) to proximal mouse Chromosome 1

    SciTech Connect

    LeBoeuf, R.C.; Oram, J.F.; Xia, Y.R.; Lusis, A.J.

    1994-09-01

    HDL binding protein (HBP) is a 150-kDa glycoprotein that is processed to smaller forms (105-110 kDa) that bind HDL. In vitro studies have shown that HBP protein mass and mRNA levels increase in cells overloaded with cholesterol, suggesting that this protein plays a role in HDL-mediated removal of cholesterol from cells, a process that may underlie the anti-atherogenic effects of HDL. The cDNA for human HBP has been identified and the gene (HDLBP) localized to chromosome 2q37. To test whether the HDL binding protein gene underlies any mutations in the mouse or whether it contributes to multigenic variations contributing to lipoprotein metabolism between inbred mouse strains, we report the chromosomal mapping in mouse for the mouse HBP gene (Hdlbp).

  10. The human and mouse receptors of hyaluronan-mediated motility, RHAMM, genes (HMMR) map to human chromosome 5q33.2-qter and mouse chromosome 11

    SciTech Connect

    Spicer, A.P.; McDonald, J.A.; Roller, M.L.; Camper, S.A.

    1995-11-01

    The gene for the receptor for hyaluronan-mediated motility, RHAAM (designated hyaluronan-mediated motility receptor, HMMR (human) and Hmmr (mouse), for mapping purposes), was localized to human chromosome 5q33.2-qter by somatic cell and radiation hybrid analyses. Investigation of two interspecific back-crosses localized the mouse RHAMM (Hmmr) locus 18 cM from the centromere of mouse chromosome 11 within a region of synteny homology with human chromosome 5q23-q35 genes. The map position of the human RHAMM gene places it in a region comparatively rich in disease-associated genes, including those for low-frequency hearing loss, dominant limb-girdle muscular dystrophy, diastrophic dysplasia, Treacher Collins syndrome, and myeloid disorders associated with the 5q-syndrome. The RHAMM gene location and its ability to transform cells when overexpressed implicate RHAMM as a possible candidate gene in the pathogenesis of the recently described t(5;14)(q33-q34;q11) acute lymphoblastic leukemias. 18 refs., 1 fig.

  11. cDNA cloning and chromosomal mapping of the mouse type VII collagen gene (Col7a1): Evidence for rapid evolutionary divergence of the gene

    SciTech Connect

    Li, Kehua; Christiano, A.M.; Chu, Mon Li; Uitto, J. Thomas Jefferson Univ., Philadelphia, PA ); Copeland, N.G.; Gilbert, D.J. )

    1993-06-01

    Type VII collagen is the major component of anchoring fibrils, critical attachment structures at the dermal-epidermal basement membrane zone. Genetic linkage analyses with recently cloned human type VII collagen cDNAs have indicated that the corresponding gene, COL7A1, is the candidate gene in the dystrophic forms of epidermolysis bullosa. To gain insight into the evolutionary conservation of COL7A1, in this study the authors have isolated mouse type VII collagen cDNAs by screening a mouse epidermal keratinocyte cDNA library with a human COL7A1 cDNA. Two overlapping mouse cDNAs were isolated, and Northern hybridization of mouse epidermal keratinocyte RNA with one of them revealed the presence of a mRNA transcript of [approximately]9.5 kb, the approximate size of the human COL7A1 mRNA. Nucleotide sequencing of the mouse cDNAs revealed a 2760-bp open reading frame that encodes the 5[prime] half of the collagenous domain and a segment of the NC-1, the noncollagenous amino-terminal domain of type VII collagen. Comparison of the mouse amino acid sequences with the corresponding human sequences deduced from cDNAs revealed 82.5% identity. The evolutionary divergence of the gene was relatively rapid in comparison to other collagen genes. Despite the high degree of sequence variation, several sequences, including the size and the position of noncollagenous imperfections and interruptions within the Gly-X-Y repeat sequence, were precisely conserved. Finally, the mouse Col7a1 gene was located by interspecific backcross mapping to mouse Chromosome 9, a region that corresponds to human chromosome 3p21, the position of human COL7Al. This assignment confirms and extends the relationship between the mouse and the human chromosomes in this region of the genome. 33 refs., 5 figs., 1 tab.

  12. Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis.

    PubMed

    Gavaldà-Navarro, Aleix; Domingo, Pere; Viñas, Octavi; Mampel, Teresa

    2015-07-01

    Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT. PMID:25817039

  13. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1993-04-01

    From 1971 to 1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF{sub 1} mice irradiated with {sup 60}Co {gamma} rays or JANUS fission-spectrum neutrons; normal and tumor tissues from mice in these studies were preserved in paraffin blocks. A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene in the paraffin-embedded tissues. Microtomed sections were used as the DNA source in PCR reaction mixtures. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments (relative to control PCR products) on a Southern blot indicated a deletion of that portion of the mRb gene. The tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice (569 cGy of {sup 60}Co {gamma} rays or 60 cGy of JANUS neutrons, doses that have been found to have approximately equal biological effectiveness in the BCF, mouse) were analyzed for mRb deletions. In all normal mouse tissues studies, all six mRb exon fragments were present on Southem blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, I of 6 tumors from {gamma}-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice had a deletion in one or both mRb alleles. All deletions detected were in the 5{prime} region of the mRb gene.

  14. PCR detection of retinoblastoma gene deletions in radiation-induced mouse lung adenocarcinomas

    SciTech Connect

    Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

    1993-01-01

    From 1971 to 1986, Argonne National Laboratory conducted a series of large-scale studies of tumor incidence in 40,000 BCF[sub 1] mice irradiated with [sup 60]Co [gamma] rays or JANUS fission-spectrum neutrons; normal and tumor tissues from mice in these studies were preserved in paraffin blocks. A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma (mRb) gene in the paraffin-embedded tissues. Microtomed sections were used as the DNA source in PCR reaction mixtures. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments (relative to control PCR products) on a Southern blot indicated a deletion of that portion of the mRb gene. The tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death in post-mortem analyses. Spontaneous tumors as well as those from irradiated mice (569 cGy of [sup 60]Co [gamma] rays or 60 cGy of JANUS neutrons, doses that have been found to have approximately equal biological effectiveness in the BCF, mouse) were analyzed for mRb deletions. In all normal mouse tissues studies, all six mRb exon fragments were present on Southem blots. Tumors in six neutron-irradiated mice also had no mRb deletions. However, I of 6 tumors from [gamma]-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice had a deletion in one or both mRb alleles. All deletions detected were in the 5[prime] region of the mRb gene.

  15. Construction and identification of an RNA interference lentiviral vector targeting the mouse TNF-? gene

    PubMed Central

    WANG, JIBO; LIANG, HONGDA; ZHAO, YINGJIE; LIU, XIANGPING; YANG, KUN; SUI, AIHUA

    2015-01-01

    The aim of this study was to construct RNA interference (RNAi) lentiviral vector particles targeting the mouse tumor necrosis factor-? (TNF-?) gene. Three types of small interfering RNA (siRNA) targeting the mouse TNF-? gene were designed, synthesized and transfected into RAW264.7 cells. Screening was performed to identify the siRNA sequence exhibiting the highest inhibition efficiency; based on this, recombinant lentiviral plasmids were constructed and co-transfected into 293T cells with packaging plasmids for the production of lentiviral particles. The screening results showed that the TNF-? mRNA expression levels of the three siRNA groups were significantly lower than those of the negative control group, with the highest inhibition rate in the siRNA2 group (83.09%). Similarly, the expression levels of TNF-? protein in the three siRNA groups were significantly lower than those of the negative control group, and the highest inhibition rate was found in the siRNA2 group (51.16%). The mRNA expression of interleukin (IL)-1? and IL-6 showed no significant difference among the siRNA groups and the negative control. The recombinant lentiviral shuttle plasmid was constructed, and electrophoresis revealed the polymerase chain reaction product to be 343 bp, while that of the empty vector was 306 bp; DNA sequencing showed partial insertion. The virus titer was calculated to be 2106 TU/l. In conclusion, RNAi lentiviral vector particles targeting the mouse TNF-? gene were successfully obtained in the present study. This method may be used to produce lentiviral vector for the in vivo study of RNAi gene therapy targeting TNF-?. PMID:26668629

  16. Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression

    PubMed Central

    Adkisson, Michael; Nava, A. J.; Kirov, Julia V.; Cipollone, Andreanna; Willis, Brandon; Rapp, Jared; de Jong, Pieter J.; Lloyd, Kent C.

    2016-01-01

    The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3’ UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels. PMID:26839965

  17. Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

    PubMed

    West, David B; Engelhard, Eric K; Adkisson, Michael; Nava, A J; Kirov, Julia V; Cipollone, Andreanna; Willis, Brandon; Rapp, Jared; de Jong, Pieter J; Lloyd, Kent C

    2016-02-01

    The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels. PMID:26839965

  18. Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells

    PubMed Central

    Lin, Zhuoheng; Feng, Xuyang; Jiang, Xue; Songyang, Zhou; Huang, Junjiu

    2015-01-01

    A recently developed strategy of sequencing alternative polyadenylation (APA) sites (SAPAS) with second-generation sequencing technology can be used to explore complete genome-wide patterns of tandem APA sites and global gene expression profiles. spermatogonial stem cells (SSCs) maintain long-term reproductive abilities in male mammals. The detailed mechanisms by which SSCs self-renew and generate mature spermatozoa are not clear. To understand the specific alternative polyadenylation pattern and global gene expression profile of male germline stem cells (GSCs, mainly referred to SSCs here), we isolated and purified mouse Thy1+ cells from testis by magnetic-activated cell sorting (MACS) and then used the SAPAS method for analysis, using pluripotent embryonic stem cells (ESCs) and differentiated mouse embryonic fibroblast cells (MEFs) as controls. As a result, we obtained 99,944 poly(A) sites, approximately 40% of which were newly detected in our experiments. These poly(A) sites originated from three mouse cell types and covered 17,499 genes, including 831 long non-coding RNA (lncRNA) genes. We observed that GSCs tend to have shorter 3'UTR lengths while MEFs tend towards longer 3'UTR lengths. We also identified 1337 genes that were highly expressed in GSCs, and these genes were highly consistent with the functional characteristics of GSCs. Our detailed bioinformatics analysis identified APA site-switching events at 3'UTRs and many new specifically expressed genes in GSCs, which we experimentally confirmed. Furthermore, qRT-PCR was performed to validate several events of the 334 genes with distal-to-proximal poly(A) switch in GSCs. Consistently APA reporter assay confirmed the total 3'UTR shortening in GSCs compared to MEFs. We also analyzed the cis elements around the proximal poly(A) site preferentially used in GSCs and found C-rich elements may contribute to this regulation. Overall, our results identified the expression level and polyadenylation site profiles and these data provide new insights into the processes potentially involved in the GSC life cycle and spermatogenesis. PMID:26713853

  19. In situ localization of mRNAs coding for mouse testicular structural genes

    SciTech Connect

    Hecht, N.B. ); Penshow, J.D. )

    1987-11-01

    In situ hybridization histochemistry has been used to localize mRNA transcripts of five nuclear and cytoplasmic structural genes in the mouse testis. The mRNAs for three nuclear structural proteins involved in chromatin transformation during spermatogenesis (the two protamine variants of the mouse and one of the testis-specific proteins) are restricted solely to postmeiotic germ cells. In contrast, mRNAs for two other structural proteins, actin and {alpha} tubulin, are detected throughout spermatogenesis. Although present in premeiotic, meiotic, and postmeiotic cell types, the mRNA levels of actin and {alpha} tubulin differ considerably during spermiogenesis, the haploid phase of spermatogenesis. Actin mRNA levels decrease markedly as the male gamete differentiates during spermiogenesis whereas {alpha}-tubulin mRNAs are equally abundant in the haploid round and elongating spermatids.

  20. Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line.

    PubMed Central

    Cavard, C; Grimber, G; Dubois, N; Chasse, J F; Bennoun, M; Minet-Thuriaux, M; Kamoun, P; Briand, P

    1988-01-01

    The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated. Images PMID:3162766

  1. Identification of an Antigen Associated with Transforming Genes of Human and Mouse Mammary Carcinomas

    NASA Astrophysics Data System (ADS)

    Becker, Dorothea; Lane, Mary-Ann; Cooper, Geoffrey M.

    1982-05-01

    Sera from tumor-bearing mice immunoprecipitated a 86,000-dalton glycoprotein from extracts of NIH cells transformed by human mammary carcinoma DNA. This antigen was not immunoprecipitated from extracts of NIH 3T3 cells, spontaneously transformed NIH cells, NIH cells transformed by normal human DNA, NIH cells transformed by human bladder carcinoma DNA, or NIH cells transformed by Rous sarcoma virus DNA. In addition, sera from mice bearing tumors induced by NIH cells transformed by either normal human DNA or human bladder carcinoma DNA did not immunoprecipitate this antigen from extracts of NIH cells transformed by human mammary carcinoma DNA. However, this antigen was immunoprecipitated by sera from mice bearing tumors induced by NIH cells transformed by mouse mammary carcinoma DNAs and from mice bearing primary mammary carcinomas. These results indicate that this glycoprotein represents an antigen that is specifically associated with expression of the transmissible transforming genes of human and mouse mammary carcinomas.

  2. Anti-metallothionein IgG and levels of metallothionein in autistic families.

    PubMed

    Russo, Anthony F

    2008-02-01

    Metallothioneins (MTs) are a family of small proteins containing 61-68 amino acids with an unusually high concentration of cysteine. MT-1, the most functional and active MT in humans, has the ability to react with and enhance the detoxification of a number of metals including zinc, mercury, copper and cadmium. MT dysfunction may result, then, in many of the aetiological syndromes observed in autistic children, such as the leaky gut. It has been proposed that allergic autoimmune reactions occurring after exposure to heavy metals, may contribute to some symptoms associated with autism. Therefore abnormalities in MT concentration and/or structure, as well as the presence of anti-MT antibodies, may be associated with autism. We used direct ELISAs to quantitate the concentration of serum anti-metallothionein IgG in 66 individuals (parents and children) from 14 families with autistic children, as well as 11 controls from families with no history of autism. We measured the concentration of serum metallothionein in 39 of the above family members from 8 families. Our results indicate that a significantly high number (23 of 66) of autistic family members had high levels of anti-metallothionein IgG, when compared to controls (1 ) and the production of these antibodies correlated with levels of metallothionein, suggesting that the production of these antibodies is inherited. However, the presence of these antibodies does not correlate with autism, types of autism, including regression, or demographics such as allergies, respiratory problems or GI disease. This suggests that the presence of anti-metallothionein antibodies is not causative to autism and may be the result of other immunological pathology seen in many autistics. PMID:18365350

  3. Abnormal social behaviors and altered gene expression rates in a mouse model for Potocki-Lupski syndrome.

    PubMed

    Molina, Jessica; Carmona-Mora, Paulina; Chrast, Jacqueline; Krall, Paola M; Canales, César P; Lupski, James R; Reymond, Alexandre; Walz, Katherina

    2008-08-15

    The Potocki-Lupski syndrome (PTLS) is associated with a microduplication of 17p11.2. Clinical features include multiple congenital and neurobehavioral abnormalities and autistic features. We have generated a PTLS mouse model, Dp(11)17/+, that recapitulates some of the physical and neurobehavioral phenotypes present in patients. Here, we investigated the social behavior and gene expression pattern of this mouse model in a pure C57BL/6-Tyr(c-Brd) genetic background. Dp(11)17/+ male mice displayed normal home-cage behavior but increased anxiety and increased dominant behavior in specific tests. A subtle impairment in the preference for a social target versus an inanimate target and abnormal preference for social novelty (the preference to explore an unfamiliar mouse versus a familiar one) was also observed. Our results indicate that these animals could provide a valuable model to identify the specific gene(s) that confer abnormal social behaviors and that map within this delimited genomic deletion interval. In a first attempt to identify candidate genes and for elucidating the mechanisms of regulation of these important phenotypes, we directly assessed the relative transcription of genes within and around this genomic interval. In this mouse model, we found that candidates genes include not only most of the duplicated genes, but also normal-copy genes that flank the engineered interval; both categories of genes showed altered expression levels in the hippocampus of Dp(11)17/+ mice. PMID:18469339

  4. Effect of Chronic Valproic Acid Treatment on Hepatic Gene Expression Profile in Wfs1 Knockout Mouse

    PubMed Central

    Sütt, Silva; Kõks, Sulev; Schalkwyk, Leonard C.; Fernandes, Catherine; Vasar, Eero

    2014-01-01

    Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300 mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0 ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype. PMID:24799886

  5. Effect of chronic valproic Acid treatment on hepatic gene expression profile in wfs1 knockout mouse.

    PubMed

    Punapart, Marite; Eltermaa, Mall; Oflijan, Julia; Sütt, Silva; Must, Anne; Kõks, Sulev; Schalkwyk, Leonard C; Fernandes, Catherine; Vasar, Eero; Soomets, Ursel; Terasmaa, Anton

    2014-01-01

    Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300 mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0 ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype. PMID:24799886

  6. High-resolution prediction of mouse brain connectivity using gene expression patterns.

    PubMed

    Fakhry, Ahmed; Ji, Shuiwang

    2015-02-01

    The brain is a multi-level system in which the high-level functions are generated by low-level genetic mechanisms. Thus, elucidating the relationship among multiple brain levels via correlative and predictive analytics is an important area in brain research. Currently, studies in multiple species have indicated that the spatiotemporal gene expression patterns are predictive of brain wiring. Specifically, results on the worm Caenorhabditis elegans have shown that the prediction of neuronal connectivity using gene expression signatures yielded statistically significant results. Recent studies on the mammalian brain produced similar results at the coarse regional level. In this study, we provide the first high-resolution, large-scale integrative analysis of the transcriptome and connectome in a single mammalian brain at a fine voxel level. By using the Allen Brain Atlas data, we predict voxel-level brain connectivity based on the gene expressions in the adult mouse brain. We employ regularized models to show that gene expression is predictive of connectivity at the voxel-level with an accuracy of 93%. We also identify a set of genes playing the most important role in connectivity prediction. We use only this small number of genes to predict the brain wiring with an accuracy over 80%. We discover that these important genes are enriched in neurons as compared to glia, and they perform connectivity-related functions. We perform several interesting correlative studies to further elucidate the transcriptome-connectome relationship. PMID:25109429

  7. Differential gene expression in mouse liver associated with the hepatoprotective effect of clofibrate

    SciTech Connect

    Moffit, Jeffrey S.; Koza-Taylor, Petra H.; Holland, Ricky D.; Thibodeau, Michael S.; Beger, Richard D.; Lawton, Michael P.; Manautou, Jose E. . E-mail: jose.manautou@uconn.edu

    2007-07-15

    Pretreatment of mice with the peroxisome proliferator clofibrate (CFB) protects against acetaminophen (APAP)-induced hepatotoxicity. Previous studies have shown that activation of the nuclear peroxisome proliferator activated receptor-alpha (PPAR{alpha}) is required for this effect. The present study utilizes gene expression profile analysis to identify potential pathways contributing to PPAR{alpha}-mediated hepatoprotection. Gene expression profiles were compared between wild type and PPAR{alpha}-null mice pretreated with vehicle or CFB (500 mg/kg, i.p., daily for 10 days) and then challenged with APAP (400 mg/kg, p.o.). Total hepatic RNA was isolated 4 h after APAP treatment and hybridized to Affymetrix Mouse Genome MGU74 v2.0 GeneChips. Gene expression analysis was performed utilizing GeneSpring (registered) software. Our analysis identified 53 genes of interest including vanin-1, cell cycle regulators, lipid-metabolizing enzymes, and aldehyde dehydrogenase 2, an acetaminophen binding protein. Vanin-1 could be important for CFB-mediated hepatoprotection because this protein is involved in the synthesis of cysteamine and cystamine. These are potent antioxidants capable of ameliorating APAP toxicity in rodents and humans. HPLC-ESI/MS/MS analysis of liver extracts indicates that enhanced vanin-1 gene expression results in elevated cystamine levels, which could be mechanistically associated with CFB-mediated hepatoprotection.

  8. Identification of genes escaping X inactivation by allelic expression analysis in a novel hybrid mouse model

    PubMed Central

    Berletch, Joel B.; Ma, Wenxiu; Yang, Fan; Shendure, Jay; Noble, William S.; Disteche, Christine M.; Deng, Xinxian

    2015-01-01

    X chromosome inactivation (XCI) is a female-specific mechanism that serves to balance gene dosage between the sexes whereby one X chromosome in females is inactivated during early development. Despite this silencing, a small portion of genes escape inactivation and remain expressed from the inactive X (Xi). Little is known about the distribution of escape from XCI in different tissues in vivo and about the mechanisms that control tissue-specific differences. Using a new binomial model in conjunction with a mouse model with identifiable alleles and skewed X inactivation we are able to survey genes that escape XCI in vivo. We show that escape from X inactivation can be a common feature of some genes, whereas others escape in a tissue specific manner. Furthermore, we characterize the chromatin environment of escape genes and show that expression from the Xi correlates with factors associated with open chromatin and that CTCF co-localizes with escape genes. Here, we provide a detailed description of the experimental design and data analysis pipeline we used to assay allele-specific expression and epigenetic characteristics of genes escaping X inactivation. The data is publicly available through the GEO database under ascension numbers GSM1014171, GSE44255, and GSE59779. Interpretation and discussion of these data are included in a previously published study (Berletch et al., 2015) [1]. PMID:26693509

  9. Expression of non-coding RNA AB063319 derived from Rian gene during mouse development.

    PubMed

    Gu, Tiantian; He, Hongjuan; Xing, Yanjiang; Liu, Qi; Gu, Ning; Kenkichi, Sugimoto; Jiang, Huijie; Wu, Qiong

    2011-04-01

    The regulatory functions of many non-coding RNAs (ncRNAs) were widely recognized. However, there are very few publications on long intronic ncRNAs. The transcriptional hierarchy driving a large amount of long and short ncRNAs originated from the maternal chromosome is not clarified in the Dlk1-Dio3 imprinted clusters of mouse distal chromosome 12. Here, we only focused on the previously identified long ncRNA AB063319 which derives from the large imprinted gene Rian and contains three retained introns of Rian, and tried to unsderstand this ncRNAs part of biological functions. We used in situ hybridization and quantitative real-time RT-PCR (QRT-PCR) to characterize the spatiotemporal expression pattern of AB063319 during mouse development. The in situ hybridization results showed that AB063319 was prominently expressed in the brain at embryonic day 10.5 (E10.5) and E11.5, and abundantly expressed in brain, muscle, liver, lung and neuroendocrine tissues at E15.5. Furthermore, quantitative analyses results showed that AB063319 was gradually up-regulated from E9.5 to E18.5 and down-regulated at E19.5 during the mouse embryonic development, and AB063319 was highly expressed in tongue and brain at E12.5, E15.5 and E18.5. Alternatively, AB063319 expression was also predominantly detected in tongue and brain at mouse postnatal day 6 (P6) by semi-quantitative RT-PCR. These results indicated that AB063319, as a stable transcriptional ncRNA, might play the important roles in the morphogenesis of diverse organs and tissues, especially associated with brain and muscle development at mouse embryonic and postnatal stages. PMID:21305344

  10. Identification of peroxisome-proliferator responsive element in the mouse HSL gene

    SciTech Connect

    Yajima, Hiroaki . E-mail: hyajima@kirin.co.jp; Kobayashi, Yumie; Kanaya, Tomoka; Horino, Yoko

    2007-01-12

    Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step of lipolysis in adipose tissue. Several studies suggest that protein phosphorylation regulates the HSL enzymatic activity. On the other hand, the precise mechanism of the transcriptional regulation of the HSL gene remains to be elucidated. Here, we identified a functional peroxisome-proliferator responsive element (PPRE) in the mouse HSL promoter by reporter assay in CV-1 cells using serial deletion and point mutants of the 5'-flanking region. Chromatin immunoprecipitation (ChIP) analysis revealed that both peroxisome-proliferator activated receptor (PPAR{gamma}) and retinoid X receptor (RXR{alpha}) interacted with the region. Binding of the PPAR{gamma}/RXR{alpha} heterodimer to the PPRE sequence was also confirmed by electrophoretic mobility shift assay. These results indicate that the HSL gene is transcriptionally regulated by PPAR{gamma}/RXR{alpha} heterodimer, and suggest that a cis-acting element regulates the HSL gene expression.

  11. Gene Expression Analysis of Mouse Embryonic Stem Cells Following Levitation in an Ultrasound Standing Wave Trap

    PubMed Central

    Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

    2011-01-01

    In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou etal. 2005a) at variable acoustic pressures (0.080.85 MPa) and times (560 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. (E-mail: Bazoud@tcd.ie) PMID:21208732

  12. Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

    PubMed Central

    Inquimbert, Perrine; Moll, Martin; Kohno, Tatsuro; Scholz, Joachim

    2013-01-01

    Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection. PMID:23542888

  13. Generation of polyclonal rabbit antisera to mouse melanoma associated antigens using gene gun immunization.

    PubMed

    Surman, D R; Irvine, K R; Shulman, E P; Allweis, T M; Rosenberg, S A; Restifo, N P

    1998-05-01

    Lymphocytes from patients with melanoma have been used to clone melanoma associated antigens which are, for the most part, nonmutated melanocyte tissue differentiation antigens. To establish a mouse model for the use of these 'self' antigens as targets for anti-tumor immune responses, we have employed the mouse homologues of the human melanoma antigens Tyrosinase, Tyrosinase Related Protein-1 (TRP-1), gp100, and MART-1. We sought to generate antisera against these proteins for use in the construction of experimental recombinant and synthetic anti-cancer vaccines, and for use in biologic studies. Using genes cloned from the B16 mouse melanoma or from murine melanocytes, we immunized rabbits with plasmid DNAs coated onto microscopic gold beads that were then delivered using a hand-held, helium-driven 'gene gun'. This strategy enabled us to generate polyclonal rabbit sera containing antibodies that specifically recognized each antigen, as measured by immunostaining of vaccinia virus infected cells. The sera that we generated specifically for TRP-1, gp100, and MART-1 recognized extracts of the spontaneous murine melanoma, B16. The identities of the recognized proteins was confirmed by Western blot analysis. The titers and specificities of these antisera were determined using ELISA. Interestingly, serum samples generated against murine MART-1 and gp100 developed antibodies that were cross-reactive with the corresponding human homologues. Recognition of human gp100 and murine Tyrosinase appeared to be dependent upon conformational epitopes since specificity was lost upon denaturation of the antigens. These antisera may be useful in the detection, purification and characterization of the mouse homologues of recently cloned human tumor associated antigens and may enable the establishment of an animal model of the immune consequences of vaccination against 'self antigens. PMID:9692858

  14. Identification of Novel SHOX Target Genes in the Developing Limb Using a Transgenic Mouse Model

    PubMed Central

    Beiser, Katja U.; Glaser, Anne; Kleinschmidt, Kerstin; Scholl, Isabell; Rth, Ralph; Li, Li; Gretz, Norbert; Mechtersheimer, Gunhild; Karperien, Marcel; Marchini, Antonio; Richter, Wiltrud; Rappold, Gudrun A.

    2014-01-01

    Deficiency of the human short stature homeobox-containing gene (SHOX) has been identified in several disorders characterized by reduced height and skeletal anomalies such as Turner syndrome, Lri-Weill dyschondrosteosis and Langer mesomelic dysplasia as well as isolated short stature. SHOX acts as a transcription factor during limb development and is expressed in chondrocytes of the growth plates. Although highly conserved in vertebrates, rodents lack a SHOX orthologue. This offers the unique opportunity to analyze the effects of human SHOX expression in transgenic mice. We have generated a mouse expressing the human SHOXa cDNA under the control of a murine Col2a1 promoter and enhancer (Tg(Col2a1-SHOX)). SHOX and marker gene expression as well as skeletal phenotypes were characterized in two transgenic lines. No significant skeletal anomalies were found in transgenic compared to wildtype mice. Quantitative and in situ hybridization analyses revealed that Tg(Col2a1-SHOX), however, affected extracellular matrix gene expression during early limb development, suggesting a role for SHOX in growth plate assembly and extracellular matrix composition during long bone development. For instance, we could show that the connective tissue growth factor gene Ctgf, a gene involved in chondrogenic and angiogenic differentiation, is transcriptionally regulated by SHOX in transgenic mice. This finding was confirmed in human NHDF and U2OS cells and chicken micromass culture, demonstrating the value of the SHOX-transgenic mouse for the characterization of SHOX-dependent genes and pathways in early limb development. PMID:24887312

  15. HER2/neu DNA vaccination by intradermal gene delivery in a mouse tumor model

    PubMed Central

    Nguyen-Hoai, Tam; Kobelt, Dennis; Hohn, Oliver; Vu, Minh D.; Schlag, Peter M.; Drken, Bernd; Norley, Steven; Lipp, Martin; Walther, Wolfgang; Pezzutto, Antonio; Westermann, Jrg

    2012-01-01

    DNA vaccines are potential tools for the induction of immune responses against both infectious disease and cancer. The dermal application of DNA vaccines is of particular interest since the epidermal and dermal layers of the skin are characterized by an abundance of antigen-presenting cells (APCs). The aim of our study was to compare tumor protection as obtained by two different methods of intradermal DNA delivery (gene gun and jet injector) in a well-established HER2/neu mouse tumor model. BALB/c mice were immunized twice with a HER2/neu-coding plasmid by gene gun or jet injector. Mice were then subcutaneously challenged with HER2/neu+ syngeneic D2F2/E2 tumor cells. Protection against subsequent challenges with tumor cells as well as humoral and T-cell immune responses induced by the vaccine were monitored. Gene gun immunization was far superior to jet injector both in terms of tumor protection and induction of HER2/neu-specific immune responses. After gene gun immunization, 60% of the mice remained tumor-free until day 140 as compared with 25% after jet injector immunization. Furthermore, gene gun vaccination was able to induce both a strong TH1-polarized T-cell response with detectable cytotoxic T-lymphocyte (CTL) activity and a humoral immune response against HER2/neu, whereas the jet injector was not. Although the disadvantages that were associated with the use of the jet injector in our model may be overcome with methodological modifications and/or in larger animals, which exhibit a thicker skin and/or subcutaneous muscle tissue, we conclude that gene gun delivery constitutes the method of choice for intradermal DNA delivery in preclinical mouse models and possibly also for the clinical development of DNA-based vaccines. PMID:23264900

  16. Vasopressin controls stanniocalcin-1 gene expression in rat and mouse kidney.

    PubMed

    Law, Alice Y; Wong, Chris K; Turner, Jeffery; Gonzalez, Alexis A; Prieto, Minolfa C; Wagner, Graham F

    2012-01-01

    Renal stanniocalcin-1 (STC-1) is made by collecting duct principal cells for autocrine and paracrine targeting of the distal nephron. While the underlying purpose of this targeting is poorly understood, increased targeting is tied to changes in extracellular fluid (ECF) balance. For example, water deprivation is a potent stimulator of renal STC-1 gene activity in both rats and mice. The effects are most evident in cortical kidney where transcript levels are increased as much as 8-fold, as compared to 2-fold in the papilla. As is now known, this gene upregulation occurs in response to the dual consequences of water deprivation; hypertonicity followed by hypovolemia. The cortical gene has proven to be uniquely responsive to hypertonicity and that in papilla to hypovolemia; the implication being that STC-1 has different roles in the two zones, both of which are somehow related to ECF balance. The role of arginine vasopressin (AVP) in maintaining ECF balance is well established. Moreover, hypertonicity and hypovolemia are, respectively, the primary and secondary stimulators of AVP release. Therefore the present study explored the hypothesis that AVP was responsible for inducing the STC-1 gene in one or both zones. The results showed that this was indeed the case. AVP had time and dose-dependent stimulatory effects on the gene in both rat and mouse cortical kidney. In the papilla, however, gene regulation was more complex, as AVP was inhibitory in rats but stimulatory in mice. Further studies on papilla revealed that angiotensin II (ANG II) was stimulatory in rats, but inhibitory in mice. Moreover, ANG II attenuated the stimulatory effects of AVP in mouse cortex and papilla. Receptor agonist studies revealed that the effects of AVP in both zones were mediated exclusively through the V2 receptor (V1a, V1b and oxytocin-specific agonists had no effect). The findings serve to further implicate STC-1 in the renal control of ECF balance. PMID:21867741

  17. Maternal gene transcription in mouse oocytes: genes implicated in oocyte maturation and fertilization.

    PubMed

    Cui, Xiang-Shun; Li, Xing-Yu; Yin, Xi-Jun; Kong, Il Keun; Kang, Jason-Jongho; Kim, Nam-Hyung

    2007-04-01

    Maternal gene expression is an important biological process in oocyte maturation and early cleavage. To gain insights into oocyte maturation and early embryo development, we used microarray analysis to compare the gene expression profiles of germinal vesicle (GV)- and metaphase II (MII)-stage oocytes. The differences in spot intensities were normalized and grouped using the Avadis Prophetic software platform. Of the 12164 genes examined, we found 1682 genes with more highly expression in GV-stage oocytes than in MII-stage oocytes, while 1936 genes were more highly expressed in MII-stage oocytes (P<0.05). The genes were grouped on the basis of the Panther classification system according to their involvement in particular biological processes. The genes that were up-regulated in GV oocytes were more likely to be involved in protein metabolism and modification, the mitotic cell cycle, electron transport, or fertilization or belong to the microtubule/cytoskeletal protein family. The genes specifically upregulated in the MII oocytes were more likely to be involved in DNA replication, amino acid metabolism, or expression of G protein-coupled receptors and signaling molecules. Identification of genes that are preferentially expressed at particular oocyte maturation stages provides insights into the complex gene regulatory networks that drive oocyte maturation and fertilization. PMID:17179655

  18. Gene organization and transcription of duplicated MBP genes of myelin deficient (shi(mld)) mutant mouse.

    PubMed Central

    Okano, H; Tamura, T; Miura, M; Aoyama, A; Ikenaka, K; Oshimura, M; Mikoshiba, K

    1988-01-01

    A hereditary dysmyelinating mutation, named myelin deficient (shi(mld)), is characterized by reduced expression of myelin basic protein (MBP). In shi(mld), the MBP gene is duplicated and its reduced expression is mainly determined by the level of mRNA. We have characterized the structure and function of the promoter regions of the duplicated MBP genes in shi(mld). Among the lambda clones containing promoter regions of the duplicated MBP genes in shi(mld), one (gene 1) had the same restriction enzyme pattern as that in control mice, but another (gene 2) had a rearrangement on a distal part of the promoter. A 712-bp nucleotide sequence upstream of the first exons of both of the duplicated MBP genes of shi(mld) was completely consistent with that of the control. Promoter activities of 1.3-kb 5'-flanking regions from respective genes of shi(mld) measured by in vitro run-off assay using HeLa whole-cell extracts were indistinguishable from that of the control MPB gene. Chromosomal mapping by in situ hybridization suggested that the duplicated MBP genes were located closely to each other at the distal part of chromosome 18. A recombinational event including the inversion seemed to have occurred within gene 1 and its possible relationship to the reduced expression of MBP is discussed. Images PMID:2452084

  19. Molecular characterization of J558 genes encoding tight-skin mouse autoantibodies: identical heavy-chain variable genes code for antibodies with different specificities.

    PubMed Central

    Kasturi, K N; Yio, X Y; Bona, C A

    1994-01-01

    Tight-skin mouse, a mutant strain with a single gene defect, develops cutaneous hyperplasia and specific autoantibodies, like humans affected by scleroderma. The autoantibodies produced in the tight-skin mouse are encoded primarily by heavy-chain variable (VH) genes from the J558 family. To understand the genetic basis of production of autoantibodies, we have analyzed the structure of J558 genes encoding these autoantibodies. The results showed that J558 genes encoding these antibodies were not derived from a selected germ-line gene(s) or a single subfamily but were derived from genes belonging to diverse J558 subfamilies. However, two prototype VH genes representing two new subfamilies were found to be repeatedly expressed in their germ-line form in eight independent clones. Autoantibodies with distinct specificities appear to be generated by pairing of similar/identical VH genes with different V kappa genes derived from the same or different families. Fourteen of 18 autoantibodies shared a conserved heptapeptide sequence motif, YNEKFKG, in the second complementarity-determining region of heavy chains. Usage of germ-line genes from diverse J558 subfamilies bearing a common motif to encode autoantibodies suggests a regulatory role for this motif. Thus, selection and expansion of the autoreactive B-cell repertoire in the tight-skin mouse appear to be VH-gene mediated. The frequency of N nucleotide addition at diversity-joining (D-JH) junctions was lower, whereas the frequency of usage of the DFL16 segment was higher. Finally, in contrast to normal and other autoimmune mouse strains, the frequencies of D-D fusions and D inversions were higher in tight-skin mouse total immunoglobulin as well as autoantibody repertoires. Images PMID:8058758

  20. Transfer of nonselectable genes into mouse teratocarcinoma cells and transcription of the transferred human. beta. -globin gene

    SciTech Connect

    Wagner, E.F.; Mintz, B.

    1982-02-01

    Teratocarcinoma (TCC) stem cells can function as vehicles for the introduction of specific recombinant genes into mice. Because most genes do not code for a selectable marker, the authors investigated the transformation efficiency of vectors with a linked selectable gene. In one series, TCC cells first selected for thymidine kinase deficiency were treated with DNA from the plasmid vector PtkH..beta..1 containing the human genomic ..beta..-globin gene and the thymidine kinase gene of herpes simplex virus. A high transformation frequency was obtained after selection in hypoxanthine-aminopterin-thymidine medium. Hybridization tests revealed that the majority of transformants had intact copies of the human gene among three to six total copies per cell. These were associated with cellular DNA sequences as judged from the presence of additional new restriction fragments and from stability of the sequences in tumors produced by injecting the cells subcutaneously. Total polyadenylate-containing RNA from cell cultures of two out of four transformants examined showed hybridization to the human gene probe: one RNA species resembled mature human ..beta..-globin mRNA transcripts; the others were of larger size. In differentiating tumors, various tissues, including hematopoietic cells of TCC provenance could be found. In a second model set of experiments, wild-type TCC cells were used to test a dominant-selection scheme with pSV-gpt vectors. Numerous transformants were isolated, and their transfected DNA was apparently stably integrated. Thus, any gene of choice can be transferred into TCC stem cells even without mutagenesis of the cells, and selected cell clones can be characterized. Cells of interest may then be introduced into early embryos to produce new mouse strains with predetermined genetic changes.

  1. Tmem79/Matt is the matted mouse gene and is a predisposing gene for atopic dermatitis in human subjects

    PubMed Central

    Saunders, Sean P.; Goh, Christabelle S.M.; Brown, Sara J.; Palmer, Colin N.A.; Porter, Rebecca M.; Cole, Christian; Campbell, Linda E.; Gierlinski, Marek; Barton, Geoffrey J.; Schneider, Georg; Balmain, Allan; Prescott, Alan R.; Weidinger, Stephan; Baurecht, Hansjrg; Kabesch, Michael; Gieger, Christian; Lee, Young-Ae; Tavendale, Roger; Mukhopadhyay, Somnath; Turner, Stephen W.; Madhok, Vishnu B.; Sullivan, Frank M.; Relton, Caroline; Burn, John; Meggitt, Simon; Smith, Catherine H.; Allen, Michael A.; Barker, Jonathan N.W. N.; Reynolds, Nick J.; Cordell, Heather J.; Irvine, Alan D.; McLean, W.H. Irwin; Sandilands, Aileen; Fallon, Padraic G.

    2013-01-01

    Background Atopic dermatitis (AD) is a major inflammatory condition of the skin caused by inherited skin barrier deficiency,with mutations in the filaggrin gene predisposing to development of AD. Support for barrier deficiency initiating AD came from flaky tail mice, which have a frameshift mutation in Flg and also carry an unknown gene, matted, causing a matted hair phenotype. Objective We sought to identify the matted mutant gene in mice and further define whether mutations in the human gene were associated with AD. Methods A mouse genetics approach was used to separate the matted and Flg mutations to produce congenic single-mutant strains for genetic and immunologic analysis. Next-generation sequencing was used to identify the matted gene. Five independently recruited AD case collections were analyzed to define associations between single nucleotide polymorphisms (SNPs) in the human gene and AD. Results The matted phenotype in flaky tail mice is due to a mutation in the Tmem79/Matt gene, with no expression of the encoded protein mattrin in the skin of mutant mice. Mattft mice spontaneously have dermatitis and atopy caused by a defective skin barrier, with mutant mice having systemic sensitization after cutaneous challenge with house dust mite allergens. Meta-analysis of 4,245 AD cases and 10,558 population-matched control subjects showed that a missense SNP, rs6694514, in the human MATT gene has a small but significant association with AD. Conclusion In mice mutations in Matt cause a defective skin barrier and spontaneous dermatitis and atopy. Acommon SNP in MATT has an association with AD in human subjects. PMID:24084074

  2. Characterization and promoter analysis of the mouse gene for transcription factor Sp4.

    PubMed

    Song, J; Mangold, M; Suske, G; Geltinger, C; Kanazawa, I; Sun, K; Yokoyama, K K

    2001-02-01

    Transcription factor Sp4 is a member of the Sp1 family. It functions differently from other members of this family, such as Sp1 and Sp3, and the gene for Sp4 is transcribed in a tissue-specific manner. Recent studies in mice suggest that Sp4 might play an important role in growth, viability, and male fertility. We report here the isolation and characterization of the gene for Sp4 from a mouse genomic library. The mouse gene for Sp4 was about 80 kb in length and it consisted of six exons and five introns. The promoter was found in a CpG island and had a high G+C content. The proximal promoter contained multiple putative binding sites for the transcription factors Sp1 and MAZ but lacked a consensus TATA box. Multiple sites for the initiation of transcription were mapped in a GC-rich region from 286 bp to 211 bp upstream of the ATG triplet at the site of initiation of translation, and all of the sites were either C or G. Transfection experiments and deletion analysis allowed us to localize the promoter to a region that was no more than 93 bp upstream from the first site of initiation of transcription. We also found that ectopic expression of Sp1 and of MAZ, but not of Sp3, suppressed expression of the Sp4 promoter in a dose-dependent manner. PMID:11245974

  3. DNA methylation map of mouse and human brain identifies target genes in Alzheimers disease

    PubMed Central

    Sanchez-Mut, Jose V.; Aso, Ester; Panayotis, Nicolas; Lott, Ira; Dierssen, Mara; Rabano, Alberto; Urdinguio, Rocio G.; Fernandez, Agustin F.; Astudillo, Aurora; Martin-Subero, Jose I.; Balint, Balazs; Fraga, Mario F.; Gomez, Antonio; Gurnot, Cecile; Roux, Jean-Christophe; Avila, Jesus; Hensch, Takao K.; Ferrer, Isidre

    2013-01-01

    The central nervous system has a pattern of gene expression that is closely regulated with respect to functional and anatomical regions. DNA methylation is a major regulator of transcriptional activity, and aberrations in the distribution of this epigenetic mark may be involved in many neurological disorders, such as Alzheimers disease. Herein, we have analysed 12 distinct mouse brain regions according to their CpG 5-end gene methylation patterns and observed their unique epigenetic landscapes. The DNA methylomes obtained from the cerebral cortex were used to identify aberrant DNA methylation changes that occurred in two mouse models of Alzheimers disease. We were able to translate these findings to patients with Alzheimers disease, identifying DNA methylation-associated silencing of three targets genes: thromboxane A2 receptor (TBXA2R), sorbin and SH3 domain containing 3 (SORBS3) and spectrin beta 4 (SPTBN4). These hypermethylation targets indicate that the cyclic AMP response element-binding protein (CREB) activation pathway and the axon initial segment could contribute to the disease. PMID:24030951

  4. Activation of Transgene-specific T Cells Following Lentivirus-mediated Gene Delivery to Mouse Lung

    PubMed Central

    Limberis, Maria P; Bell, Christie L; Heath, Jack; Wilson, James M

    2009-01-01

    Integrating lentiviral vectors based on the human immunodeficiency virus type-1 (HIV-1) can transduce quiescent cells, which in lung account for almost 95% of the epithelial cell population. Pseudotyping lentiviral vectors with the envelope glycoprotein from the Ebola Zaire virus, the lymphocytic choriomeningitis virus (LCMV), the Mokola virus, and the vesicular stomatitis virus (VSV-G) resulted in transduction of mouse alveolar epithelium, but gene expression in the lung of C57BL/6 and BALB/c mice waned within 90 days of vector injection. Intratracheal delivery of the four pseudotyped lentiviral vectors resulted in transgene-specific T-cell activation in both mouse strains, albeit lower than that achieved by intramuscular injection of the vectors. We performed an adoptive transfer of luciferase-specific T cells, isolated from spleen or lung of donor mice injected with VSV-G-pseudotyped lentivirus vector expressing luciferase into the muscle or lung, respectively, into recipient recombination-activating gene (RAG)deficient mice transduced in lung with adenovirus expressing firefly luciferase (ffluc2). Gene expression declined within 7 days of adoptive transfer approaching background levels by day 36. Taken together, our results suggest that the loss of transduced cells in lung is due to VSV-G.HIV vectormediated activation of transgene-specific T cells rather than as result of normal turnover of airway cells. PMID:19724265

  5. Delimiting the Location of the Scrapie Prion Incubation Time Gene on Chromosome 2 of the Mouse

    PubMed Central

    Carlson, G. A.; Ebeling, C.; Torchia, M.; Westaway, D.; Prusiner, S. B.

    1993-01-01

    Scrapie is a transmissible neurodegenerative disease caused by unusual pathogens called prions. The interval between inoculation and illness for experimental mouse scrapie is dramatically influenced by an incubation time gene (Prn-i) that is linked to Prn-p, the structural gene for prion protein (PrP). Although prion proteins from mouse strains with short and long scrapie incubation times differ by two amino acids, mice with discordant disease phenotype and Prn-p genotype occur in segregating crosses, suggesting recombination between Prn-p and a distinct incubation time locus. In addition, expression of Prn-p(b) transgenes from long incubation time mice shortened, rather than prolonged, incubation time. In this study, mice carrying chromosomes with meiotic crossovers near Prn-p were analyzed for scrapie incubation time phenotype. The results indicated that Prn-i (should it exist) must lie within an interval 0.67 cM proximal and 0.22 cM distal to Prn-p. The results also suggest that the cumulative effects of other genes, rather than meiotic recombination, were responsible for the putative recombinants of earlier studies. However, the effect of Prn-p(b) transgene expression in abbreviating scrapie incubation time was mitigated when the transgenes were transferred to mice with an endogenous long incubation time allele. Thus, Prn-p(b) transgenes and Prn-i may modulate scrapie pathogenesis by different mechanisms. PMID:8462855

  6. Microarray analysis of gene expression in mouse (strain 129) embryonic stem cells after typical synthetic musk exposure.

    PubMed

    Shi, Jiachen; Li, Ming; Jiao, Zhihao; Zhang, Jing; Feng, Yixing; Shao, Bing

    2013-01-01

    Synthetic musks are widely used in personal-care products and can readily accumulate in the adipose tissue, breast milk, and blood of humans. In this study, the Affymetrix Mouse Genome GeneChip was used to identify alterations in gene expression of embryonic stem cells from the 129 strain of the laboratory mouse after treatment with the synthetic musk tonalide (AHTN). Among the 45,037 transcripts in the microarray, 2,879 genes were differentially expressed. According to the microarray analysis, the potential influence of AHTN on the development to embryo should be of concern, and the toxicological effects of it and related musk compounds should be studied further. PMID:23099888

  7. The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes.

    PubMed

    Czosnek, H H; Bienz, B; Givol, D; Zakut-Houri, R; Pravtcheva, D D; Ruddle, F H; Oren, M

    1984-08-01

    The chromosomal assignments of the two genes encoding the murine p53 cellular tumor antigen were determined by using a panel of mouse-Chinese hamster somatic cell hybrid clones and a mouse p53-specific cDNA clone. One gene, probably the functional member of the family, was found to be on chromosome 11. The other gene, which is probably a processed pseudogene, was assigned to chromosome 14. The potential relevance of these findings to documented cases of chromosome 11 trisomy are also discussed. PMID:6387444

  8. Tissue-Specific Evolution of Protein Coding Genes in Human and Mouse

    PubMed Central

    Kryuchkova-Mostacci, Nadezda; Robinson-Rechavi, Marc

    2015-01-01

    Protein-coding genes evolve at different rates, and the influence of different parameters, from gene size to expression level, has been extensively studied. While in yeast gene expression level is the major causal factor of gene evolutionary rate, the situation is more complex in animals. Here we investigate these relations further, especially taking in account gene expression in different organs as well as indirect correlations between parameters. We used RNA-seq data from two large datasets, covering 22 mouse tissues and 27 human tissues. Over all tissues, evolutionary rate only correlates weakly with levels and breadth of expression. The strongest explanatory factors of purifying selection are GC content, expression in many developmental stages, and expression in brain tissues. While the main component of evolutionary rate is purifying selection, we also find tissue-specific patterns for sites under neutral evolution and for positive selection. We observe fast evolution of genes expressed in testis, but also in other tissues, notably liver, which are explained by weak purifying selection rather than by positive selection. PMID:26121354

  9. Structure, sequence, and chromosomal location of the gene for USF2 transcription factors in mouse

    SciTech Connect

    Henrion, A.A.; Martinez, A.; Kahn, A.

    1995-01-01

    The ubiquitously expressed upstream stimulatory factor (USF) involved in the transcription of a wide variety of cellular genes is defined as dimers of c-myc-related proteins, composed of a basic helix-loop-helix/leucine zipper region. The USF family consists of different members that split into two groups: MLTF or USF1 and USF2 or FIR. We present here evidence that USF1 and USF2 are distinct closely related genes in human, rat, and mouse. Based on the recent cloning of rat and human new cDNAs, we have isolated genomic clones encompassing the murine USF2 gene, which consists of at least 10 exons spanning a minimum of 10 kb of genomic DNA. Unexpectedly, the organization of USF2 appears very split up by introns (0.08 to over 6 kb in size), compared to the myc gene structure. The entire gene (but the larger intron) and 1.6 kb of the 5{prime} flanking region were sequenced. This 5{prime} flanking region is GC-rich, contains several putative transcription binding sites, and has no apparent TATA box. Gene mapping of murine USF2 and USF1 has been determined by in situ hybridization, indicating the localization of USF2 on chromosome 7 and of USF1 on chromosomes 1 and 11. 35 refs., 4 figs., 1 tab.

  10. FACS-array gene expression analysis during early development of mouse telencephalic interneurons.

    PubMed

    Marsh, Eric D; Minarcik, Jennifer; Campbell, Kenneth; Brooks-Kayal, Amy R; Golden, Jeffrey A

    2008-03-01

    Cortical interneuron dysfunction has been implicated in multiple human disorders including forms of epilepsy, mental retardation, and autism. Although significant advances have been made, understanding the biologic basis of these disorders will require a level of anatomic, molecular, and genetic detail of interneuron development that currently does not exist. To further delineate the pathways modulating interneuron development we performed fluorescent activated cell sorting (FACs) on genetically engineered mouse embryos that selectively express green fluorescent protein (GFP) in developing interneurons followed by whole genome microarray expression profiling on the isolated cells. Bioinformatics analysis revealed expression of both predicted and unexpected genes in developing cortical interneurons. Two unanticipated pathways discovered to be up regulated prior to interneurons differentiating in the cortex were ion channels/neurotransmitters and synaptic/vesicular related genes. A significant association of neurological disease related genes to the population of developing interneurons was found. These results have defined new and potentially important data on gene expression changes during the development of cortical interneurons. In addition, these data can be mined to uncover numerous novel genes involved in the generation of interneurons and may suggest genes/pathways potentially involved in a number of human neurological disorders. PMID:18172891

  11. Venezuelan equine encephalitis virus infection causes modulation of inflammatory and immune response genes in mouse brain

    PubMed Central

    Sharma, Anuj; Bhattacharya, Bhaskar; Puri, Raj K; Maheshwari, Radha K

    2008-01-01

    Background Neurovirulent Venezuelan equine encephalitis virus (VEEV) causes lethal encephalitis in equines and is transmitted to humans by mosquitoes. VEEV is highly infectious when transmitted by aerosol and has been developed as a bio-warfare agent, making it an important pathogen to study from a military and civilian standpoint. Molecular mechanisms of VEE pathogenesis are poorly understood. To study these, the gene expression profile of VEEV infected mouse brains was investigated. Changes in gene expression were correlated with histological changes in the brain. In addition, a molecular framework of changes in gene expression associated with progression of the disease was studied. Results Our results demonstrate that genes related to important immune pathways such as antigen presentation, inflammation, apoptosis and response to virus (Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27 Oas1b, Fcerg1,Mif, Clusterin and MHC class II) were upregulated as a result of virus infection. The number of over-expressed genes (>1.5-fold level) increased as the disease progressed (from 197, 296, 400, to 1086 at 24, 48, 72 and 96 hours post infection, respectively). Conclusion Identification of differentially expressed genes in brain will help in the understanding of VEEV-induced pathogenesis and selection of biomarkers for diagnosis and targeted therapy of VEEV-induced neurodegeneration. PMID:18558011

  12. Mapping of Heavy Chain Genes for Mouse Immunoglobulins M and D

    NASA Astrophysics Data System (ADS)

    Liu, Chih-Ping; Tucker, Philip W.; Mushinski, J. Frederic; Blattner, Frederick R.

    1980-09-01

    A single DNA fragment containing both μ and δ immunoglobulin heavy chain genes has been cloned from normal BALB/c mouse liver DNA with a new λ phage vector Charon 28. The physical distance between the membrane terminal exon of μ and the first domain of δ is 2466 base pairs, with δ on the 3' side of μ . A single transcript could contain a variable region and both μ and δ constant regions. The dual expression of immunoglobulins M and D on spleen B cells may be due to alternate splicing of this transcript.

  13. Erythrocyte metallothionein as an index of zinc status in humans

    SciTech Connect

    Grider, A.; Bailey, L.B.; Cousins, R.J. )

    1990-02-01

    Metallothionein concentrations in erythrocyte lysates derived from human subjects were measured by an ELISA procedure. IgG obtained from serum of sheep injected with human metallothionein 1 was used in this competitive assay. Subjects were fed a semipurified zinc-deficient diet for an 8-day depletion period after 3 days of acclimation. Fasting plasma zinc concentrations were reduced {approx}7%. Metallothionein in the erythrocyte lysates was significantly decreased to 59% of the initial level by the end of the depletion period. Supplementation of these depleted subjects with zinc did not increase erythrocyte metallothionein levels within 24 hr. Daily supplementation of control subjects with zinc increased erythrocyte metallothionein to a 7-fold maximum within 7 days. These levels were reduced by 61% within 14 days after zinc supplementation was terminated. Incubation of rat ({sup 35}S)metallothionein with human erythrocyte lysate showed a time-dependent increase in {sup 35}S soluble in 20% trichloroacetic acid, indicating degradation of the labeled protein, presumably via protease activity in the lysate. It is proposed that zinc supplementation induces erythrocyte metallothionein during erythropoiesis and that low zinc intake decreases synthesis and/or accelerates degradation of the protein in reticulocytes/erythrocytes. Metallothionein levels in erythrocytes may provide a useful index upon which to assess zinc status in humans.

  14. Application of a modified 203Hg binding assay for metallothionein.

    PubMed

    Patierno, S R; Pellis, N R; Evans, R M; Costa, M

    1983-04-01

    A sensitive and rapid method to estimate concentrations of functional metallothionein in small biological samples, based upon the acid stability of 203Hg binding and solubility of this protein in trichloroacetic acid is described. Sephadex G-10 minicolumns supported in centrifuge tubes afforded separation and quantitation of isotope bound metallothionein from unbound metal. Elution of metallothionein bound 203Hg was achieved by short term-low speed centrifugation that segregated chelator-ligand complex into the eluate while unbound ligand remained in the gel. A well characterized standard of pure metallothionein protein was utilized to verify the specificity and sensitivity of the modified assay. Metallothionein levels were estimated by 203Hg binding in extracts of wild type and cadmium resistant Chinese hamster ovary cells treated with maximum tolerable concentrations of CdCl2. Similar separation methods demonstrated [35S]-cysteine incorporation into induced metallothionein. Additionally, induction of metallothionein was observed after treatment with particulate CdS but not crystalline NiS particles. These results demonstrate that the modified assay system is easily applied to serial measurement of metallothionein levels in multiple small biological samples. PMID:6835007

  15. Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences

    PubMed Central

    Feng, Weiguo; Leach, Sonia M.; Tipney, Hannah; Phang, Tzulip; Geraci, Mark; Spritz, Richard A.; Hunter, Lawrence E.; Williams, Trevor

    2009-01-01

    Orofacial malformations resulting from genetic and/or environmental causes are frequent human birth defects yet their etiology is often unclear because of insufficient information concerning the molecular, cellular and morphogenetic processes responsible for normal facial development. We have, therefore, derived a comprehensive expression dataset for mouse orofacial development, interrogating three distinct regions – the mandibular, maxillary and frontonasal prominences. To capture the dynamic changes in the transcriptome during face formation, we sampled five time points between E10.5–E12.5, spanning the developmental period from establishment of the prominences to their fusion to form the mature facial platform. Seven independent biological replicates were used for each sample ensuring robustness and quality of the dataset. Here, we provide a general overview of the dataset, characterizing aspects of gene expression changes at both the spatial and temporal level. Considerable coordinate regulation occurs across the three prominences during this period of facial growth and morphogenesis, with a switch from expression of genes involved in cell proliferation to those associated with differentiation. An accompanying shift in the expression of polycomb and trithorax genes presumably maintains appropriate patterns of gene expression in precursor or differentiated cells, respectively. Superimposed on the many coordinated changes are prominence-specific differences in the expression of genes encoding transcription factors, extracellular matrix components, and signaling molecules. Thus, the elaboration of each prominence will be driven by particular combinations of transcription factors coupled with specific cell:cell and cell:matrix interactions. The dataset also reveals several prominence-specific genes not previously associated with orofacial development, a subset of which we externally validate. Several of these latter genes are components of bidirectional transcription units that likely share cis-acting sequences with well-characterized genes. Overall, our studies provide a valuable resource for probing orofacial development and a robust dataset for bioinformatic analysis of spatial and temporal gene expression changes during embryogenesis. PMID:20016822

  16. Multiple mechanisms regulate imprinting of the mouse distal chromosome 7 gene cluster.

    PubMed

    Caspary, T; Cleary, M A; Baker, C C; Guan, X J; Tilghman, S M

    1998-06-01

    Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57(Kip2), as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57(Kip2) during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57(Kip2), and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57(Kip2) have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57(Kip2) is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s). PMID:9584186

  17. Identification of genes and networks driving cardiovascular and metabolic phenotypes in a mouse F2 intercross.

    PubMed

    Derry, Jonathan M J; Zhong, Hua; Molony, Cliona; MacNeil, Doug; Guhathakurta, Debraj; Zhang, Bin; Mudgett, John; Small, Kersten; El Fertak, Lahcen; Guimond, Alain; Selloum, Mohammed; Zhao, Wenqing; Champy, Marie France; Monassier, Laurent; Vogt, Tom; Cully, Doris; Kasarskis, Andrew; Schadt, Eric E

    2010-01-01

    To identify the genes and pathways that underlie cardiovascular and metabolic phenotypes we performed an integrated analysis of a mouse C57BL/6JxA/J F2 (B6AF2) cross by relating genome-wide gene expression data from adipose, kidney, and liver tissues to physiological endpoints measured in the population. We have identified a large number of trait QTLs including loci driving variation in cardiac function on chromosomes 2 and 6 and a hotspot for adiposity, energy metabolism, and glucose traits on chromosome 8. Integration of adipose gene expression data identified a core set of genes that drive the chromosome 8 adiposity QTL. This chromosome 8 trans eQTL signature contains genes associated with mitochondrial function and oxidative phosphorylation and maps to a subnetwork with conserved function in humans that was previously implicated in human obesity. In addition, human eSNPs corresponding to orthologous genes from the signature show enrichment for association to type II diabetes in the DIAGRAM cohort, supporting the idea that the chromosome 8 locus perturbs a molecular network that in humans senses variations in DNA and in turn affects metabolic disease risk. We functionally validate predictions from this approach by demonstrating metabolic phenotypes in knockout mice for three genes from the trans eQTL signature, Akr1b8, Emr1, and Rgs2. In addition we show that the transcriptional signatures for knockout of two of these genes, Akr1b8 and Rgs2, map to the F2 network modules associated with the chromosome 8 trans eQTL signature and that these modules are in turn very significantly correlated with adiposity in the F2 population. Overall this study demonstrates how integrating gene expression data with QTL analysis in a network-based framework can aid in the elucidation of the molecular drivers of disease that can be translated from mice to humans. PMID:21179467

  18. Identification of Pax6-Dependent Gene Regulatory Networks in the Mouse Lens

    PubMed Central

    Wolf, Louise V.; Yang, Ying; Wang, Jinhua; Xie, Qing; Braunger, Barbara; Tamm, Ernst R.; Zavadil, Jiri; Cvekl, Ales

    2009-01-01

    Lineage-specific DNA-binding transcription factors regulate development by activating and repressing particular set of genes required for the acquisition of a specific cell type. Pax6 is a paired domain and homeodomain-containing transcription factor essential for development of central nervous, olfactory and visual systems, as well as endocrine pancreas. Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis. Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development. Using DNA microarrays, we have identified 559 genes expressed differentially between 1-day old mouse Pax6 heterozygous and wild type lenses. Of these, 178 (31.8%) were similarly increased and decreased in Pax6 homozygous embryonic telencephalon [Holm PC, Mader MT, Haubst N, Wizenmann A, Sigvardsson M, Gtz M (2007) Loss- and gain-of-function analyses reveals targets of Pax6 in the developing mouse telencephalon. Mol Cell Neurosci 34: 99119]. In contrast, 381 (68.2%) genes were differently regulated between the lens and embryonic telencephalon. Differential expression of nine genes implicated in lens development and homeostasis: Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2, was confirmed by quantitative RT-PCR, with five of these genes: Cspg2, Mab21l2, Olfm3, Spag5 and Tgfb2, identified as candidate direct Pax6 target genes by quantitative chromatin immunoprecipitation (qChIP). In Mab21l2 and Tgfb2 promoter regions, twelve putative individual Pax6-binding sites were tested by electrophoretic mobility shift assays (EMSAs) with recombinant Pax6 proteins. This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs. Collectively, the present studies represent an integrative genome-wide approach to identify downstream networks controlled by Pax6 that control mouse lens and forebrain development. PMID:19132093

  19. Mapping of the NEP receptor tyrosine kinase gene to human chromosome 6p21.3 and mouse chromosome 17C

    SciTech Connect

    Edelhoff, S.; Disteche, C.M.; Sweetser, D.A.

    1995-01-01

    The mouse receptor tyrosine kinase (RTK) NEP, also called Ptk-3, is widely expressed, with high levels in proliferating neuroepithelia of mouse embryos. The recently described human discoidin domain receptor (DDR) has a predicted amino acid sequence 93% identical to that of murine NEP and may be its human homologue. We have mapped the gene encoding NEP in human and mouse by fluorescence in situ hybridization using a mouse cDNA probe. The NEP/Nep gene maps to human chromosome 6p21.3 and mouse chromosome 17C, respectively. This places the NEP/Nep gene at, or near, the major histocompatibility (MHC) locus-HLA in human and H2 in mouse, respectively. Based on its pattern of expression during development, NEP and Nep represent candidate genes for several MHC-linked developmental abnormalities in human and mouse. 19 refs., 1 fig.

  20. Molecular cloning and chromosomal mapping of the mouse gene encoding cyclin-dependent kinase 5 regulatory subunit p35

    SciTech Connect

    Ohshima, Toshio; Kozak, C.A.; Nagle, J.W.

    1996-07-15

    A neural-specific activating subunit, p35, of cyclin-dependent kinase 5 (Cdk5) was recently reported to differ from other mammalian cyclins, suggesting a new type of regulatory subunit for Cdk activity. The mouse gene encoding p35, Cdk5r, was isolated from a mouse 129/SvJ genomic library, and the genomic structure of Cdk5r was characterized. The most notable features of Cdk5r are the absence of introns in the amino acid coding region and the high homology of amino acid sequence among species. The 5{prime}-flanking region of Cdk5r contained no canonical TATA or CAAT box but had several putative promoter elements, including Sp1, AP2, MRE, and NGFIA. The mouse Cdk5r transcript was detected only in the brain by Northern blot analysis. Mouse Cdk5r was mapped to a position on mouse chromosome 11. 14 refs., 2 figs.

  1. Joint mouse-human phenome-wide association to test gene function and disease risk.

    PubMed

    Wang, Xusheng; Pandey, Ashutosh K; Mulligan, Megan K; Williams, Evan G; Mozhui, Khyobeni; Li, Zhengsheng; Jovaisaite, Virginija; Quarles, L Darryl; Xiao, Zhousheng; Huang, Jinsong; Capra, John A; Chen, Zugen; Taylor, William L; Bastarache, Lisa; Niu, Xinnan; Pollard, Katherine S; Ciobanu, Daniel C; Reznik, Alexander O; Tishkov, Artem V; Zhulin, Igor B; Peng, Junmin; Nelson, Stanley F; Denny, Joshua C; Auwerx, Johan; Lu, Lu; Williams, Robert W

    2016-01-01

    Phenome-wide association is a novel reverse genetic strategy to analyze genome-to-phenome relations in human clinical cohorts. Here we test this approach using a large murine population segregating for ∼5 million sequence variants, and we compare our results to those extracted from a matched analysis of gene variants in a large human cohort. For the mouse cohort, we amassed a deep and broad open-access phenome consisting of ∼4,500 metabolic, physiological, pharmacological and behavioural traits, and more than 90 independent expression quantitative trait locus (QTL), transcriptome, proteome, metagenome and metabolome data sets-by far the largest coherent phenome for any experimental cohort (www.genenetwork.org). We tested downstream effects of subsets of variants and discovered several novel associations, including a missense mutation in fumarate hydratase that controls variation in the mitochondrial unfolded protein response in both mouse and Caenorhabditis elegans, and missense mutations in Col6a5 that underlies variation in bone mineral density in both mouse and human. PMID:26833085

  2. Hepatocellular carcinoma mouse models: Hepatitis B virus-associated hepatocarcinogenesis and haploinsufficient tumor suppressor genes.

    PubMed

    Teng, Yuan-Chi; Shen, Zhao-Qing; Kao, Cheng-Heng; Tsai, Ting-Fen

    2016-01-01

    The multifactorial and multistage pathogenesis of hepatocellular carcinoma (HCC) has fascinated a wide spectrum of scientists for decades. While a number of major risk factors have been identified, their mechanistic roles in hepatocarcinogenesis still need to be elucidated. Many tumor suppressor genes (TSGs) have been identified as being involved in HCC. These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors: the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele. Hepatitis B virus (HBV) is one of the most important risk factors associated with HCC. Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor, one advantage of mouse models for HBV/HCC research is the numerous and powerful genetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs. Here, we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner. Discoveries obtained using mouse models will have a great impact on HCC translational medicine. PMID:26755878

  3. Chromosomal location of the genes encoding complement components C5 and factor H in the mouse.

    PubMed

    D'Eustachio, P; Kristensen, T; Wetsel, R A; Riblet, R; Taylor, B A; Tack, B F

    1986-12-15

    Complementary DNA probes corresponding to the factor H and C5 polypeptides have been used to determine the chromosomal localizations of these two complement components. Both probes revealed complex and polymorphic arrays of DNA fragments in Southern blot analysis of mouse genomic DNA. Following the distribution of these bands in panels of somatic cell hybrids carrying various combinations of mouse chromosomes on a constant rat or Chinese hamster background allowed the localization of the C5-associated fragments to proximal chromosome 2 and the localization of the factor H-associated fragments to chromosome 1 or chromosome 3. Following the inheritance of DNA restriction fragment-length polymorphisms revealed by the probes in recombinant inbred mouse strains allowed the factor H-associated fragments to be mapped to Sas-1 on chromosome 1, and the C5-associated fragments to be mapped to Hc. Analysis of three-point crosses, in turn, placed the latter locus 19 cM distal to Sd on chromosome 2. We have designated the two loci Cfh and C5, respectively. This genetic analysis raises the possibility that C5 and factor H are both encoded by complex loci composed of distinct structural and regulatory genes. PMID:2878046

  4. Hepatocellular carcinoma mouse models: Hepatitis B virus-associated hepatocarcinogenesis and haploinsufficient tumor suppressor genes

    PubMed Central

    Teng, Yuan-Chi; Shen, Zhao-Qing; Kao, Cheng-Heng; Tsai, Ting-Fen

    2016-01-01

    The multifactorial and multistage pathogenesis of hepatocellular carcinoma (HCC) has fascinated a wide spectrum of scientists for decades. While a number of major risk factors have been identified, their mechanistic roles in hepatocarcinogenesis still need to be elucidated. Many tumor suppressor genes (TSGs) have been identified as being involved in HCC. These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors: the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele. Hepatitis B virus (HBV) is one of the most important risk factors associated with HCC. Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor, one advantage of mouse models for HBV/HCC research is the numerous and powerful genetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs. Here, we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner. Discoveries obtained using mouse models will have a great impact on HCC translational medicine. PMID:26755878

  5. The mouse and human excitatory amino acid transporter gene (EAAT1) maps to mouse chromosome 15 and a region of syntenic homology on human chromosome 5

    SciTech Connect

    Kirschner, M.A.; Arriza, J.L.; Amara, S.G.

    1994-08-01

    The gene for human excitatory amino acid transporter (EAAT1) was localized to the distal region of human chromosome 5p13 by in situ hybridization of metaphase chromosome spreads. Interspecific backcross analysis identified the mouse Eaat1 locus in a region of 5p13 homology on mouse chromosome 15. Markers that are linked with EAAT1 on both human and mouse chromosomes include the receptors for leukemia inhibitory factor, interleukin-7, and prolactin. The Eaat1 locus appears not be linked to the epilepsy mutant stg locus, which is also on chromosome 15. The EAAT1 locus is located in a region of 5p deletions that have been associated with mental retardation and microcephaly. 22 refs., 2 figs.

  6. The rhombotin gene family encode related LIM-domain proteins whose differing expression suggests multiple roles in mouse development.

    PubMed

    Foroni, L; Boehm, T; White, L; Forster, A; Sherrington, P; Liao, X B; Brannan, C I; Jenkins, N A; Copeland, N G; Rabbitts, T H

    1992-08-01

    The rhombotin (RBTN1 or Ttg-1) gene was first identified at a chromosome translocation in a T-cell acute leukaemia and later used to isolate two related genes (RBTN2 or Ttg-2 and RBTN3). Complete characterization of these genes in man and mouse shows that all three encode cysteine-rich proteins with typical LIM domains. RBTN1 and RBTN3-derived proteins have 98% identity in the LIM domains but are located on separate chromosomes in man and in mouse while RBTN1 and RBTN2, both located on human chromosome 11p but are on separate chromosomes in mouse, are only 48% identical in this part of the protein. The exon organization of RBTN1 and RBTN3 genes are similar, both having an intron, absent from the RBTN2 gene, in the LIM2-encoding region. The remarkable similarity between rbtn-1 and rbtn-3 proteins is parallelled in their expression patterns in mouse development, since both genes show high expression in restricted areas of the brain, but little lymphoid expression. rbtn-2 expression, however, is more ubiquitous. This gene shows a low level of thymus expression but high expression in fetal liver, adult spleen and B-cell lines, consistent with a role in B-cell development. These results suggest multiple cellular targets for the action of these proteins during development. PMID:1507224

  7. NON-MAMMALIAN FAT-1 GENE PREVENTS NEOPLASIA WHEN INTRODUCED TO A MOUSE HEPATOCARCINOGENESIS MODEL

    PubMed Central

    Griffitts, J.; Saunders, D.; Tesiram, Y.A.; Reid, G.E.; Salih, A.; Liu, S.; Lydic, T.A.; Busik, J.V.; Kang, J.X.; Towner, R.A.

    2010-01-01

    We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis model. Mice containing double mutations (DM) in c-myc and TGF-? (transforming growth factor-?), leading to liver neoplasia, were crossed with mice containing omega-3 desaturase. MRI analysis of triple mutant (TM) mice showed the absence of neoplasia at all time points for 92% of mice in the study. Pathological changes of TM (TGF?/c-myc/fat-1) mouse liver tissue was similar to control mouse liver tissue. Magnetic resonance spectroscopy (MRS) measurements of unsaturated fatty acids found a significant difference (p<0.005) between DM and TM transgenic (Tg) mice at 34 and 40 weeks of age. HPLC analysis of mouse liver tissue revealed markedly decreased levels of omega-6 fatty acids in TM mice when compared to DM (TGF?/c-myc) and control (CD1) mice. Mass spectrometry (MS) analysis indicated significantly decreased 16:0/20:4 and 18:1/20:4 and elevated 16:0/22:6 fatty acyl groups in both GPCho and GPEtn, and elevated 16:0/20:5, 18:0/18:2, 18:0/18:1 and 18:0/22:6 in GPCho, within TM mice compared to DM mice. Total fatty acid analysis indicated a significant decrease in 18:1n9 in TM mice compared to DM mice. Western blot analysis of liver tissue showed a significant (p<0.05) decrease in NF-?B (nuclear factor- ?B) levels at 40 weeks of age in TM mice compared to DM mice. Microarray analysis of TM versus DM mice livers at 40 weeks revealed alterations in genes involved in cell cycle regulation, cell-to-cell signaling, p53 signaling, and arachidonic acid (20:4) metabolism. Endogenous omega-3 fatty acids were found to prevent HCC development in mice. PMID:20620224

  8. A Mutation in the Mouse Ttc26 Gene Leads to Impaired Hedgehog Signaling

    PubMed Central

    Swiderski, Ruth E.; Nakano, Yoko; Mullins, Robert F.; Seo, Seongjin; Bnfi, Botond

    2014-01-01

    The phenotype of the spontaneous mutant mouse hop-sterile (hop) is characterized by a hopping gait, polydactyly, hydrocephalus, and male sterility. Previous analyses of the hop mouse revealed a deficiency of inner dynein arms in motile cilia and a lack of sperm flagella, potentially accounting for the hydrocephalus and male sterility. The etiology of the other phenotypes and the location of the hop mutation remained unexplored. Here we show that the hop mutation is located in the Ttc26 gene and impairs Hedgehog (Hh) signaling. Expression analysis showed that this mutation led to dramatically reduced levels of the Ttc26 protein, and protein-protein interaction assays demonstrated that wild-type Ttc26 binds directly to the Ift46 subunit of Intraflagellar Transport (IFT) complex B. Although IFT is required for ciliogenesis, the Ttc26 defect did not result in a decrease in the number or length of primary cilia. Nevertheless, Hh signaling was reduced in the hop mouse, as revealed by impaired activation of Gli transcription factors in embryonic fibroblasts and abnormal patterning of the neural tube. Unlike the previously characterized mutations that affect IFT complex B, hop did not interfere with Hh-induced accumulation of Gli at the tip of the primary cilium, but rather with the subsequent dissociation of Gli from its negative regulator, Sufu. Our analysis of the hop mouse line provides novel insights into Hh signaling, demonstrating that Ttc26 is necessary for efficient coupling between the accumulation of Gli at the ciliary tip and its dissociation from Sufu. PMID:25340710

  9. Enriched Environment-induced Maternal Weight Loss Reprograms Metabolic Gene Expression in Mouse Offspring*

    PubMed Central

    Wei, Yanchang; Yang, Cai-Rong; Wei, Yan-Ping; Ge, Zhao-Jia; Zhao, Zhen-Ao; Zhang, Bing; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2015-01-01

    The global prevalence of weight loss is increasing, especially in young women. However, the extent and mechanisms by which maternal weight loss affects the offspring is still poorly understood. Here, using an enriched environment (EE)-induced weight loss model, we show that maternal weight loss improves general health and reprograms metabolic gene expression in mouse offspring, and the epigenetic alterations can be inherited for at least two generations. EE in mothers induced weight loss and its associated physiological and metabolic changes such as decreased adiposity and improved glucose tolerance and insulin sensitivity. Relative to controls, their offspring exhibited improved general health such as reduced fat accumulation, decreased plasma and hepatic lipid levels, and improved glucose tolerance and insulin sensitivity. Maternal weight loss altered gene expression patterns in the liver of offspring with coherent down-regulation of genes involved in lipid and cholesterol biosynthesis. Epigenomic profiling of offspring livers revealed numerous changes in cytosine methylation depending on maternal weight loss, including reproducible changes in promoter methylation over several key lipid biosynthesis genes, correlated with their expression patterns. Embryo transfer studies indicated that oocyte alteration in response to maternal metabolic conditions is a strong factor in determining metabolic and epigenetic changes in offspring. Several important lipid metabolism-related genes have been identified to partially inherit methylated alleles from oocytes. Our study reveals a molecular and mechanistic basis of how maternal lifestyle modification affects metabolic changes in the offspring. PMID:25555918

  10. Specificity and Heterogeneity of Terahertz Radiation Effect on Gene Expression in Mouse Mesenchymal Stem Cells

    PubMed Central

    Alexandrov, Boian S.; Phipps, M. Lisa; Alexandrov, Ludmil B.; Booshehri, Layla G.; Erat, Anna; Zabolotny, Janice; Mielke, Charles H.; Chen, Hou-Tong; Rodriguez, George; Rasmussen, Kim .; Martinez, Jennifer S.; Bishop, Alan R.; Usheva, Anny

    2013-01-01

    We report that terahertz (THz) irradiation of mouse mesenchymal stem cells (mMSCs) with a single-frequency (SF) 2.52?THz laser or pulsed broadband (centered at 10?THz) source results in irradiation specific heterogenic changes in gene expression. The THz effect depends on irradiation parameters such as the duration and type of THz source, and on the degree of stem cell differentiation. Our microarray survey and RT-PCR experiments demonstrate that prolonged broadband THz irradiation drives mMSCs toward differentiation, while 2-hour irradiation (regardless of THz sources) affects genes transcriptionally active in pluripotent stem cells. The strictly controlled experimental environment indicates minimal temperature changes and the absence of any discernable response to heat shock and cellular stress genes imply a non-thermal response. Computer simulations of the core promoters of two pluripotency markers reveal association between gene upregulation and propensity for DNA breathing. We propose that THz radiation has potential for non-contact control of cellular gene expression. PMID:23378916

  11. Ex vivo magnetofection: A novel strategy for the study of gene function in mouse organogenesis

    PubMed Central

    Svingen, Terje; Wilhelm, Dagmar; Combes, Alexander N.; Hosking, Brett; Harley, Vincent R.; Sinclair, Andrew H.; Koopman, Peter

    2010-01-01

    Gene function during mouse development is often studied through the production and analysis of transgenic and knock-out models. However, these techniques are time- and resource-consuming, and require specialized equipment and expertise. We have established a new protocol for functional studies that combines organ culture of explanted fetal tissues with micro-injection and magnetically-induced transfection (magnetofection) of gene expression constructs. As proof-of-principle, we magnetofected cDNA constructs into genital ridge tissue as a means of gain-of-function analysis, and shRNA constructs for loss-of-function analysis. Ectopic expression of Sry induced female-to-male sex-reversal, whereas knockdown of Sox9 expression caused male-to-female sex-reversal, consistent with the known functions of these genes. Further, ectopic expression of Tmem184a, a gene of unknown function, in female genital ridges, resulted