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Sample records for genetically manipulated mice

  1. Flanking gene and genetic background problems in genetically manipulated mice.

    PubMed

    Crusio, Wim E

    2004-09-15

    Mice carrying engineered genetic modifications have become an indispensable tool in the study of gene functioning. The interpretation of results obtained with targeted mutants is not completely straightforward, however, because of genetic complications due to linkage and epistasis. Effects of closely linked genes flanking the targeted locus might sometimes be responsible for phenotypic changes ascribed to the null mutation. The effects of the latter might also be modified by the general genetic background. This review presents some examples and discusses some simple strategies to deal with these complications.

  2. Genetic Manipulation

    ERIC Educational Resources Information Center

    Klein, David

    1973-01-01

    Knowledge of genetic manipulations opens the door to ambitious possibilities of inhabiting the world with genetically perfect human beings. Legal, technological and social problems are involved. Attempts must be made to identify hereditary complaints in individuals. (PS)

  3. Generation of mice with longer and better preserved telomeres in the absence of genetic manipulations

    PubMed Central

    Varela, Elisa; Muñoz-Lorente, Miguel A.; Tejera, Agueda M.; Ortega, Sagrario; Blasco, Maria A.

    2016-01-01

    Although telomere length is genetically determined, mouse embryonic stem (ES) cells with telomeres of twice the normal size have been generated. Here, we use such ES cells with ‘hyper-long' telomeres, which also express green fluorescent protein (GFP), to generate chimaeric mice containing cells with both hyper-long and normal telomeres. We show that chimaeric mice contain GFP-positive cells in all mouse tissues, display normal tissue histology and normal survival. Both hyper-long and normal telomeres shorten with age, but GFP-positive cells retain longer telomeres as mice age. Chimaeric mice with hyper-long telomeres also accumulate fewer cells with short telomeres and less DNA damage with age, and express lower levels of p53. In highly renewing compartments, such as the blood, cells with hyper-long telomeres are longitudinally maintained or enriched with age. We further show that wound-healing rates in the skin are increased in chimaeric mice. Our work demonstrates that mice with functional, longer and better preserved telomeres can be generated without the need for genetic manipulations, such as TERT overexpression. PMID:27252083

  4. Modeling socially anhedonic syndromes: genetic and pharmacological manipulation of opioid neurotransmission in mice.

    PubMed

    Cinque, C; Pondiki, S; Oddi, D; Di Certo, M G; Marinelli, S; Troisi, A; Moles, A; D'Amato, F R

    2012-08-28

    Social anhedonia, or the diminished capacity to experience pleasure and reward from social affiliation, is a major symptom of different psychiatric disorders, including some forms of infantile autism and schizophrenia spectrum disorders. The brain opioid hypothesis of social attachment is a promising model for achieving insights into how neurobiological and developmental factors contribute to the regulation of social reward. In this study, genetic knocking-out and naltrexone (NTRX) treatment during the first 4 days of life were used to disrupt opioid neurotransmission in mouse pups and their attachment relationships with the mother. Both permanent (genetic) and transient (pharmacological) manipulations of opioid neurotransmission exerted long-term effects on social affiliation. When juveniles, both μ-opioid receptor knockout mice and NTRX-treated pups showed reduced interest in peers and no preference for socially rewarding environment. These results demonstrate that sociability in juvenile mice is highly dependent on the establishment during infancy of a positive affective relationship with their mothers and that opioid neurotransmission has a major role in the regulation of social hedonic capacity. If the validity of this animal model will be confirmed by future research, translational studies focusing on the interaction between early experience and opioid neurotransmission could provide useful insights for identifying endophenotypes of human psychiatric disorders associated with social anhedonia.

  5. Update: Biochemistry of Genetic Manipulation.

    ERIC Educational Resources Information Center

    Barker, G. R.

    1983-01-01

    Various topics on the biochemistry of genetic manipulation are discussed. These include genetic transformation and DNA; genetic expression; DNA replication, repair, and mutation; technology of genetic manipulation; and applications of genetic manipulation. Other techniques employed are also considered. (JN)

  6. Update: Biochemistry of Genetic Manipulation.

    ERIC Educational Resources Information Center

    Barker, G. R.

    1983-01-01

    Various topics on the biochemistry of genetic manipulation are discussed. These include genetic transformation and DNA; genetic expression; DNA replication, repair, and mutation; technology of genetic manipulation; and applications of genetic manipulation. Other techniques employed are also considered. (JN)

  7. Genetic manipulation of insulin action and beta-cell function in mice.

    PubMed

    Lamothe, B; Duvillié, B; Cordonnier, N; Baudry, A; Saint-Just, S; Bucchini, D; Jami, J; Joshi, R L

    1998-05-01

    Transgenic and gene targeting approaches have now been applied to a number of genes in order to investigate the metabolic disorders that would result by manipulating insulin action or pancreatic beta-cell function in the mouse. The availability of such mutant mice will allow in the future to develop animal models in which the pathophysiologies resulting from polygenic defects might be reconstituted and studied in detail. Such animal models hopefully will lead to better understanding of complex polygenic diseases such as non-insulin-dependent diabetes mellitus (NIDDM).

  8. Genetic Manipulations in Dermatophytes.

    PubMed

    Alshahni, Mohamed Mahdi; Yamada, Tsuyoshi

    2017-02-01

    Dermatophytes are a group of closely related fungi that nourish on keratinized materials for their survival. They infect stratum corneum, nails, and hair of human and animals, accounting the largest portion of fungi causing superficial mycoses. Huge populations are suffering from dermatophytoses, though the biology of these fungi is largely unknown yet. Reasons are partially attributed to the poor amenability of dermatophytes to genetic manipulation. However, advancements in this field over the last decade made it possible to conduct genetic studies to satisfying extents. These included genetic transformation methods, indispensable molecular tools, i.e., dominant selectable markers, inducible promoter, and marker recycling system, along with improving homologous recombination frequency and gene silencing. Furthermore, annotated genome sequences of several dermatophytic species have recently been available, ensuring an optimal recruitment of the molecular tools to expand our knowledge on these fungi. In conclusion, the establishment of basic molecular tools and the availability of genomic data will open a new era that might change our understanding on the biology and pathogenicity of this fungal group.

  9. Manipulating Genetic Material in Bacteria

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Lisa Crawford, a graduate research assistant from the University of Toledo, works with Laurel Karr of Marshall Space Flight Center (MSFC) in the molecular biology laboratory. They are donducting genetic manipulation of bacteria and yeast for the production of large amount of desired protein. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  10. Manipulating Genetic Material in Bacteria

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Lisa Crawford, a graduate research assistant from the University of Toledo, works with Laurel Karr of Marshall Space Flight Center (MSFC) in the molecular biology laboratory. They are donducting genetic manipulation of bacteria and yeast for the production of large amount of desired protein. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  11. Genetic manipulation of Coxiella burnetii.

    PubMed

    Beare, Paul A

    2012-01-01

    Until very recently, Coxiella burnetii was viewed and studied as an obligate intracellular bacterium that relied exclusively on a eucaryotic host cell for growth. Other medically relevant obligate intracellular bacteria reside in the genera Anaplasma, Chlamydia, Ehrlichia, Orientia, and Rickettsia. An obligate intracellular lifestyle presents a significant obstacle to genetic transformation. Procedures that are straightforward with free-living bacteria, such as antibiotic selection and cloning, can be very difficult when growth of transformants is restricted to a host cell. Long-term passage in host cells to expand small transformant populations can further complicate the procedure. Despite these and other obstacles, at least rudimentary systems are currently available for genetic transformation of most obligate intracellular bacterial pathogens. Dramatically aiding the development of new genetic methods for C. burnetii is the recent discovery of a medium that supports host cell-free growth of the organism in liquid, and importantly, on solid media as clonal colonies. The expanded C. burnetii genetics toolbox now includes transposon systems for random mutagenesis and single-copy, site-specific chromosomal gene knock-ins, as well as a shuttle vector for heterologous gene expression and in trans complementation. A reliable method of targeted gene inactivation remains a challenge. Advances in C. burnetii genetic manipulation will allow identification of genes essential for intracellular parasitism and disease pathogenesis, and undoubtedly fuel new interest in this minimally studied bacterial pathogen.

  12. Functional MRI to assess alterations of functional networks in response to pharmacological or genetic manipulations of the serotonergic system in mice.

    PubMed

    Razoux, Florence; Baltes, Christof; Mueggler, Thomas; Seuwen, Aline; Russig, Holger; Mansuy, Isabelle; Rudin, Markus

    2013-07-01

    Imaging methods that enable the investigation of functional networks both in human and animal brain provide important insights into mechanisms underlying pathologies including psychiatric disorders. Since the serotonergic receptor 1A (5-HT(1A)-R) has been strongly implicated in the pathophysiology of depressive and anxiety disorders, as well as in the action of antidepressant drugs, we investigated brain connectivity related to the 5-HT(1A)-R system by use of pharmacological functional magnetic resonance imaging in mice. We characterized functional connectivity elicited by activation of 5-HT(1A)-R and investigated how pharmacological and genetic manipulations of its function may modulate the evoked connectivity. Functional connectivity elicited by administration of the 5-HT(1A)-R agonist 8-OH-DPAT can be described by networks characterized by small-world attributes with nodes displaying highly concerted response patterns. Circuits identified comprised the brain structures known to be involved in stress-related disorders (e.g. prefrontal cortex, amygdala and hippocampus). The results also highlight the dorsomedial thalamus, a structure associated with fear processing, as a hub of the 5-HT(1A)-R functional network. Administration of a specific 5-HT(1A)-R antagonist or use of heterozygous 5-HT(1A)-R knockout mice significantly reduced functional connectivity elicited by 8-OH-DPAT. Whole brain functional connectivity analysis constitutes an attractive tool to characterize impairments in neurotransmission and the efficacy of pharmacological treatment in a comprehensive manner.

  13. Genetic manipulation of Giardia lamblia.

    PubMed

    Davis-Hayman, Sara R; Nash, Theodore E

    2002-06-01

    Giardia lamblia is a flagellated protozoan that infects several species including humans and is a major agent of waterborne outbreaks of diarrhea. G. lamblia is also important in the study of basic eukaryotic molecular biology and evolution; however, it has been difficult to employ standard genetic methods in the study of Giardia. Over the past 6 years, two transfection systems were developed and used for the genetic manipulation of G. lamblia. Both systems allow transient or stable transfection of Giardia and/or foreign genes. The DNA-based transfection system allows electroporation of circular or linear plasmid DNA into trophozoites. The RNA virus-based transfection system requires electroporation of in vitro transcribed RNA into GLV-infected trophozoites. Because G. lamblia is one of the most rudimentary eukaryotes, its processes of transcription, translation and protein transport, as well as its metabolic and biochemical pathways, are of interest. Study of these areas will continue to be advanced using transfection in combination with cellular and molecular tools. Several groups have combined these technologies with other techniques to study protein transport and the transcriptional and post-transcriptional regulation of Giardia genes, including encystation-specific and variant surface protein genes. In addition, coupling antisense techniques with transfection has permitted functional knockout of Giardia metabolic genes, allowing Giardia metabolic pathways to be studied. In the near future, both transfection systems will be potent tools in our investigations of the perplexing questions in Giardia biology.

  14. Pharmacological activation and genetic manipulation of cystathionine beta-synthase alter circulating levels of homocysteine and hydrogen sulfide in mice.

    PubMed

    Jensen, Kristian K; Geoghagen, Neil S; Jin, Lan; Holt, Tom G; Luo, Qi; Malkowitz, Lorraine; Ni, Weihua; Quan, Shuo; Waters, M Gerard; Zhang, Aiwu; Zhou, Heather H; Cheng, Kang; Luo, Ming-Juan

    2011-01-10

    Hydrogen sulfide (H(2)S) is a recently discovered gasotransmitter found in mammalian tissues and blood. Treatment with H(2)S donor molecules has shown promising results in preclinical models of inflammatory and cardiovascular diseases. Augmentation of H(2)S levels thus holds promise as a novel therapeutic approach for treatment of disease in man. Cystathionine β-synthase (CBS) has been shown to catalyze H(2)S production in vitro. CBS enzyme activity is allosterically regulated by the endogenous activator S-adenosyl methionine. This mode of regulation suggests the possibility for designing a small molecule activator of CBS to enhance H(2)S production. This hypothesis, however, has not been directly tested in vivo. We show here that CBS contributes significantly to endogenous H(2)S production in mice: adenovirus mediated over expression of CBS in the liver significantly increased circulating levels of H(2)S, whereas CBS deficiency resulted in reduced levels. We demonstrate that CBS enzyme from endogenous sources can be activated by S-adenosyl methionine to a greater extent compared to recombinant enzyme, suggesting greater potential for activation than previously anticipated. Importantly, we show that circulating H(2)S levels are increased by pharmacological activation of CBS in vivo; i.e. in the presence of the endogenous activator. Together, our data demonstrate that CBS activity partially regulates endogenous H(2)S in mice, and suggest that pharmacological activation of CBS is a promising approach for enhancing endogenous production of H(2)S for the treatment of cardiovascular and other diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. What is morally new in genetic manipulation?

    PubMed

    Keenan, J F

    1990-01-01

    The investigation into the specific moral issues of genetic manipulation requires us to determine exactly the new moral issues of genetic manipulation. But even that determination requires us to consider whether the context in which we live and the method of moral reflection which we use is adequate enough to address genetic manipulation. Given the liberalist context in which we live, this paper argues that an act-oriented ethics is inadequate and that only a virtue-oriented ethics enables us to recognize and resolve the new problems ahead of us in genetic manipulation. Moreover, those problems have a common root, that is, that through genetics we will be in danger of objectifying the human subject.

  16. Genetic manipulation of acidophilic bacteria

    SciTech Connect

    Ward, T.E.; Rowland, M.L.; Glenn, A.W.; Watkins, C.S.; Bruhn, D.F.; Bulmer, D.; Roberto, F.F.

    1989-01-01

    Thiobacillus ferrooxidans is important in leaching of metals from mineral ores and in the removal of pyritic sulfur from coal. It is also intimately involved in production of acid mine drainage. Other acidophilic bacteria, including members of the genus Acidiphilium, are usually present in the same environments as T. ferrooxidans, and there is evidence to suggest that these acidophilic heterotrophs may increase the rate of T. ferrooxidans' attack on inorganic sulfides. Our laboratory is studying the genetic characteristics of these acidophilic bacteria and developing techniques for introducing desirable genes into them. Several endogenous plasmids from Acidiphilium strains have been cloned into E. coli vectors. Some of the resulting plasmids are able to confer antibiotic resistance to Acidiphilium after transformation by electroporation. In addition, a broad-host range plasmid conferring resistance to tetracycline has been introduced into Acidiphilium strains by electroporation. This same plasmid, has also been transferred to Acidiphilium from E. coli directly by conjugation. A temperate bacteriophage which infects a number of Acidiphilium isolates has been discovered and partially characterized. It has a lambdoid morphology and a genome of approximately 97 kb, comprised of double-stranded DNA which is probably modified. 16 refs., 2 figs., 4 tabs.

  17. Genetic manipulation of cardiac ageing.

    PubMed

    Cannon, Leah; Bodmer, Rolf

    2016-04-15

    Ageing in humans is associated with a significant increase in the prevalence of cardiovascular disease. We still do not fully understand the molecular mechanisms underpinning this correlation. However, a number of insights into which genes control cardiac ageing have come from studying hearts of the fruit fly, Drosophila melanogaster. The fly's simple heart tube has similar molecular structure and basic physiology to the human heart. Also, both fly and human hearts experience significant age-related morphological and functional decline. Studies on the fly heart have highlighted the involvement of key nutrient sensing, ion channel and sarcomeric genes in cardiac ageing. Many of these genes have also been implicated in ageing of the mammalian heart. Genes that increase oxidative stress, or are linked to cardiac hypertrophy or neurodegenerative diseases in mammals also affect cardiac ageing in the fruit fly. Moreover, fly studies have demonstrated the potential of exercise and statins to treat age-related cardiac disease. These results show the value of Drosophila as a model to discover the genetic causes of human cardiac ageing. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  18. Molecular Genetic Manipulation of Vector Mosquitoes

    PubMed Central

    Terenius, Olle; Marinotti, Osvaldo; Sieglaff, Douglas; James, Anthony A.

    2008-01-01

    Genetic strategies for reducing populations of vector mosquitoes or replacing them with those that are not able to transmit pathogens benefit greatly from molecular tools that allow gene manipulation and transgenesis. Mosquito genome sequences and associated EST (Expressed Sequence Tags) databases enable large-scale investigations to provide new insights into evolutionary, biochemical, genetic, metabolic and physiological pathways. Additionally, comparative genomics reveals the bases for evolutionary mechanisms with particular focus on specific interactions between vectors and pathogens. We discuss how this information may be exploited for the optimization of transgenes that interfere with the propagation and development of pathogens in their mosquito hosts. PMID:18996342

  19. REVIEW: GENETIC MANIPULATION OF THE RODENT PLACENTA

    PubMed Central

    Renaud, Stephen J.; Rumi, M.A. Karim; Soares, Michael J.

    2011-01-01

    The principal role of the placenta is the maintenance of pregnancy and promotion of fetal growth and viability. The use of transgenic rodents has greatly enhanced our understanding of placental development and function. However, embryonic lethality is often a confounding variable in determining whether a genetic modification adversely affected placental development. In these cases, it is beneficial to specifically manipulate the placental genome. The purpose of this review is to summarize available methodologies for specific genetic modification of the rodent placenta. By restricting genetic alterations to the trophoblast lineage, it is possible to gain a deeper understanding of placental development that perhaps will lead to gene-targeted therapies to rescue irregular placentation in transgenic animals or in women at high-risk for placenta-associated pregnancy complications. PMID:21256588

  20. Molecular genetic manipulation of mosquito vectors.

    PubMed

    Carlson, J; Olson, K; Higgs, S; Beaty, B

    1995-01-01

    Despite their central role in disease transmission, relatively little is known of the molecular biology of arthropod vectors. Modern molecular approaches will undoubtedly provide considerable information about gene regulation and expression in vectors and consequently a much better understanding of the biology and molecular biology of vectors. Such knowledge is essential for developing effective control strategies for vector-borne diseases. In this review, we focus upon techniques and approaches used at the Arthropod-Borne and Infectious Diseases Laboratory (AIDL) at Colorado State University to bioengineer mosquitoes with reduced vector competence. We have developed technologies and procedures that allow genetic manipulation of mosquitoes, including RNA and DNA virus gene-delivery vehicles and efficacious antiviral constructs, which will facilitate the development of pathogen-resistant, transformed mosquitoes. Many of the approaches, constructs, and technologies developed at AIDL will be applicable to molecular manipulation of other arthropod genomes.

  1. Genetic Manipulation of Neurofilament Protein Phosphorylation.

    PubMed

    Jones, Maria R; Villalón, Eric; Garcia, Michael L

    2016-01-01

    Neurofilament biology is important to understanding structural properties of axons, such as establishment of axonal diameter by radial growth. In order to study the function of neurofilaments, a series of genetically modified mice have been generated. Here, we describe a brief history of genetic modifications used to study neurofilaments, as well as an overview of the steps required to generate a gene-targeted mouse. In addition, we describe steps utilized to analyze neurofilament phosphorylation status using immunoblotting. Taken together, these provide comprehensive analysis of neurofilament function in vivo, which can be applied to many systems.

  2. Genetic Tools for Identifying and Manipulating Fibroblasts in the Mouse

    PubMed Central

    Swonger, Jessica M.; Liu, Jocelyn S.; Ivey, Malina J.; Tallquist, Michelle D.

    2016-01-01

    The use of mouse genetic tools to track and manipulate fibroblasts has provided invaluable in vivo information regarding the activities of these cells. Recently, many new mouse strains have been described for the specific purpose of studying fibroblast behavior. Colorimetric reporter mice and lines expressing Cre are available for the study of fibroblasts in the organs prone to fibrosis, including heart, kidney, liver, lung, and skeletal muscle. In this review we summarize the current state of the models that have been used to define tissue resident fibroblast populations. While these complex genetic reagents provide unique insights into the process of fibrosis, they also require a thorough understanding of the caveats and limitations. Here, we discuss the specificity and efficiency of the available genetic models and briefly describe how they have been used to document the mechanisms of fibrosis. PMID:27342817

  3. Genetic manipulation of lignocellulosic biomass for bioenergy.

    PubMed

    Wang, Peng; Dudareva, Natalia; Morgan, John A; Chapple, Clint

    2015-12-01

    Lignocellulosic biomass represents an abundant and sustainable raw material for biofuel production. The recalcitrance of biomass to degradation increases the estimated cost of biofuel production and limits its competitiveness in the market. Genetic engineering of lignin, a major recalcitrance factor, improves saccharification and thus the potential yield of biofuels. Recently, our understanding of lignification and its regulation has been advanced by new studies in various systems, all of which further enhances our ability to manipulate the biosynthesis and deposition of lignin in energy crops for producing cost-effective second generation biofuels.

  4. Diet selection in immunologically manipulated mice.

    PubMed

    Teixeira, Gerlinde; Paschoal, Patrícia Olaya; de Oliveira, Vivian Leite; Pedruzzi, Monique M B; Campos, Sylvia M N; Andrade, Luiz; Nóbrega, Alberto

    2008-01-01

    Diet selection is a complex problem that animals in wildlife have to deal with daily. In their natural environment, these animals meet a great variety of foods some of which they are able and prepared to eat, yet, not all of it is eaten. In addition to the biological factors, some of which we shall discuss deeper in this paper, an important factor in food preference is social contact. Alterations in the physiology of mammals can have profound effects on the choice or preference for certain foods. On the other hand the decline of taste and smell perception in the elderly, the degree of food restriction, the sensorial properties of foods (such as presentation, taste, and smell) can be considered factors that influence feeding behavior leading to aversion. Many species, including man, learn to associate nausea with taste, and as a consequence avoid its specific intake, which has been shown to be persistent. Conditioned taste aversion is a form of associative learning in which animals display an aversion to the taste of a food that has previously been paired with illness. Our group has investigated the pattern of ingestion of foods that are frequently eaten by mice in wildlife and are potentially allergenic to humans in order to study the immunological consequences to these foods such as oral tolerance and inflammatory processes of the gut. We have chosen two seeds, peanuts (Arachis hypogea) and cashew nuts (Anacardium occidentale), as our source of antigens as the first is considered to be one of the most potent food allergens and for the second there seems to be very little allergy in the human setting. We used male and female, normal, adult CBA/J, A/J, C57BL/6 and Balb/c mice 2-3 months old and hybrid (C57Bl/6xBalb/c) F1, (Balb/cxC57Bl/6) F1), (C57Bl/6xDBA2) F1 mice. Food preference appeared to be strain-specific. Animals tolerized to a determined seed, then immunized with its protein extract and re-exposed to the seed in natura alter their feeding pattern. We

  5. Pharmacological and Genetic Manipulation of p53 in Brown Fat at Adult But Not Embryonic Stages Regulates Thermogenesis and Body Weight in Male Mice.

    PubMed

    Al-Massadi, Omar; Porteiro, Begoña; Kuhlow, Doreen; Köhler, Markus; Gonzalez-Rellan, María J; Garcia-Lavandeira, Montserrat; Díaz-Rodríguez, Esther; Quiñones, Mar; Senra, Ana; Alvarez, Clara V; López, Miguel; Diéguez, Carlos; Schulz, Tim J; Nogueiras, Rubén

    2016-07-01

    p53 is a well-known tumor suppressor that plays multiple biological roles, including the capacity to modulate metabolism at different levels. However, its metabolic role in brown adipose tissue (BAT) remains largely unknown. Herein we sought to investigate the physiological role of endogenous p53 in BAT and its implication on BAT thermogenic activity and energy balance. To this end, we generated and characterized global p53-null mice and mice lacking p53 specifically in BAT. Additionally we performed gain-and-loss-of-function experiments in the BAT of adult mice using virogenetic and pharmacological approaches. BAT was collected and analyzed by immunohistochemistry, thermography, real-time PCR, and Western blot. p53-deficient mice were resistant to diet-induced obesity due to increased energy expenditure and BAT activity. However, the deletion of p53 in BAT using a Myf5-Cre driven p53 knockout did not show any changes in body weight or the expression of thermogenic markers. The acute inhibition of p53 in the BAT of adult mice slightly increased body weight and inhibited BAT thermogenesis, whereas its overexpression in the BAT of diet-induced obese mice reduced body weight and increased thermogenesis. On the other hand, pharmacological activation of p53 improves body weight gain due to increased BAT thermogenesis by sympathetic nervous system in obese adult wild-type mice but not in p53(-/-) animals. These results reveal that p53 regulates BAT metabolism by coordinating body weight and thermogenesis, but these metabolic actions are tissue specific and also dependent on the developmental stage.

  6. Genetic manipulation of the ghrelin signaling system in male mice reveals bone compartment specificity of acylated and unacylated ghrelin in the regulation of bone remodeling

    USDA-ARS?s Scientific Manuscript database

    Ghrelin receptor-deficient (Ghsr-/-) mice that lack acylated ghrelin (AG) signaling retain a metabolic response to unacylated ghrelin (UAG). Recently, we showed that Ghsr-deficiency affects bone metabolism. The aim of this study was to further establish the impact of AG and UAG on bone metabolism. W...

  7. Platelet gene therapy corrects the hemophilic phenotype in immunocompromised hemophilia A mice transplanted with genetically manipulated human cord blood stem cells.

    PubMed

    Shi, Qizhen; Kuether, Erin L; Chen, Yingyu; Schroeder, Jocelyn A; Fahs, Scot A; Montgomery, Robert R

    2014-01-16

    Our previous studies have demonstrated that platelet FVIII (2bF8) gene therapy can improve hemostasis in hemophilia A mice, even in the presence of inhibitory antibodies, but none of our studies has targeted human cells. Here, we evaluated the feasibility for lentivirus (LV)-mediated human platelet gene therapy of hemophilia A. Human platelet FVIII expression was introduced by 2bF8LV-mediated transduction of human cord blood (hCB) CD34(+) cells followed by xenotransplantation into immunocompromised NSG mice or NSG mice in an FVIII(null) background (NSGF8KO). Platelet FVIII was detected in all recipients that received 2bF8LV-transduced hCB cells as long as human platelet chimerism persisted. All NSGF8KO recipients (n = 7) that received 2bF8LV-transduced hCB cells survived tail clipping if animals had greater than 2% of platelets derived from 2bF8LV-transduced hCB cells, whereas 5 of 7 survived when human platelets were 0.3% to 2%. Whole blood clotting time analysis confirmed that hemostasis was improved in NSGF8KO mice that received 2bF8LV-transduced hCB cells. We demonstrate, for the first time, the feasibility of 2bF8LV gene delivery to human hematopoietic stem cells to introduce FVIII expression in human platelets and that human platelet-derived FVIII can improve hemostasis in hemophilia A.

  8. Platelet gene therapy corrects the hemophilic phenotype in immunocompromised hemophilia A mice transplanted with genetically manipulated human cord blood stem cells

    PubMed Central

    Kuether, Erin L.; Chen, Yingyu; Schroeder, Jocelyn A.; Fahs, Scot A.; Montgomery, Robert R.

    2014-01-01

    Our previous studies have demonstrated that platelet FVIII (2bF8) gene therapy can improve hemostasis in hemophilia A mice, even in the presence of inhibitory antibodies, but none of our studies has targeted human cells. Here, we evaluated the feasibility for lentivirus (LV)-mediated human platelet gene therapy of hemophilia A. Human platelet FVIII expression was introduced by 2bF8LV-mediated transduction of human cord blood (hCB) CD34+ cells followed by xenotransplantation into immunocompromised NSG mice or NSG mice in an FVIIInull background (NSGF8KO). Platelet FVIII was detected in all recipients that received 2bF8LV-transduced hCB cells as long as human platelet chimerism persisted. All NSGF8KO recipients (n = 7) that received 2bF8LV-transduced hCB cells survived tail clipping if animals had greater than 2% of platelets derived from 2bF8LV-transduced hCB cells, whereas 5 of 7 survived when human platelets were 0.3% to 2%. Whole blood clotting time analysis confirmed that hemostasis was improved in NSGF8KO mice that received 2bF8LV-transduced hCB cells. We demonstrate, for the first time, the feasibility of 2bF8LV gene delivery to human hematopoietic stem cells to introduce FVIII expression in human platelets and that human platelet–derived FVIII can improve hemostasis in hemophilia A. PMID:24269957

  9. Genetic manipulation of sinusoidal endothelial cells.

    PubMed

    Takei, Yoshiyuki; Maruyama, Atsushi; Ikejima, Kenichi; Enomoto, Nobuyuki; Yamashina, Shunhei; Lemasters, John J; Sato, Nobuhiro

    2007-06-01

    Altered gene expression in liver sinusoidal endothelial cells (SEC) is associated with a variety of aspects of liver pathophysiology. It is, therefore, possible to envision a new therapeutic strategy for treatment of intractable liver diseases and achievement of graft-specific immunotolerance through modulation of SEC functions by genetic engineering. The SEC possesses unique hyaluronan receptors that recognize and internalize hyaluronic acid (HA). This characteristic was used in the development of a system for targeting foreign DNA to SEC. A gene carrier system was prepared by coupling HA oligomers to poly L-lysine (PLL) in a 1:1 weight ratio by reductive amination reaction. The resulting copolymer (PLL-g-HA) was mixed with various amounts of DNA in 154 mM NaCl. Inter-polyelectrolyte complex formation between PLL-g-HA and DNA exhibited minimal self-aggregation, explaining the highly soluble nature of the complex. Complex formation between PLL-g-HA and DNA was further assessed with a gel retardation assay. The titration point representing the minimum proportion of PLL-g-HA required to retard the DNA completely occurred at a 1:1 copolymer (based on PLL) to DNA charge ratio. Following intravenous injection of (32)P-labeled pSV beta-Gal plasmid complexed to PLL-g-HA in Wistar rats, >90% of the injected counts were shown to be taken up by the liver. Further, it was shown that the PLL-g-HA/DNA complex was distributed exclusively in the SEC. At 72 h after injection of 90 mug of pSV beta-Gal in a PLL-g-HA-complexed form, a large number of SEC expressing beta-galactosidase were detected. So, the PLL-g-HA/DNA system permits targeted delivery of exogenous nucleotide agents selectively to the liver SEC, providing a novel strategy for manipulation of SEC functions.

  10. Dietary manipulations influence sucrose acceptance in diet induced obese mice.

    PubMed

    Johnson, Alexander W

    2012-02-01

    The current studies examined the influence of a high fat diet on sucrose acceptance in diet induced obese (DIO) mice. C57BL/6J mice were placed on either a 45 kcal% fat diet (group DIO), or a control 10% kcal fat diet (group control) for 12 weeks followed by sucrose consumption tests and dietary manipulations. After 12 weeks exposure, body weights of DIO mice significantly exceeded those of the control mice. During subsequent sucrose consumption tests, DIO mice showed suppression in the total number of licks relative to controls. In a second experiment, consumption tests with water and a variety of sucrose concentrations revealed a hypophagic phenotype in naïve DIO mice. Licking microstructure analyses were conducted on the licking behavior of all mice, which revealed a reduction in burst size and number for DIO mice. Subsequently, we examined whether 10 days exposure to regular lab chow would alter sucrose consumption and taste evaluation in DIO mice. As a result of this dietary switch, all mice showed comparable licking behavior suggesting that exposure to the high-fat diet and diet-induced obesity may reduce preferences for other tastants in C57BL/6J mice. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Genetic Manipulation of a Naturally Competent Bacterium, Helicobacter pylori

    PubMed Central

    Noto, Jennifer M.; Peek, Richard M.

    2013-01-01

    Genetic manipulation of Helicobacter pylori facilitates characterization and functional analysis of individual H. pylori genes. This chapter discusses the methods involved in H. pylori chromosomal DNA isolation, mutagenesis of individual genes, and natural transformation. PMID:23015491

  12. Effect of Enrichment Devices on Aggression in Manipulated Nude Mice.

    PubMed

    Lockworth, Cynthia R; Kim, Sun-Jin; Liu, Jun; Palla, Shana L; Craig, Suzanne L

    2015-11-01

    Agonistic behavior in group-housed male mice is a recurring problem in many animal research facilities. Common management procedures, such as the removal of aggressors, are moderately successful but often fail, owing to recurrence of aggressive behavior among cagemates. Studies have incorporated enrichment devices to attenuate aggression, but such devices have had mixed results. However, these studies did not include research manipulations when assessing the benefits of various enrichment devices. We obtained 100 male athymic nude mice and studied the efficacy of various enrichment devices, including cotton squares, paper rolls, shredded paper, nylon bones, and a mouse house and wheel combination in the reduction of fighting during an ongoing study that involved randomization followed by prostate and intratibial injections. Groups were evaluated according to a numerical grading system for wound assessment. Examination of the data revealed that the enrichment devices had no effect on the presence of wounds, thus none of the devices tested affected fighting in nude mice. However, when mice began experimental use, fight wounds increased significantly at cage change and after randomization, reflecting a disruption of existing social hierarchies. Therefore, in the context of an actual research study that involves common manipulations, the specific enrichment device had less effect on aggression in male nude mice than did the destruction and reconstruction of social structures within each group.

  13. Using genetically engineered mice for radiation research.

    PubMed

    Kirsch, David G

    2011-09-01

    The laboratory mouse has been used for many decades as a model system for radiation research. Recent advances in genetic engineering now allow scientists to delete genes in specific cell types at different stages of development. The ability to manipulate genes in the mouse with spatial and temporal control opens new opportunities to investigate the role of genes in regulating the response of normal tissues and tumors to radiation. Currently, we are using the Cre-loxP system to delete genes, such as p53, in a cell-type specific manner in mice to study mechanisms of acute radiation injury and late effects of radiation. Our results demonstrate that p53 is required in the gastrointestinal (GI) epithelium to prevent radiation-induced GI syndrome and in endothelial and/or hematopoietic cells to prevent late effects of radiation. We have also used these genetic tools to generate primary tumors in mice to study tumor response to radiation therapy. These advances in genetic engineering provide a powerful model system to dissect both the mechanisms of normal tissue injury after irradiation and the mechanisms by which radiation cures cancer.

  14. Improved Wood Properties Through Genetic Manipulation

    SciTech Connect

    2006-10-01

    This factsheet describes a research project to replacing the more chemically resistant guaiacyl (G) lignin with the less resistant hardwood guaiacyl (G)-syringyl (S) lignin genes. Achieving this genetic change would reduce the energy, chemical, and bleaching required in Kraft pulp production of softwoods.

  15. Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)

    PubMed Central

    Le Breton, Yoann; McIver, Kevin S.

    2013-01-01

    Streptococcus pyogenes (the group A streptococcus, GAS) is a Gram-positive bacterium responsible for a wide spectrum of diseases ranging from mild superficial infections (pharyngitis, impetigo) to severe often life-threatening invasive diseases (necrotizing fasciitis, streptococcal toxic shock syndrome) in humans. This unit describes molecular techniques for the genetic manipulation of S. pyogenes with detailed protocols for transformation, gene disruption, allelic exchange, transposon mutagenesis, and genetic complementation. PMID:24510894

  16. Genetic manipulation of Streptococcus pyogenes (the Group A Streptococcus, GAS).

    PubMed

    Le Breton, Yoann; McIver, Kevin S

    2013-10-02

    Streptococcus pyogenes (the Group A Streptococcus, GAS) is a Gram-positive bacterium responsible for a wide spectrum of diseases ranging from mild superficial infections (pharyngitis, impetigo) to severe, often life-threatening invasive diseases (necrotizing fasciitis, streptococcal toxic shock syndrome) in humans. This unit describes molecular techniques for the genetic manipulation of S. pyogenes with detailed protocols for transformation, gene disruption, allelic exchange, transposon mutagenesis, and genetic complementation.

  17. Murine Norovirus: Propagation, Quantification and Genetic Manipulation

    PubMed Central

    Hwang, Seungmin; Alhatlani, Bader; Arias, Armando; Caddy, Sarah L; Christodoulou, Constantina; Cunha, Juliana; Emmott, Ed; Gonzalez-Hernandez, Marta; Kolawole, Abimbola; Lu, Jia; Rippinger, Christine; Sorgeloos, Frédéric; Thorne, Lucy; Vashist, Surender; Goodfellow, Ian

    2014-01-01

    Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. It is the most common pathogen in biomedical research colonies. MNV is also related to the human noroviruses, which cause the majority of non-bacterial gastroenteritis worldwide. Like the human noroviruses, MNV is an enteric virus that replicates in the intestine and is transmitted by the fecal-oral route. MNV replicates in murine macrophages and dendritic cells in cells in culture and in the murine host. This virus is often used to study mechanisms in norovirus biology, because the human noroviruses are refractory to growth in cell culture. MNV combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Herein, we describe a panel of techniques that are commonly used to study MNV biology. PMID:24789596

  18. Genetically manipulated virulence of Yersinia enterocolitica.

    PubMed Central

    Heesemann, J; Algermissen, B; Laufs, R

    1984-01-01

    Mobilizable virulence plasmids of Yersinia enterocolitica of serotypes O:3 and O:9 were constructed by cointegration of a mobilizable vector into the virulence plasmids. The obtained cointegrates were mobilized into plasmidless Y. enterocolitica strains of serotypes O:3, O:5, O:8, and O:9. The transfer experiments revealed the existence of two different subgroups of plasmid-associated traits. (i) Animal virulence functions (mouse lethality and conjuctivitis provocation) were only transferable to plasmid-cured derivatives of virulent parent strains (serotypes O:3, O:8, and O:9), but they were not transferable to Y. enterocolitica antigen reference strains (serotypes O:3 and O:8) or to a plasmidless clinical isolate of serotype O:5. A further striking result was that a serotype O:8 strain regained the mouse lethality trait after receipt of a plasmid from a strain not lethal to mice. These results demonstrate that plasmid-mediated animal virulence functions are not uniformly expressed within Y. enterocolitica. (ii) The second subgroup of plasmid-mediated traits (calcium dependency, surface agglutinogens, HEp-2 cell adherence, and protein release) were transferable to all Y. enterocolitica recipient strains tested (serotypes O:3, O:5, O:8, and O:9 of different origin). For the first time HEp-2 cell adherence and temperature-induced release of five major protein species are described as transferable traits. Images PMID:6480101

  19. Public Attitudes toward Human Genetic Manipulation: A Revitalization of Eugenics?

    ERIC Educational Resources Information Center

    Veglia, Geremia; And Others

    The purpose of this investigation was to measure the attitudes of college students across the United States concerning the possible use of genetic manipulation, especially in terms of enhancing human physical and intellectual characteristics. The instrument used was divided into three general areas of inquiry: the first, designed to measure the…

  20. Public Attitudes toward Human Genetic Manipulation: A Revitalization of Eugenics?

    ERIC Educational Resources Information Center

    Veglia, Geremia; And Others

    The purpose of this investigation was to measure the attitudes of college students across the United States concerning the possible use of genetic manipulation, especially in terms of enhancing human physical and intellectual characteristics. The instrument used was divided into three general areas of inquiry: the first, designed to measure the…

  1. Genetic manipulation for inherited neurodegenerative diseases: myth or reality?

    PubMed Central

    Yu-Wai-Man, Patrick

    2016-01-01

    Rare genetic diseases affect about 7% of the general population and over 7000 distinct clinical syndromes have been described with the majority being due to single gene defects. This review will provide a critical overview of genetic strategies that are being pioneered to halt or reverse disease progression in inherited neurodegenerative diseases. This field of research covers a vast area and only the most promising treatment paradigms will be discussed with a particular focus on inherited eye diseases, which have paved the way for innovative gene therapy paradigms, and mitochondrial diseases, which are currently generating a lot of debate centred on the bioethics of germline manipulation. PMID:27002113

  2. Genetic manipulation of poxviruses using bacterial artificial chromosome recombineering.

    PubMed

    Cottingham, Matthew G

    2012-01-01

    Traditional methods for genetic manipulation of poxviruses rely on low-frequency natural recombination in virus-infected cells. Although these powerful systems represent the technical foundation of current knowledge and applications of poxviruses, they require long (≥ 500 bp) flanking sequences for homologous recombination, an efficient viral selection method, and burdensome, time-consuming plaque purification. The beginning of the twenty-first century has seen the application of bacterial artificial chromosome (BAC) technology to poxviruses as an alternative method for their genetic manipulation, following the invention of a long-sought-after method for deriving a BAC clone of vaccinia virus (VAC-BAC) by Arban Domi and Bernard Moss. The key advantages of the BAC system are the ease and versatility of performing genetic manipulation using bacteriophage λ Red recombination (recombineering), which requires only ∼50 bp homology arms that can be easily created by PCR, and which allows seamless mutations lacking any marker gene without having to perform transient-dominant selection. On the other hand, there are disadvantages, including the significant setup time, the risk of contamination of the cloned genome with bacterial insertion sequences, and the nontrivial issue of removal of the BAC cassette from derived viruses. These must be carefully weighed to decide whether the use of BACs will be advantageous for a particular application, making pox-BAC systems likely to complement, rather than supplant, traditional methods in most laboratories.

  3. Human cytomegalovirus: bacterial artificial chromosome (BAC) cloning and genetic manipulation.

    PubMed

    Paredes, Anne M; Yu, Dong

    2012-02-01

    The understanding of human cytomegalovirus (HCMV) biology was long hindered by the inability to perform efficient viral genetic analysis. This hurdle was recently overcome when the genomes of multiple HCMV strains were cloned as infectious bacterial artificial chromosomes (BACs). The BAC system takes advantage of the single-copy F plasmid of E. coli that can stably carry large pieces of foreign DNA. In this system, a recombinant HCMV virus carrying a modified F plasmid is first generated in eukaryotic cells. Recombinant viral genomes are then isolated and recovered in E. coli as BAC clones. BAC-captured viral genomes can be manipulated using prokaryotic genetics, and recombinant virus can be reconstituted from BAC transfection in eukaryotic cells. The BAC reverse genetic system provides a reliable and efficient method to introduce genetic alterations into the viral genome in E.coli and subsequently analyze their effects on virus biology in eukaryotic cells.

  4. Genetically manipulated mouse models of lung disease: potential and pitfalls

    PubMed Central

    Choi, Alexander J. S.; Owen, Caroline A.; Choi, Augustine M. K.

    2012-01-01

    Gene targeting in mice (transgenic and knockout) has provided investigators with an unparalleled armamentarium in recent decades to dissect the cellular and molecular basis of critical pathophysiological states. Fruitful information has been derived from studies using these genetically engineered mice with significant impact on our understanding, not only of specific biological processes spanning cell proliferation to cell death, but also of critical molecular events involved in the pathogenesis of human disease. This review will focus on the use of gene-targeted mice to study various models of lung disease including airways diseases such as asthma and chronic obstructive pulmonary disease, and parenchymal lung diseases including idiopathic pulmonary fibrosis, pulmonary hypertension, pneumonia, and acute lung injury. We will attempt to review the current technological approaches of generating gene-targeted mice and the enormous dataset derived from these studies, providing a template for lung investigators. PMID:22198907

  5. Kanamycin Resistance Cassette for Genetic Manipulation of Treponema denticola.

    PubMed

    Li, Yuebin; Ruby, John; Wu, Hui

    2015-07-01

    Treponema denticola has been recognized as an important oral pathogen of the "red complex" bacterial consortium that is associated with the pathogenesis of endodontal and periodontal diseases. However, little is known about the virulence of T. denticola due to its recalcitrant genetic system. The difficulty in genetically manipulating oral spirochetes is partially due to the lack of antibiotic resistance cassettes that are useful for gene complementation following allelic replacement mutagenesis. In this study, a kanamycin resistance cassette was identified and developed for the genetic manipulation of T. denticola ATCC 35405. Compared to the widely used ermF-ermAM cassette, the kanamycin cassette used in the transformation experiments gave rise to additional antibiotic-resistant T. denticola colonies. The kanamycin cassette is effective for allelic replacement mutagenesis as demonstrated by inactivation of two open reading frames of T. denticola, TDE1430 and TDE0911. In addition, the cassette is also functional in trans-chromosomal complementation. This was determined by functional rescue of a periplasmic flagellum (PF)-deficient mutant that had the flgE gene coding for PF hook protein inactivated. The integration of the full-length flgE gene into the genome of the flgE mutant rescued all of the defects associated with the flgE mutant that included the lack of PF filament and spirochetal motility. Taken together, we demonstrate that the kanamycin resistance gene is a suitable cassette for the genetic manipulation of T. denticola that will facilitate the characterization of virulence factors attributed to this important oral pathogen.

  6. Development of Genetic Tools for the Manipulation of the Planctomycetes

    PubMed Central

    Rivas-Marín, Elena; Canosa, Inés; Santero, Eduardo; Devos, Damien P.

    2016-01-01

    Bacteria belonging to the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum are of interest for biotechnology, evolutionary cell biology, ecology, and human health. Some PVC species lack a number of typical bacterial features while others possess characteristics that are usually more associated to eukaryotes or archaea. For example, the Planctomycetes phylum is atypical for the absence of the FtsZ protein and for the presence of a developed endomembrane system. Studies of the cellular and molecular biology of these infrequent characteristics are currently limited due to the lack of genetic tools for most of the species. So far, genetic manipulation in Planctomycetes has been described in Planctopirus limnophila only. Here, we show a simple approach that allows mutagenesis by homologous recombination in three different planctomycetes species (i.e., Gemmata obscuriglobus, Gimesia maris, and Blastopirellula marina), in addition to P. limnophila, thus extending the repertoire of genetically modifiable organisms in this superphylum. Although the Planctomycetes show high resistance to most antibiotics, we have used kanamycin resistance genes in G. obscuriglobus, P. limnophila, and G. maris, and tetracycline resistance genes in B. marina, as markers for mutant selection. In all cases, plasmids were introduced in the strains by mating or electroporation, and the genetic modification was verified by Southern Blotting analysis. In addition, we show that the green fluorescent protein (gfp) is expressed in all four backgrounds from an Escherichia coli promoter. The genetic manipulation achievement in four phylogenetically diverse planctomycetes will enable molecular studies in these strains, and opens the door to developing genetic approaches not only in other planctomycetes but also other species of the superphylum, such as the Lentisphaerae. PMID:27379046

  7. Development of Genetic Tools for the Manipulation of the Planctomycetes.

    PubMed

    Rivas-Marín, Elena; Canosa, Inés; Santero, Eduardo; Devos, Damien P

    2016-01-01

    Bacteria belonging to the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum are of interest for biotechnology, evolutionary cell biology, ecology, and human health. Some PVC species lack a number of typical bacterial features while others possess characteristics that are usually more associated to eukaryotes or archaea. For example, the Planctomycetes phylum is atypical for the absence of the FtsZ protein and for the presence of a developed endomembrane system. Studies of the cellular and molecular biology of these infrequent characteristics are currently limited due to the lack of genetic tools for most of the species. So far, genetic manipulation in Planctomycetes has been described in Planctopirus limnophila only. Here, we show a simple approach that allows mutagenesis by homologous recombination in three different planctomycetes species (i.e., Gemmata obscuriglobus, Gimesia maris, and Blastopirellula marina), in addition to P. limnophila, thus extending the repertoire of genetically modifiable organisms in this superphylum. Although the Planctomycetes show high resistance to most antibiotics, we have used kanamycin resistance genes in G. obscuriglobus, P. limnophila, and G. maris, and tetracycline resistance genes in B. marina, as markers for mutant selection. In all cases, plasmids were introduced in the strains by mating or electroporation, and the genetic modification was verified by Southern Blotting analysis. In addition, we show that the green fluorescent protein (gfp) is expressed in all four backgrounds from an Escherichia coli promoter. The genetic manipulation achievement in four phylogenetically diverse planctomycetes will enable molecular studies in these strains, and opens the door to developing genetic approaches not only in other planctomycetes but also other species of the superphylum, such as the Lentisphaerae.

  8. Genetic manipulation of Xanthophyllomyces dendrorhous and Phaffia rhodozyma.

    PubMed

    Lin, Guangyun; Bultman, Joanna; Johnson, Eric A; Fell, Jack W

    2012-01-01

    The yeasts Xanthophyllomyces dendrorhous (teleomorph) and Phaffia rhodozyma (anamorph) are of basidiomycetous affinity and have the unique property among yeasts of producing the carotenoid pigment astaxanthin. Astaxanthin imparts the attractive coloration to salmonids, crustaceans, and several birds such as the flamingo, and it has considerable economic value. Microbiological and genetic techniques for manipulation are rudimentary in the yeast, while their utility would be valuable for strain development including hypermutants that overproduce astaxanthin. Here we describe methods for manipulation of the yeast, including induction of the sexual stage with basidiospore formation, methods for isolation of mutants (particularly mutants affected in carotenoid biosynthesis) as well as techniques for isolation and analysis of carotenoids. These methods are valuable for understanding the biology and enhancing the biotechnology value of the yeast.

  9. Genetic-based motion planning for articulated robotic manipulators

    NASA Astrophysics Data System (ADS)

    Chan, Kwong K.; Zalzala, Ali M. S.

    1993-05-01

    A genetic based algorithm for the minimum time trajectory planning of articulated robotic manipulators is proposed. The planning procedure is performed in the configuration space and respects all physical constraints imposed on the manipulator design, including the limits on the torque values applied to the motor of each joint of the arm. The complete nonlinear dynamic robot model is incorporated in the formulation. The purpose of the algorithm is to tessellate joint space into a grid of possible motion nodes, where at each option node, given the position and velocity at the previous node, possible velocity values are constrained by the time optimality together with the dynamics of the arm. The algorithm is proven to be more efficient than conventional heuristic search technique with a reduction of the execution time.

  10. Genetic manipulation of Staphylococci—breaking through the barrier

    PubMed Central

    Monk, Ian R.; Foster, Timothy J.

    2012-01-01

    Most strains of Staphylococcus aureus and Staphylococcus epidermidis possess a strong restriction barrier that hinders exchange of DNA. Recently, major advances have been made in identifying and characterizing the restriction-modification (RM) systems involved. In particular a novel type IV restriction enzyme that recognizes cytosine methylated DNA has been shown to be the major barrier to transfer of plasmid DNA from Escherichia coli into S. aureus and S. epidermidis. While the conserved type I RM system provides a further barrier. Here we review the recent advances in understanding of restriction systems in staphylococci and highlight how this has been exploited to improve our ability to manipulate genetically previously untransformable strains. PMID:22919640

  11. Genetic regulation and manipulation for natural product discovery.

    PubMed

    Chen, Jianwei; Wu, Qihao; Hawas, Usama W; Wang, Hong

    2016-04-01

    Natural products are an important source of modern medical development, e.g., antibiotics, anticancers, immune modulators, etc. and will continue to be a powerful driving force for the discovery of novel potential drugs. In the heterologous hosts, natural products are biosynthesized using dedicated metabolic networks. By gene engineering, pathway reconstructing, and enzyme engineering, metabolic networks can be modified to synthesize novel compounds containing enhanced structural feature or produce a large quantity of known valuable bioactive compounds. The review introduces some important technical platforms and relevant examples of genetic regulation and manipulation to improve natural product titers or drive novel secondary metabolite discoveries.

  12. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells.

    PubMed

    Reichert, Maximilian; Takano, Shigetsugu; Heeg, Steffen; Bakir, Basil; Botta, Gregory P; Rustgi, Anil K

    2013-01-01

    The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles duct cells morphologically and, to some extent, at a molecular level. Recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and ductlike cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. This approach is fast (~4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas.

  13. Genetic manipulation for inherited neurodegenerative diseases: myth or reality?

    PubMed

    Yu-Wai-Man, Patrick

    2016-10-01

    Rare genetic diseases affect about 7% of the general population and over 7000 distinct clinical syndromes have been described with the majority being due to single gene defects. This review will provide a critical overview of genetic strategies that are being pioneered to halt or reverse disease progression in inherited neurodegenerative diseases. This field of research covers a vast area and only the most promising treatment paradigms will be discussed with a particular focus on inherited eye diseases, which have paved the way for innovative gene therapy paradigms, and mitochondrial diseases, which are currently generating a lot of debate centred on the bioethics of germline manipulation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  14. Genetic manipulation in nutrition, metabolism, and obesity research.

    PubMed

    Campión, Javier; Milagro, Fermín I; Martínez, J Alfredo

    2004-08-01

    We summarize the current standard methods for overexpressing, inactivating, or manipulating genes, with special focus on nutritional and obesity research. These molecular biology procedures can be carried out with the maintenance of the genetic information to subsequent generations (transgenic technology) or devised to exclusively transfer the genetic material to a given target animal, which cannot be transmitted to the future progeny (gene therapy). On the other hand, the RNA interference (RNAi) approach allows for the creation of new experimental models by transient ablation of gene expression by degrading specific mRNA, which can be applied to assess different biological functions and mechanisms. The combination of these technologies contributes to the study of the function and regulation of different metabolism- and obesity-related genes as well as the identification of new pharmacologic targets for nutritional and therapeutic approaches.

  15. Genetic Manipulation of Pathogenicity Loci in Non-Typhimurium Salmonella

    PubMed Central

    Butela, Kristen; Lawrence, Jeffrey G.

    2012-01-01

    The traditional genetic tools used in Salmonella enterica serovar Typhimurium rely heavily on a high-transducing mutant of bacteriophage P22. P22 recognizes its hosts by the structure of their O-antigens, which vary among serovars of Salmonella; therefore, it cannot be used in most non-Typhimurium Salmonella, including the majority of those causing food-borne illnesses in both humans and livestock. Bacteriophage P1 infects a variety of enteric bacteria, including galE mutants of serovar Typhimurium; however, the degree to which the presence of coimmune prophages, the lack of required attachment sites or the lack of host factors act as barriers to using phage P1 in natural isolates of Salmonella is unknown. Here, we show that recombineering can be used to make virtually any serovar of Salmonella susceptible to P1 infection; as a result, P1 can be utilized for facile genetic manipulation of non-Typhimurium Salmonella, including movement of very large pathogenicity islands. A toolkit for easy manipulation of non-Typhimurium serovars of Salmonella is described. PMID:23041268

  16. [Genetic manipulation and the study of the protozoan parasite Leishmania].

    PubMed

    Cortázar, Tania M; Walker, John

    2004-12-01

    During the last 15 years, many aspects of the functional genomics of Leishmania have been revealed due to advances in DNA transfection, gene disruption and complementation through homologous recombination, and efficient strategies for the selection of transfected cells. These strategies have provided information about gene expression and protein function in the context of the intact parasite. The genome of Leishmania shows a marked deficiency of known transcription initiation factors, and gene expression is regulated almost entirely at the post-transcriptional level through trans-splicing of mRNAs and novel control mechanisms involving differential processing of 3'-untranslated regions (3'-UTRs) of mRNAs. Therefore, gene transfection represents a useful tool for the identification and functional analysis of genes of interest as well as the mechanisms that direct their regulation. The development of genetic manipulation systems has provided opportunities for the study of genes involved in virulence, intracellular survival and drug resistance of Leishmania, as well as for the functional validation of specific parasite proteins as new chemo- and immunotherapeutic targets. The current review presents recent advances in genetic manipulations that permit structural, functional and phenotypic analyses and by means of gene deletion and complementation using the methods of gene transfection.

  17. Growth and Survival of Genetically Manipulated Lactobacillus plantarum in Silage

    PubMed Central

    Sharp, R.; O'Donnell, A. G.; Gilbert, H. G.; Hazlewood, G. P.

    1992-01-01

    The growth and persistence of two genetically manipulated forms of Lactobacillus plantarum NCDO (National Collection of Dairy Organisms) 1193 have been monitored in grass silage. Both recombinants contained pSA3, a shuttle vector for gram-positive organisms that encodes erythromycin resistance. In one of the recombinants, pSA3 was integrated onto the chromosome, whereas in the other, a pSA3 derivative designated pM25, which contains a Clostridium thermocellum cellulase gene cloned into pSA3, was maintained as an extrachromosomal element. This extrachromosomal element is a plasmid. Rifampin-resistant mutants were selected for the recombinants and the parent strain. When applied to minisilos at a rate of 106 CFU/g of grass, both the recombinants and the parent strain proliferated to dominate the epiphytic microflora and induced an increase in the decline in pH compared with that of the noninoculated silos. The presence of extra genetic material did not appear to disadvantage the bacterium in comparison with the parent strain. The selective recovery of both strains by using rifampin and erythromycin was confirmed by Southern hybridization. Interestingly, the free plasmid (pM25) appeared more stable in silage than was expected from studies in MRS broth. The plasmid was retained by 85% of the rifampin-resistant L. plantarum colonies isolated from a day 30 silo. These data answer an important question by showing that genetically manipulated recombinants of L. plantarum can proliferate and compete with epiphytic lactic acid bacteria in silage. Images PMID:16348752

  18. Analyzing the metabolic capabilities of Desulfovibrio speciesthrough genetic manipulation.

    SciTech Connect

    Bender, K.; Yen, H.-C.; Wall, J.D.

    2005-12-31

    Sulfate-reducing bacteria (SRB) are an environmentallysignificant group belonging to the anaerobic delta-Proteobacteria thatrespire sulfate for growth. From an industrial stand point, SRB pose athreat through corrosion of ferrous metals and production of toxicsulfides. The more positive aspects of the metabolism of the SRB includea robust but poorly understood hydrogen metabolism that is of interest toalternative energy studies. SRB also immobilize a number of heavy metalsthrough sulfide precipitation or through changing the redox state of themetal and thus its solubility. When metals are made less soluble, as isthe case with chromium (Cr(VI) to Cr(III)) or uranium (U(VI) to U(IV)),toxicity is reduced by limiting biological availability. Despite theeconomic and environmental impacts associated with SRB activities, ourcurrent knowledge of their metabolism is inadequate. Among the SRB,members of the Desulfovibrio genus have received most attention becausethese strains are most readily grown in pure culture. Therefore,Desulfovibrio strains have been the focus of biochemical and biophysicalanalyses, however, genetic studies have been more difficult. Over thelast 15 years, progress has been made in developing techniques for DNAtransformation, gene mutagenesis and over expression, and proteintagging. Since the last genetics of SRB review by van Dongen, 10 yearshave passed (van Dongen, 1995) and the complete genome sequences of a fewstrains are now available (Heidelberg, et al., 2004). This reviewhighlights the current advances in the genetic manipulation ofDesulfovibrio species and the potential use of these tools inunderstanding the metabolism of sulfate reducers for biotechnologicalpurposes.

  19. The genetic manipulation of medicinal and aromatic plants.

    PubMed

    Gómez-Galera, Sonia; Pelacho, Ana M; Gené, Anna; Capell, Teresa; Christou, Paul

    2007-10-01

    Medicinal and aromatic plants have always been intimately linked with human health and culture. Plant-derived medicines constitute a substantial component of present day human healthcare systems in industrialized as well as developing countries. They are products of plant secondary metabolism and are involved in many other aspects of a plant's interaction with its immediate environment. The genetic manipulation of plants together with the establishment of in vitro plant regeneration systems facilitates efforts to engineer secondary product metabolic pathways. Advances in the cloning of genes involved in relevant pathways, the development of high throughput screening systems for chemical and biological activity, genomics tools and resources, and the recognition of a higher order of regulation of secondary plant metabolism operating at the whole plant level facilitate strategies for the effective manipulation of secondary products in plants. Here, we discuss advances in engineering metabolic pathways for specific classes of compounds in medicinal and aromatic plants and we identify remaining constraints and future prospects in the field. In particular we focus on indole, tropane, nicotine, isoquinoline alcaloids, monoterpenoids such as menthol and related compounds, diterpenoids such as taxol, sequiterpenoids such as artemisinin and aromatic amino acids.

  20. Tools for the genetic manipulation of Zygosaccharomyces rouxii.

    PubMed

    Pribylova, Lenka; de Montigny, Jacky; Sychrova, Hana

    2007-12-01

    A set of tools for the genetic manipulation of the osmotolerant yeast Zygosaccharomyces rouxii was developed. Auxotrophic mutants (ura3 leu2, ura3 ade2, ura3 leu2 ade2) derived from the CBS 732 type strain were prepared. Centromeric and episomal Z. rouxii/Escherichia coli shuttle plasmids with different marker genes (ScURA3, ZrLEU2, ZrADE2) and with multiple cloning sites were constructed, together with a plasmid enabling green fluorescent protein-tagging. A system for repeatable targeted gene deletion in Z. rouxii was established, involving first the integration of a PCR-generated loxP-kanMX-loxP cassette and second the removal of kanMX from the genome using a Z. rouxii plasmid harbouring cre recombinase.

  1. Carotenoids in Staple Cereals: Metabolism, Regulation, and Genetic Manipulation

    PubMed Central

    Zhai, Shengnan; Xia, Xianchun; He, Zhonghu

    2016-01-01

    Carotenoids play a critical role in animal and human health. Animals and humans are unable to synthesize carotenoids de novo, and therefore rely upon diet as sources of these compounds. However, major staple cereals often contain only small amounts of carotenoids in their grains. Consequently, there is considerable interest in genetic manipulation of carotenoid content in cereal grain. In this review, we focus on carotenoid metabolism and regulation in non-green plant tissues, as well as genetic manipulation in staple cereals such as rice, maize, and wheat. Significant progress has been made in three aspects: (1) seven carotenogenes play vital roles in carotenoid regulation in non-green plant tissues, including 1-deoxyxylulose-5-phosphate synthase influencing isoprenoid precursor supply, phytoene synthase, β-cyclase, and ε-cyclase controlling biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase and carotenoid cleavage dioxygenases responsible for degradation, and orange gene conditioning sequestration sink; (2) provitamin A-biofortified crops, such as rice and maize, were developed by either metabolic engineering or marker-assisted breeding; (3) quantitative trait loci for carotenoid content on chromosomes 3B, 7A, and 7B were consistently identified, eight carotenogenes including 23 loci were detected, and 10 gene-specific markers for carotenoid accumulation were developed and applied in wheat improvement. A comprehensive and deeper understanding of the regulatory mechanisms of carotenoid metabolism in crops will be beneficial in improving our precision in improving carotenoid contents. Genomic selection and gene editing are emerging as transformative technologies for provitamin A biofortification. PMID:27559339

  2. Electroporation-Based Genetic Manipulation in Type I Methanotrophs

    PubMed Central

    Chu, Frances; Puri, Aaron W.; Fu, Yanfen; Lidstrom, Mary E.

    2016-01-01

    Methane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. strain LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency of M. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) and sacB counterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains. PMID:26801578

  3. 512-Channel and 13-Region Simultaneous Recordings Coupled with Optogenetic Manipulation in Freely Behaving Mice

    PubMed Central

    Xie, Kun; Fox, Grace E.; Liu, Jun; Tsien, Joe Z.

    2016-01-01

    The development of technologies capable of recording both single-unit activity and local field potentials (LFPs) over a wide range of brain circuits in freely behaving animals is the key to constructing brain activity maps. Although mice are the most popular mammalian genetic model, in vivo neural recording has been traditionally limited to smaller channel count and fewer brain structures because of the mouse’s small size and thin skull. Here, we describe a 512-channel tetrode system that allows us to record simultaneously over a dozen cortical and subcortical structures in behaving mice. This new technique offers two major advantages – namely, the ultra-low cost and the do-it-yourself flexibility for targeting any combination of many brain areas. We show the successful recordings of both single units and LFPs from 13 distinct neural circuits of the mouse brain, including subregions of the anterior cingulate cortices, retrosplenial cortices, somatosensory cortices, secondary auditory cortex, hippocampal CA1, dentate gyrus, subiculum, lateral entorhinal cortex, perirhinal cortex, and prelimbic cortex. This 512-channel system can also be combined with Cre-lox neurogenetics and optogenetics to further examine interactions between genes, cell types, and circuit dynamics across a wide range of brain structures. Finally, we demonstrate that complex stimuli – such as an earthquake and fear-inducing foot-shock – trigger firing changes in all of the 13 brain regions recorded, supporting the notion that neural code is highly distributed. In addition, we show that localized optogenetic manipulation in any given brain region could disrupt network oscillations and caused changes in single-unit firing patterns in a brain-wide manner, thereby raising the cautionary note of the interpretation of optogenetically manipulated behaviors. PMID:27378865

  4. 512-Channel and 13-Region Simultaneous Recordings Coupled with Optogenetic Manipulation in Freely Behaving Mice.

    PubMed

    Xie, Kun; Fox, Grace E; Liu, Jun; Tsien, Joe Z

    2016-01-01

    The development of technologies capable of recording both single-unit activity and local field potentials (LFPs) over a wide range of brain circuits in freely behaving animals is the key to constructing brain activity maps. Although mice are the most popular mammalian genetic model, in vivo neural recording has been traditionally limited to smaller channel count and fewer brain structures because of the mouse's small size and thin skull. Here, we describe a 512-channel tetrode system that allows us to record simultaneously over a dozen cortical and subcortical structures in behaving mice. This new technique offers two major advantages - namely, the ultra-low cost and the do-it-yourself flexibility for targeting any combination of many brain areas. We show the successful recordings of both single units and LFPs from 13 distinct neural circuits of the mouse brain, including subregions of the anterior cingulate cortices, retrosplenial cortices, somatosensory cortices, secondary auditory cortex, hippocampal CA1, dentate gyrus, subiculum, lateral entorhinal cortex, perirhinal cortex, and prelimbic cortex. This 512-channel system can also be combined with Cre-lox neurogenetics and optogenetics to further examine interactions between genes, cell types, and circuit dynamics across a wide range of brain structures. Finally, we demonstrate that complex stimuli - such as an earthquake and fear-inducing foot-shock - trigger firing changes in all of the 13 brain regions recorded, supporting the notion that neural code is highly distributed. In addition, we show that localized optogenetic manipulation in any given brain region could disrupt network oscillations and caused changes in single-unit firing patterns in a brain-wide manner, thereby raising the cautionary note of the interpretation of optogenetically manipulated behaviors.

  5. [Genetic manipulation in farm animals: how and why?].

    PubMed

    van der Zijpp, A J

    1990-06-15

    A review of the history of the knowledge of the development of DNA was presented on the symposium 'Biotechnology'. Entirely in agreement with expectations, genetic manipulation became suitable for use, also in farm animals, approximately thirty years after the discovery of the double helix. The technology available for transfection is limited and is only successful in a small number of cases: less than one per cent. In addition, gene constructions give rise to a large number of problems as they are not tissue-specific and fail to function at the correct time in the course of development. The knowledge of interesting genes (at DNA level) in farm animals is of vital importance. Detecting these genes will undoubtedly still require considerable effort. In view of the technical state of things, medical and physiological studies using transfection will obviously have to provide a new insight prior to use. This is in agreement with the memorandum on 'Ethics and Biotechnology in Animals'. A 'no, unless' procedure is recommended in this note, room being left for 'good' objectives of research following ethical consideration.

  6. Genetic Manipulation of Condensed Tannins in Higher Plants1

    PubMed Central

    Robbins, Mark P.; Bavage, Adrian D.; Strudwicke, Catherine; Morris, Phillip

    1998-01-01

    We have produced and analyzed transgenic birdsfoot trefoil (Lotus corniculatus L.) plants harboring antisense dihydroflavonol reductase (AS-DFR) sequences. In initial experiments the effect of introducing three different antisense Antirrhinum majus L. DFR constructs into a single recipient genotype (S50) was assessed. There were no obvious effects on plant biomass, but levels of condensed tannins showed a statistical reduction in leaf, stem, and root tissues of some of the antisense lines. Transformation events were also found, which resulted in increased levels of condensed tannins. In subsequent experiments a detailed study of AS-DFR phenotypes was carried out in genotype S33 using pMAJ2 (an antisense construct comprising the 5′ half of the A. majus cDNA). In this case, reduced tannin levels were found in leaf and stem tissues and in juvenile shoot tissues. Analysis of soluble flavonoids and isoflavonoids in tannin down-regulated shoot tissues indicated few obvious default products. When two S33 AS-DFR lines were outcrossed, there was an underrepresentation of transgene sequences in progeny plants and no examples of inheritance of an antisense phenotype were observed. To our knowledge, this is the first report of the genetic manipulation of condensed tannin biosynthesis in higher plants. PMID:9501146

  7. Isolation, Genetic Manipulation, and Transplantation of Canine Spermatogonial Stem Cells: Progress Toward Transgenesis Through the Male Germ Line

    PubMed Central

    Harkey, Michael A.; Asano, Atsushi; Zoulas, Mary Ellen; Torok-Storb, Beverly; Nagashima, Jennifer; Travis, Alexander

    2013-01-01

    The dog is recognized as a highly predictive model for pre-clinical research. Its size, life span, physiology and genetics more closely match human parameters than do those of the mouse model. Investigations of the genetic basis of disease and of new regenerative treatments have frequently taken advantage of canine models. However, full utility of this model hasn’t been realized because of the lack of easy transgenesis. Blastocyst-mediated transgenic technology developed in mice has been very slow to translate to larger animals, and somatic cell nuclear transfer remains technically challenging, expensive, and low yield. Spermatogonial stem cell (SSC) transplantation, which does not involve manipulation of ova or blastocysts, has proven to be an effective alternative approach for generating transgenic offspring in rodents, and in some large animals. Our recent demonstration that canine testis cells can engraft in a host testis, and generate donor-derived sperm, suggests that SSC transplantation may offer a similar avenue to transgenesis in the canine model. Here, we explore the potential of SSC transplantation in dogs as a means of generating canine transgenic models for pre-clinical models of genetic diseases. Specifically, we 1) established markers for identification and tracking canine spermatogonial cells; 2) established methods for enrichment and genetic manipulation of these cells; 3) described their behavior in culture; and 4) demonstrated engraftment of genetically manipulated SSC, and production of transgenic sperm. These findings help set the stage for generation of transgenic canine models via SSC transplantation. PMID:23690628

  8. Isolation, genetic manipulation, and transplantation of canine spermatogonial stem cells: progress toward transgenesis through the male germ-line.

    PubMed

    Harkey, Michael A; Asano, Atsushi; Zoulas, Mary Ellen; Torok-Storb, Beverly; Nagashima, Jennifer; Travis, Alexander

    2013-07-01

    The dog is recognized as a highly predictive model for preclinical research. Its size, life span, physiology, and genetics more closely match human parameters than do those of the mouse model. Investigations of the genetic basis of disease and of new regenerative treatments have frequently taken advantage of canine models. However, full utility of this model has not been realized because of the lack of easy transgenesis. Blastocyst-mediated transgenic technology developed in mice has been very slow to translate to larger animals, and somatic cell nuclear transfer remains technically challenging, expensive, and low yield. Spermatogonial stem cell (SSC) transplantation, which does not involve manipulation of ova or blastocysts, has proven to be an effective alternative approach for generating transgenic offspring in rodents and in some large animals. Our recent demonstration that canine testis cells can engraft in a host testis, and generate donor-derived sperm, suggests that SSC transplantation may offer a similar avenue to transgenesis in the canine model. Here, we explore the potential of SSC transplantation in dogs as a means of generating canine transgenic models for preclinical models of genetic diseases. Specifically, we i) established markers for identification and tracking canine spermatogonial cells; ii) established methods for enrichment and genetic manipulation of these cells; iii) described their behavior in culture; and iv) demonstrated engraftment of genetically manipulated SSC and production of transgenic sperm. These findings help to set the stage for generation of transgenic canine models via SSC transplantation.

  9. Of mice and microflora: considerations for genetically engineered mice.

    PubMed

    Treuting, P M; Clifford, C B; Sellers, R S; Brayton, C F

    2012-01-01

    The phenotype of genetically engineered mice is a combination of both genetic and environmental factors that include the microflora of the mouse. The impact a particular microbe has on a mouse reflects the host-microbe interaction within the context of the mouse genotype and environment. Although often considered a confounding variable, many host-microbe interactions have resulted in the generation of novel model systems and characterization of new microbial agents. Microbes associated with overt disease in mice have been the historical focus of the laboratory animal medical and pathology community and literature. The advent of genetic engineering and the complex of mouse models have revealed previously unknown or disregarded agents that now oblige the attention of the biomedical research community. The purpose of this article is to describe and illustrate how phenotypes can be affected by microflora by focusing on the infectious diseases present in genetically engineered mouse (GEM) colonies of our collective institutions and by reviewing important agents that are rarely seen in most research facilities today. The goal is to introduce the concept of the role of microflora on phenotypes and in translational research using GEM models.

  10. Chemical Genetics: receptor-ligand pairs for rapid manipulation of neuronal activity

    PubMed Central

    Wulff, Peer; Arenkiel, Benjamin R.

    2012-01-01

    Towards the functional dissection of neuronal circuits, a number of new genetic tools have been developed that enable rapid and reversible manipulation of genetically defined neuronal subtypes in intact mammalian brain circuits. Alongside the breakthrough technology of optogenetics, receptor-ligand pairs provide complementary approaches to modulate neuronal activity using chemical-genetics. PMID:22119143

  11. Wildland Collection, Population Development, and Genetic Manipulation of Native Rangeland Grasses in the Intermountain West USA

    USDA-ARS?s Scientific Manuscript database

    In the Intermountain West USA, a high demand for native plant materials exists, but customer expectations for native plant materials may be contradictory (Jones, 2003). Some customers spurn genetically manipulated or non-local plant materials, while others accept manipulation or non-local origin wh...

  12. Demodex musculi Infestation in Genetically Immunomodulated Mice

    PubMed Central

    Smith, Peter C; Zeiss, Caroline J; Beck, Amanda P; Scholz, Jodi A

    2016-01-01

    Demodex musculi, a prostigmatid mite that has been reported infrequently in laboratory mice, has been identified with increasing frequency in contemporary colonies of immunodeficient mice. Here we describe 2 episodes of D. musculi infestation with associated clinical signs in various genetically engineered mouse strains, as well as treatment strategies and an investigation into transmissibility and host susceptibility. The first case involved D. musculi associated with clinical signs and pathologic lesions in BALB/c-Tg(DO11.10)Il13tm mice, which have a defect in type 2 helper T cell (Th2) immunity. Subsequent investigation revealed mite transmission to both parental strains (BALB/c-Tg[DO11.10] and BALB/c-Il13tm), BALB/c-Il13/Il4tm, and wild-type BALB/c. All Tg(DO11.10)Il13tm mice remained infested throughout the investigation, and D. musculi were recovered from all strains when they were cohoused with BALB/c-Tg(DO11.10)Il13tm index mice. However, only Il13tm and Il13/Il4tm mice demonstrated persistent infestation after index mice were removed. Only BALB/c-Tg(DO11.10)Il13tm showed clinical signs, suggesting that the phenotypic dysfunction of Th2 immunity is sufficient for persistent infestation, whereas clinical disease associated with D. musculi appears to be genotype-specific. This pattern was further exemplified in the second case, which involved NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) and C;129S4 Rag2tm1.1Flv Il2rgtm1.1Flv/J mice with varying degrees of blepharitis, conjunctivitis, and facial pruritis. Topical amitraz decreased mite burden but did not eliminate infestation or markedly ameliorate clinical signs. Furthermore, mite burden began to increase by 1 mo posttreatment, suggesting that topical amitraz is an ineffective treatment for D. musculi. These experiences illustrate the need for vigilance regarding opportunistic and uncommon pathogens in rodent colonies, especially among mice with immunologic deficits. PMID:27538858

  13. Demodex musculi Infestation in Genetically Immunomodulated Mice.

    PubMed

    Smith, Peter C; Zeiss, Caroline J; Beck, Amanda P; Scholz, Jodi A

    2016-01-01

    Demodex musculi, a prostigmatid mite that has been reported infrequently in laboratory mice, has been identified with increasing frequency in contemporary colonies of immunodeficient mice. Here we describe 2 episodes of D. musculi infestation with associated clinical signs in various genetically engineered mouse strains, as well as treatment strategies and an investigation into transmissibility and host susceptibility. The first case involved D. musculi associated with clinical signs and pathologic lesions in BALB/c-Tg(DO11.10)Il13(tm) mice, which have a defect in type 2 helper T cell (Th2) immunity. Subsequent investigation revealed mite transmission to both parental strains (BALB/c-Tg[DO11.10] and BALB/c-Il13(tm)), BALB/c-Il13/Il4(tm), and wild-type BALB/c. All Tg(DO11.10)Il13(tm) mice remained infested throughout the investigation, and D. musculi were recovered from all strains when they were cohoused with BALB/c-Tg(DO11.10)Il13(tm) index mice. However, only Il13(tm) and Il13/Il4(tm) mice demonstrated persistent infestation after index mice were removed. Only BALB/c-Tg(DO11.10)Il13(tm) showed clinical signs, suggesting that the phenotypic dysfunction of Th2 immunity is sufficient for persistent infestation, whereas clinical disease associated with D. musculi appears to be genotype-specific. This pattern was further exemplified in the second case, which involved NOD.Cg-Prkdc(scid)Il2r(tm1Wjl)/SzJ (NSG) and C;129S4 Rag2(tm1.1Flv) Il2rg(tm1.1Flv)/J mice with varying degrees of blepharitis, conjunctivitis, and facial pruritis. Topical amitraz decreased mite burden but did not eliminate infestation or markedly ameliorate clinical signs. Furthermore, mite burden began to increase by 1 mo posttreatment, suggesting that topical amitraz is an ineffective treatment for D. musculi. These experiences illustrate the need for vigilance regarding opportunistic and uncommon pathogens in rodent colonies, especially among mice with immunologic deficits.

  14. Genetics-based manipulation of adipose tissue sympathetic innervation.

    PubMed

    François, Marie; Qualls-Creekmore, Emily; Berthoud, Hans-Rudolf; Münzberg, Heike; Yu, Sangho

    2017-08-28

    There is renewed interest in leveraging the thermogenic capacity of brown adipose tissue (BAT) and browning of white adipose tissue (WAT) to improve energy balance and prevent obesity. In addition to these effects on energy expenditure, both BAT and WAT secrete large numbers of hormones and cytokines that play important roles in maintaining metabolic health. Both BAT and WAT are densely innervated by the sympathetic nervous system (SNS) and this innervation is crucial for BAT thermogenesis and WAT browning, making it a potentially interesting target for manipulating energy balance and treatment of obesity and metabolic disease. Peripheral neuromodulation in the form of electrical manipulation of the SNS and parasympathetic nervous system (PSNS) has been used for the management of pain and many other conditions, but progress is hampered by lack of detailed knowledge of function-specific neurons and nerves innervating particular organs and tissues. Therefore, the goal of the National Institutes of Health (NIH) Common Fund project "Stimulating Peripheral Activity to Relieve Conditions (SPARC)" is to comprehensively map both anatomical and neurochemical aspects of the peripheral nervous system in animal model systems to ultimately guide optimal neuromodulation strategies in humans. Compared to electrical manipulation, neuron-specific opto- and chemogenetic manipulation, now being extensively used to decode the function of brain circuits, will further increase the functional specificity of peripheral neuromodulation. Copyright © 2017. Published by Elsevier Inc.

  15. Genetics of meiosis and recombination in mice.

    PubMed

    Bolcun-Filas, Ewelina; Schimenti, John C

    2012-01-01

    Meiosis is one of the most critical developmental processes in sexually reproducing organisms. One round of DNA replication followed by two rounds of cell divisions results in generation of haploid gametes (sperm and eggs in mammals). Meiotic failure typically leads to infertility in mammals. In the process of meiotic recombination, maternal and paternal genomes are shuffled, creating new allelic combinations and thus genetic variety. However, in order to achieve this, meiotic cells must self-inflict DNA damage in the form of programmed double-strand breaks (DSBs). Complex processes evolved to ensure proper DSB repair, and to do so in a way that favors interhomolog reciprocal recombination and crossovers. The hallmark of meiosis, a structurally conserved proteinaceous structure called the synaptonemal complex, is found only in meiotic cells. Conversely, meiotic homologous recombination is an adaptation of the mitotic DNA repair process but involving specialized proteins. In this chapter, we summarize current developments in mammalian meiosis enabled by genetically modified mice.

  16. Molecular genetic techniques for gene manipulation in Candida albicans

    PubMed Central

    Xu, Qiu-Rong; Yan, Lan; Lv, Quan-Zhen; Zhou, Mi; Sui, Xue; Cao, Yong-Bing; Jiang, Yuan-Ying

    2014-01-01

    Candida albicans is one of the most common fungal pathogen in humans due to its high frequency as an opportunistic and pathogenic fungus causing superficial as well as invasive infections in immunocompromised patients. An understanding of gene function in C. albicans is necessary to study the molecular basis of its pathogenesis, virulence and drug resistance. Several manipulation techniques have been used for investigation of gene function in C. albicans, including gene disruption, controlled gene expression, protein tagging, gene reintegration, and overexpression. In this review, the main cassettes containing selectable markers used for gene manipulation in C. albicans are summarized; the advantages and limitations of these cassettes are discussed concerning the influences on the target gene expression and the virulence of the mutant strains. PMID:24759671

  17. Improved and expanded Q-system reagents for genetic manipulations

    PubMed Central

    Riabinina, Olena; Luginbuhl, David; Marr, Elizabeth; Liu, Sha; Wu, Mark N.; Luo, Liqun; Potter, Christopher J.

    2014-01-01

    The Q-system is a repressible binary expression system for transgenic manipulations in living organisms. Through protein engineering and in vivo functional tests, we report here new variants of the Q-system transcriptional activator, including QF2, that allows the Q-system to drive strong and ubiquitous expression for the first time in all tissues. Our new QF2, GAL4QF and LexAQF chimeric transcriptional activators substantially enrich the toolkit available for transgenic regulation in Drosophila. PMID:25581800

  18. Plasmid vectors and molecular building blocks for the development of genetic manipulation tools for Trypanosoma cruzi.

    PubMed

    Bouvier, León A; Cámara, María de los Milagros; Canepa, Gaspar E; Miranda, Mariana R; Pereira, Claudio A

    2013-01-01

    The post genomic era revealed the need for developing better performing, easier to use and more sophisticated genetic manipulation tools for the study of Trypanosoma cruzi, the etiological agent of Chagas disease. In this work a series of plasmids that allow genetic manipulation of this protozoan parasite were developed. First of all we focused on useful tools to establish selection strategies for different strains and which can be employed as expression vectors. On the other hand molecular building blocks in the form of diverse selectable markers, modifiable fluorescent protein and epitope-tag coding sequences were produced. Both types of modules were harboured in backbone molecules conceived to offer multiple construction and sub-cloning strategies. These can be used to confer new properties to already available genetic manipulation tools or as starting points for whole novel designs. The performance of each plasmid and building block was determined independently. For illustration purposes, some simple direct practical applications were conducted.

  19. Software for analysis and manipulation of genetic linkage data.

    PubMed Central

    Weaver, R; Helms, C; Mishra, S K; Donis-Keller, H

    1992-01-01

    We present eight computer programs written in the C programming language that are designed to analyze genotypic data and to support existing software used to construct genetic linkage maps. Although each program has a unique purpose, they all share the common goals of affording a greater understanding of genetic linkage data and of automating tasks to make computers more effective tools for map building. The PIC/HET and FAMINFO programs automate calculation of relevant quantities such as heterozygosity, PIC, allele frequencies, and informativeness of markers and pedigrees. PREINPUT simplifies data submissions to the Centre d'Etude du Polymorphisme Humain (CEPH) data base by creating a file with genotype assignments that CEPH's INPUT program would otherwise require to be input manually. INHERIT is a program written specifically for mapping the X chromosome: by assigning a dummy allele to males, in the nonpseudoautosomal region, it eliminates falsely perceived noninheritances in the data set. The remaining four programs complement the previously published genetic linkage mapping software CRI-MAP and LINKAGE. TWOTABLE produces a more readable format for the output of CRI-MAP two-point calculations; UNMERGE is the converse to CRI-MAP's merge option; and GENLINK and LINKGEN automatically convert between the genotypic data file formats required by these packages. All eight applications read input from the same types of data files that are used by CRI-MAP and LINKAGE. Their use has simplified the management of data, has increased knowledge of the content of information in pedigrees, and has reduced the amount of time needed to construct genetic linkage maps of chromosomes. PMID:1598906

  20. Metabolic engineering: techniques for analysis of targets for genetic manipulations.

    PubMed

    Nielsen, J

    Metabolic engineering has been defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. With this definition metabolic engineering includes: (1) inserting new pathways in microorganisms with the aim of producing novel metabolites, e.g., production of polyketides by Streptomyces; (2) production of heterologous peptides, e.g., production of human insulin, erythropoitin, and tPA; and (3) improvement of both new and existing processes, e.g., production of antibiotics and industrial enzymes. Metabolic engineering is a multidisciplinary approach, which involves input from chemical engineers, molecular biologists, biochemists, physiologists, and analytical chemists. Obviously, molecular biology is central in the production of novel products, as well as in the improvement of existing processes. However, in the latter case, input from other disciplines is pivotal in order to target the genetic modifications; with the rapid developments in molecular biology, progress in the field is likely to be limited by procedures to identify the optimal genetic changes. Identification of the optimal genetic changes often requires a meticulous mapping of the cellular metabolism at different operating conditions, and the application of metabolic engineering to process optimization is, therefore, expected mainly to have an impact on the improvement of processes where yield, productivity, and titer are important design factors, i.e., in the production of metabolites and industrial enzymes. Despite the prospect of obtaining major improvement through metabolic engineering, this approach is, however, not expected to completely replace the classical approach to strain improvement-random mutagenesis followed by screening. Identification of the optimal genetic changes for improvement of a given process requires analysis of the underlying mechanisms, at best, at the molecular level. To reveal these mechanisms a number of different techniques may be applied

  1. [Direct genetic manipulation and criminal code in Venezuela: absolute criminal law void?].

    PubMed

    Cermeño Zambrano, Fernando G De J

    2002-01-01

    The judicial regulation of genetic biotechnology applied to the human genome is of big relevance currently in Venezuela due to the drafting of an innovative bioethical law in the country's parliament. This article will highlight the constitutional normative of Venezuela's 1999 Constitution regarding this subject, as it establishes the framework from which this matter will be legally regulated. The approach this article makes towards the genetic biotechnology applied to the human genome is made taking into account the Venezuelan penal law and by highlighting the violent genetic manipulations that have criminal relevance. The genetic biotechnology applied to the human genome has another important relevance as a consequence of the reformulation of the Venezuelan Penal Code discussed by the country's National Assembly. Therefore, a concise study of the country's penal code will be made in this article to better understand what judicial-penal properties have been protected by the Venezuelan penal legislation. This last step will enable us to identify the penal tools Venezuela counts on to face direct genetic manipulations. We will equally indicate the existing punitive loophole and that should be covered by the penal legislator. In conclusion, this essay concerns criminal policy, referred to the direct genetic manipulations on the human genome that haven't been typified in Venezuelan law, thus discovering a genetic biotechnology paradise.

  2. The Cre/loxP system in Giardia lamblia: genetic manipulations in a binucleate tetraploid protozoan.

    PubMed

    Wampfler, Petra B; Faso, Carmen; Hehl, Adrian B

    2014-07-01

    The bacteriophage-derived Cre/loxP system is a valuable tool that has revolutionised genetic and cell biological research in many organisms. We implemented this system in the intestinal parasite Giardia lamblia, an evolutionarily diverged protozoan whose binucleate and tetraploid genome organisation severely limits the application of reverse genetic approaches. We show that Cre-recombinase is functionally expressed in G. lamblia and demonstrate "recycling" of selectable markers. Providing the means for more complex and versatile genetic modifications, this technique massively increases the scope of functional investigations in G. lamblia and other protozoa with similar limitations with respect to genetic manipulation.

  3. Noninheritable Maternal Factors Useful for Genetic Manipulation in Mammals.

    PubMed

    Sakurai, Takayuki; Shindo, Takayuki; Sato, Masahiro

    2017-01-01

    Mammalian early embryogenesis is supported by maternal factors, such as messenger RNA (mRNA) and proteins, produced and accumulated during oogenesis at least up to the stage when zygotic activation commences. These maternal factors are involved in biologically important events such as epigenetic activation, reprogramming, and mitochondrial growth. Most of these maternal mRNAs are degraded by the 2-cell to 4 ~ 8-cell stages. Maternal proteins, which are produced during oogenesis or by the maternal mRNAs, are degraded by the 4 ~ 8-cell stage. In other words, the maternal factors exist during specific stages of early embryogenesis. In this chapter, we will briefly summarize the property of these maternal factors and mention possible applications of these factors for developing new reproduction engineering-related technologies and producing genetically modified animals. More specifically, we will show the usefulness of maternally accumulated Cas9 protein as a promising tool for CRISPR-/Cas9-based simultaneous genetic modification of multiple loci in mammals.

  4. Genetic manipulation of the obligate chemolithoautotrophic bacterium Thiobacillus denitrificans

    SciTech Connect

    Beller, H.R.; Legler, T.C.; Kane, S.R.

    2011-07-15

    Chemolithoautotrophic bacteria can be of industrial and environmental importance, but they present a challenge for systems biology studies, as their central metabolism deviates from that of model organisms and there is a much less extensive experimental basis for their gene annotation than for typical organoheterotrophs. For microbes with sequenced genomes but unconventional metabolism, the ability to create knockout mutations can be a powerful tool for functional genomics and thereby render an organism more amenable to systems biology approaches. In this chapter, we describe a genetic system for Thiobacillus denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. Insertion mutations are generated by in vitro transposition, the mutated genes are amplified by the PCR, and the amplicons are introduced into T. denitrificans by electroporation. Use of a complementation vector, pTL2, based on the IncP plasmid pRR10 is also addressed.

  5. Genetic manipulation of a cyanobacterium for heavy metal detoxivication

    SciTech Connect

    McCormick, P.; Cannon, G.; Heinhorst, S.

    1995-12-31

    Increasing heavy metal contamination of soil and water has produced a need for economical and effective methods to reduce toxic buildup of these materials. Biological systems use metallothionein proteins to sequester such metals as Cu, Cd, and Zn. Studies are underway to genetically engineer a cyanobacteria strain with increased ability for metallothionein production and increased sequestration capacity. Cyanobacteria require only sunlight and CO{sub 2}. Vector constructs are being developed in a naturally competent, unicellular cyanobacterium Anacystis nidulans R2. Closed copies of a yeast copper metallothionein gene have been inserted into a cyanobacterial shuttle vector as well as a vector designed for genomic integration. Transformation studies have produced recombinant cyanobacteria from both of these systems, and work is currently underway to assess the organism`s ability to withstand increasing Cu, Cd, and Zn concentrations.

  6. Notice of release of Charleston Peak Germplasm: selected class, genetically manipulated track pre-variety germplasm

    USDA-ARS?s Scientific Manuscript database

    The USDA-Agricultural Research Service (ARS) announces the release of Charleston Peak Germplasm slender wheatgrass [Elymus trachycaulus (Link) Gould ex Shinners] as a selected class, genetically manipulated track pre-variety germplasm selected directly from collection D-3269. This collection is uni...

  7. Attitudes of Prairie Bible College Students toward Human Genetic Manipulation. A Survey and Comparative Study.

    ERIC Educational Resources Information Center

    Jordahl, Ron, Ed.

    This document reports a survey instituted to compare the attitudes of students at a Christian college (Prairie Bible College) in Alberta, Canada with those of college students in general concerning the possible use of genetic manipulation. Comparison was made with the findings of a 1990 study by Geremia Veglia, et al., "Public Attitudes…

  8. Abortion and genetic manipulation: breaking with reasoning founded on disrespect for life and human dignity.

    PubMed

    Dijon, X

    1993-01-01

    Scientists often base their claim to the right to carry out experiments with embryos on the freedom of women to have abortions. In this article the attempts made by two French jurists to counter this claim are studied. In my view, their juridical line of reasoning needs to be extended from the ban on genetic manipulation to a ban on abortion.

  9. Idiotypic manipulation in mice: BALB/c mice can express the crossreactive idiotype of A/J mice.

    PubMed Central

    Moser, M; Leo, O; Hiernaux, J; Urbain, J

    1983-01-01

    The response of A/J mice to arsonate-coupled keyhole limpet hemocyanin is characterized by a crossreactive idiotype (CRIA). CRIA+ antibodies are restricted to the Igh-Ic haplotype and are never expressed in BALB/c mice after immunization with antigen. Studies at the DNA level suggest that the gene encoding the CRIA heavy chain in A/J mice is probably absent in the genome of BALB/c mice. Despite this, using the immunization cascade tool, we have been able to induce the expression of CRIA+ antibodies in BALB/c mice. These studies led to an apparent paradox, whose understanding will provide new insights into the regulatory mechanisms of the immune system. We suggest that clones secreting CRIA-like Igs in BALB/c mice are "somatic variants" that could arise from gene conversion events. PMID:6576348

  10. Utility of genetically modified mice for understanding the neurobiology of substance use disorders.

    PubMed

    Fowler, Christie D; Kenny, Paul J

    2012-06-01

    Advances in our ability to modify the mouse genome have enhanced our understanding of the genetic and neurobiological mechanisms contributing to addiction-related behaviors underlying substance use and abuse. These experimentally induced manipulations permit greater spatial and temporal specificity for modification of gene expression within specific cellular populations and during select developmental time periods. In this review, we consider the current mouse genetic model systems that have been employed to understand aspects of addiction and highlight significant conceptual advances achieved related to substance use and abuse. The mouse models reviewed herein include conventional knock-out and knock-in, conditional knockout, transgenic, inducible transgenic, mice suitable for optogenetic control of discrete neuronal populations, and phenotype-selected mice. By establishing a reciprocal investigatory relationship between genetic findings in humans and genomic manipulations in mice, a far better understanding of the discrete neuromechanisms underlying addiction can be achieved, which is likely to provide a strong foundation for developing and validating novel therapeutics for the treatment of substance abuse disorders.

  11. Transplantation of bone marrow genetically engineered to express proinsulin II protects against autoimmune insulitis in NOD mice.

    PubMed

    Chan, James; Clements, Warren; Field, Judith; Nasa, Zeyad; Lock, Peter; Yap, Felicia; Toh, Ban-Hock; Alderuccio, Frank

    2006-11-01

    Type 1 diabetes (T1D) is a T-cell-dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin-producing pancreatic beta-cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice. HSCs were isolated from 6-8-week-old NOD female mice and transduced in vitro with retrovirus encoding enhanced green fluorescent protein (EGFP) and either proinsulin II or control autoantigen. Additional control groups included mice transferred with non-manipulated bone marrow and mice which did not receive bone marrow transfer. EGFP-sorted or non-sorted HSCs were transferred into pre-conditioned 3-4-week-old female NOD mice and insulitis was assessed 8 weeks post-transfer. Chimerism was established in all major lymphoid tissues, ranging from 5-15% in non-sorted bone marrow transplants to 20-45% in EGFP-sorted bone marrow transplants. The incidence and degree of insulitis was significantly reduced in mice receiving proinsulin II bone marrow compared to controls. However, the incidence of sialitis in mice receiving proinsulin II bone marrow and control mice was not altered, indicating protection from insulitis was antigen specific. We show for the first time that ex vivo genetic manipulation of HSCs to express proinsulin II followed by transplantation to NOD mice can establish molecular chimerism and protect from destructive insulitis in an antigen-specific manner.

  12. Oral Immunotherapy for Pollen Allergy Using T-Cell Epitope-Containing Egg White Derived from Genetically Manipulated Chickens

    PubMed Central

    Kawabe, Yoshinori; Hayashida, Yuuki; Numata, Kensaku; Harada, Shota; Hayashida, Yoshifumi; Ito, Akira; Kamihira, Masamichi

    2012-01-01

    Peptide immunotherapy using T-cell epitopes is expected to be an effective treatment for allergic diseases such as Japanese cedar (Cryptomeria japonica; Cj) pollinosis. To develop a treatment for pollen allergy by inducing oral tolerance, we generated genetically manipulated (GM) chickens by retroviral gene transduction, to produce a fusion protein of chicken egg white lysozyme and a peptide derived from seven dominant human T-cell epitopes of Japanese cedar pollen allergens (cLys-7crp). The transgene sequence was detected in all chickens transduced with the retroviral vector. Transduction efficiency in blood cells correlated to transgene expression. Western blot analysis revealed that cLys-7crp was expressed in the egg white of GM hens. Mice induced to develop allergic rhinitis by Cj pollinosis were fed with cLys-7crp-containing egg white produced by GM chickens. Total and Cj allergen (Cry j 1)-specific IgE levels were significantly decreased in allergic mice fed with cLys-7crp-containing egg white compared with allergic mice fed with normal egg white. These results suggest that oral administration of T-cell epitope-containing egg white derived from GM chickens is effective for the induction of immune tolerance as an allergy therapy. PMID:23144766

  13. The post-humanist embryo: genetic manipulation, assisted reproductive technologies and the Principle of Procreative Beneficence.

    PubMed

    Güell Pelayo, Francisco

    2014-01-01

    Drawing from Julian Savulescu's argument for the obligation to use technological interventions for the enhancement human life, the Principle of Procreative Beneficence (PPB) states that parents have a moral obligation to use available reproductive technologies, including techniques of genetic manipulation, to create children who have the best chance of enjoying the best possible life. The aim of this study is to analyse the extent to which the possibility of using genetic manipulation to promote specific personality traits and thereby enhance human life is actually supported by current scientific knowledge and to determine whether the techniques employed in embryo selection comply with the PPB. In light of this analysis, the importance of involving the scientific community in the enhancement debate will be made clear. Moreover, when current knowledge of genetic and epigenetic processes and evidence of the risks of assisted reproductive technologies are taken into account, we find sufficient reason - even when guided by the PPB - to abstain from the use of current techniques of genetic manipulation and embryonic selection.

  14. Alterations of social interaction through genetic and environmental manipulation of the 22q11.2 gene Sept5 in the mouse brain.

    PubMed

    Harper, Kathryn M; Hiramoto, Takeshi; Tanigaki, Kenji; Kang, Gina; Suzuki, Go; Trimble, William; Hiroi, Noboru

    2012-08-01

    Social behavior dysfunction is a symptomatic element of schizophrenia and autism spectrum disorder (ASD). Although altered activities in numerous brain regions are associated with defective social cognition and perception, the causative relationship between these altered activities and social cognition and perception-and their genetic underpinnings-are not known in humans. To address these issues, we took advantage of the link between hemizygous deletion of human chromosome 22q11.2 and high rates of social behavior dysfunction, schizophrenia and ASD. We genetically manipulated Sept5, a 22q11.2 gene, and evaluated its role in social interaction in mice. Sept5 deficiency, against a high degree of homogeneity in a congenic genetic background, selectively impaired active affiliative social interaction in mice. Conversely, virally guided overexpression of Sept5 in the hippocampus or, to a lesser extent, the amygdala elevated levels of active affiliative social interaction in C57BL/6J mice. Congenic knockout mice and mice overexpressing Sept5 in the hippocampus or amygdala were indistinguishable from control mice in novelty and olfactory responses, anxiety or motor activity. Moreover, post-weaning individual housing, an environmental condition designed to reduce stress in male mice, selectively raised levels of Sept5 protein in the amygdala and increased active affiliative social interaction in C57BL/6J mice. These findings identify this 22q11.2 gene in the hippocampus and amygdala as a determinant of social interaction and suggest that defective social interaction seen in 22q11.2-associated schizophrenia and ASD can be genetically and environmentally modified by altering this 22q11.2 gene.

  15. Improving cellular properties for genetic manipulation by dispersed growing mutagenesis in Myxococcus fulvus HW-1.

    PubMed

    Zhang, Cui-ying; Cai, Ke; Wu, Zhi-hong; Li, Yue-zhong

    2010-06-01

    One of the key limitations to genetic manipulation in myxobacteria is that the cells grow in clumps in liquid. A salt-tolerant strain HW-1 of Myxococcus fulvus was treated with UV irradiation and produced a completely dispersedly growing mutant UV684. There were no significant differences between the parent HW-1 and the mutant UV684 in terms of salt-tolerant growth. The mutant UV684 and the parent strain had the similar abilities of the fruiting body formation and S motility. Interestingly, the mutant exhibited high transformation/transposition efficiency with 10(5)-10(6) colony-forming units per microg DNA, which was about 10(3)-10(5) fold higher than HW-1. The results indicate that the mutation that led to dispersed growth in the UV684 mutant strain had a few impacts on social behavior, but greatly facilitated molecular genetic manipulation.

  16. Methods for human embryonic stem cells derived cardiomyocytes cultivation, genetic manipulation, and transplantation.

    PubMed

    Arbel, Gil; Caspi, Oren; Huber, Irit; Gepstein, Amira; Weiler-Sagie, Michal; Gepstein, Lior

    2010-01-01

    A decade has passed since the initial derivation of human embryonic stem cells (hESC). The ensuing years have witnessed a significant progress in the development of methodologies allowing cell cultivation, differentiation, genetic manipulation, and in vivo transplantation. Specifically, the potential to derive human cardiomyocytes from the hESC lines, which can be used for several basic and applied cardiovascular research areas including in the emerging field of cardiac regenerative medicine, attracted significant attention from the scientific community. This resulted in the development of protocols for the cultivation of hESC and their successful differentiation toward the cardiomyocyte lineage fate. In this chapter, we will describe in detail methods related to the cultivation, genetic manipulation, selection, and in vivo transplantation of hESC-derived cardiomyocytes.

  17. CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method.

    PubMed

    Ma, Xiaolong; Chen, Chao; Veevers, Jennifer; Zhou, XinMin; Ross, Robert S; Feng, Wei; Chen, Ju

    2017-02-08

    Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate.

  18. CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method

    PubMed Central

    Ma, Xiaolong; Chen, Chao; Veevers, Jennifer; Zhou, XinMin; Ross, Robert S.; Feng, Wei; Chen, Ju

    2017-01-01

    Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate. PMID:28176880

  19. Reclamation of Ampicillin Sensitivity for the Genetic Manipulation of Legionella pneumophila

    PubMed Central

    Sutherland, Molly C.

    2012-01-01

    Research on Legionella pneumophila, the causative agent of Legionnaires' disease, has been hampered due to the lack of selectable markers for genetic manipulation. We report the construction of a mutant strain of L. pneumophila lacking loxA, a chromosomally encoded β-lactamase, that has enhanced sensitivity to ampicillin. Also described are a method for converting Legionella strains to ampicillin sensitivity and conditions for utilizing bla as a selectable marker. PMID:22635996

  20. Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

    PubMed Central

    Bastidas, Robert J.

    2016-01-01

    SUMMARY Chlamydia species infect millions of individuals worldwide and are important etiological agents of sexually transmitted disease, infertility, and blinding trachoma. Historically, the genetic intractability of this intracellular pathogen has hindered the molecular dissection of virulence factors contributing to its pathogenesis. The obligate intracellular life cycle of Chlamydia and restrictions on the use of antibiotics as selectable markers have impeded the development of molecular tools to genetically manipulate these pathogens. However, recent developments in the field have resulted in significant gains in our ability to alter the genome of Chlamydia, which will expedite the elucidation of virulence mechanisms. In this review, we discuss the challenges affecting the development of molecular genetic tools for Chlamydia and the work that laid the foundation for recent advancements in the genetic analysis of this recalcitrant pathogen. PMID:27030552

  1. Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen.

    PubMed

    Bastidas, Robert J; Valdivia, Raphael H

    2016-06-01

    Chlamydia species infect millions of individuals worldwide and are important etiological agents of sexually transmitted disease, infertility, and blinding trachoma. Historically, the genetic intractability of this intracellular pathogen has hindered the molecular dissection of virulence factors contributing to its pathogenesis. The obligate intracellular life cycle of Chlamydia and restrictions on the use of antibiotics as selectable markers have impeded the development of molecular tools to genetically manipulate these pathogens. However, recent developments in the field have resulted in significant gains in our ability to alter the genome of Chlamydia, which will expedite the elucidation of virulence mechanisms. In this review, we discuss the challenges affecting the development of molecular genetic tools for Chlamydia and the work that laid the foundation for recent advancements in the genetic analysis of this recalcitrant pathogen. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. Genetic modification of corneal neovascularization in Dstncorn1 mice

    PubMed Central

    Kawakami-Schulz, Sharolyn V.; Sattler, Shannon G.; Doebley, Anna-Lisa; Ikeda, Akihiro; Ikeda, Sakae

    2013-01-01

    Mutations in the gene for destrin (Dstn), an actin depolymerizing factor, lead to corneal abnormalities in mice. A null mutation in Dstn, termed Dstncorn1, isolated and maintained in the A.BY background (A.BY Dstncorn1), results in corneal epithelial hyperproliferation, inflammation and neovascularization. We previously reported that neovascularization in the cornea of Dstncorn1 on the C57BL/6 background (B6.A.BY-Dstncorn1) mice is significantly reduced when compared to A.BY Dstncorn1 mice, suggesting the existence of genetic modifier(s). The purpose of this study is to identify the genetic basis of difference in cornea neovascularization between A.BY Dstncorn1 and B6.A.BY-Dstncorn1 mice. We generated N2 mice for a whole genome scan by backcrossing F1 progeny (A.BY Dstncorn1 X B6.A.BY-Dstncorn1) to B6.A.BY-Dstncorn1 mice. N2 progeny were quantitatively phenotyped for the extent of corneal neovascularization and genotyped for markers across the mouse genome. We identified significant association of variability in corneal neovascularization with a locus on chromosome 3 (Chr3). The validity of identified quantitative trait locus (QTL) was tested using B6 consomic mice carrying Chr3 from A/J mice. Dstncorn1 mice from F1 and F2 intercrosses (B6.A.BY- Dstncorn1 x C57BL/6J-Chr 3A/J/NaJ) were phenotyped for the extent of corneal neovascularization. This analysis showed that mice carrying the A/J allele at the QTL show significantly increased neovascularization. Our results indicate the existence of a modifier that genetically interacts with the Dstn gene. This modifier demonstrates allelic differences between C57BL6 and A.BY or A/J. The modifier is sufficient to increase neovascularization in Dstncorn1 mice. PMID:23929036

  3. Genetic manipulation in Δku80 strains for functional genomic analysis of Toxoplasma gondii.

    PubMed

    Rommereim, Leah M; Hortua Triana, Miryam A; Falla, Alejandra; Sanders, Kiah L; Guevara, Rebekah B; Bzik, David J; Fox, Barbara A

    2013-07-12

    Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination. Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein(1,2). The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale(1-4). Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human

  4. Genetics of Rapid and Extreme Size Evolution in Island Mice.

    PubMed

    Gray, Melissa M; Parmenter, Michelle D; Hogan, Caley A; Ford, Irene; Cuthbert, Richard J; Ryan, Peter G; Broman, Karl W; Payseur, Bret A

    2015-09-01

    Organisms on islands provide a revealing window into the process of adaptation. Populations that colonize islands often evolve substantial differences in body size from their mainland relatives. Although the ecological drivers of this phenomenon have received considerable attention, its genetic basis remains poorly understood. We use house mice (subspecies: Mus musculus domesticus) from remote Gough Island to provide a genetic portrait of rapid and extreme size evolution. In just a few hundred generations, Gough Island mice evolved the largest body size among wild house mice from around the world. Through comparisons with a smaller-bodied wild-derived strain from the same subspecies (WSB/EiJ), we demonstrate that Gough Island mice achieve their exceptional body weight primarily by growing faster during the 6 weeks after birth. We use genetic mapping in large F(2) intercrosses between Gough Island mice and WSB/EiJ to identify 19 quantitative trait loci (QTL) responsible for the evolution of 16-week weight trajectories: 8 QTL for body weight and 11 QTL for growth rate. QTL exhibit modest effects that are mostly additive. We conclude that body size evolution on islands can be genetically complex, even when substantial size changes occur rapidly. In comparisons to published studies of laboratory strains of mice that were artificially selected for divergent body sizes, we discover that the overall genetic profile of size evolution in nature and in the laboratory is similar, but many contributing loci are distinct. Our results underscore the power of genetically characterizing the entire growth trajectory in wild populations and lay the foundation necessary for identifying the mutations responsible for extreme body size evolution in nature. Copyright © 2015 by the Genetics Society of America.

  5. Genetic control of BCG-induced granulomatous inflammation in mice.

    PubMed

    Sternick, J L; Schrier, D J; Moore, V L

    1983-11-01

    The genetics of BCG-induced pulmonary granulomatous inflammation (PGI) and splenomegaly was studied by breeding experiments and by the use of BXD recombinant inbred (RI) and allotype-congenic mice. Breeding studies indicated that the genetic control was multifactorial; this observation was confirmed using BXD RI mice. In addition, studies with BXD RI mice suggested that genes linked to the Igh complex influence responsiveness. The influence of the Igh-linked genes was studied further using allotype-congenic mice; SJL mice (Ighb) developed significantly greater PGI than their congenic partner, SJA/9 (Igha). Data from BALB.Igb, CB-20, and BAB-14 mice suggested that Igh-linked genes influencing PGI were a considerable distance from Igh-1. Igh-linked genes that influence BCG-induced splenomegaly were located on the centrometric side of the Igh-1 locus. This was shown by data in which splenomegaly in BALB.Igb and CB-20, but not BAB-14, mice was significantly augmented over BALB/c mice. Further studies are necessary to understand the significance of these observations.

  6. Genetic manipulation of STEP reverses behavioral abnormalities in a fragile X syndrome mouse model

    PubMed Central

    Goebel-Goody, Susan M.; Wilson-Wallis, Evan D.; Royston, Sara; Tagliatela, Stephanie; Naegele, Janice R.; Lombroso, Paul J.

    2014-01-01

    Fragile X syndrome (FXS), the most common inherited form of intellectual disability and prevailing known genetic basis of autism, is caused by an expansion in the Fmr1 gene that prevents transcription and translation of fragile X mental retardation protein (FMRP). FMRP binds to and controls translation of mRNAs downstream of metabotropic glutamate receptor (mGluR) activation. Recent work identified striatal-enriched protein tyrosine phosphatase (STEP) as an FMRP target mRNA. STEP opposes synaptic strengthening and promotes synaptic weakening by dephosphorylating its substrates, including ERK1/2, p38, Fyn, Pyk2, and subunits of NMDA and AMPA receptors. Here we demonstrate that STEP translation is dysregulated in Fmr1KO mice, resulting in elevated basal levels of STEP with a concomitant loss of mGluR-dependent STEP translation. We hypothesized that the weakened synaptic strength and behavioral abnormalities reported in FXS may be linked to excess levels of STEP. To test this hypothesis, we reduced or eliminated STEP genetically in Fmr1KO mice. In addition to attenuating audiogenic seizures and seizure-induced c-Fos activation in the periaqueductal gray, genetically reducing STEP in Fmr1KO mice reversed characteristic social abnormalities, including approach, investigation, novelty-induced hyperactivity and anxiety. Loss of STEP also corrected select non-social anxiety-related behaviors in Fmr1KO mice, such as open arm exploration in the elevated plus maze. Our findings indicate that genetically reducing STEP significantly diminishes seizures and restores social and non-social anxiety-related behaviors in Fmr1KO mice, suggesting that strategies to inhibit STEP activity may be effective for treating patients with FXS. PMID:22405502

  7. Genetic Analysis of Daily Activity in Humans and Mice

    DTIC Science & Technology

    2007-11-02

    of the technical developments that have made such genetic dissections a productive force in the mouse , have, when combined with innovations in...and Mice AFOSR grant F49620-97-1-0321 Joseph S. Takahashi Dept. of Neurobiology & Physiology Northwestern University 2153 North Campus Dr. Evanston...Activity in Humans and Mice Unclassified 5c. PROGRAM ELEMENT NUMBER 5d. PROJECT NUMBER 5e. TASK NUMBER 6. AUTHOR(S) Takahashi, Joseph S. ; 5f. WORK

  8. Genetic manipulation of genes and cells in the nervous system of the fruit fly

    PubMed Central

    Venken, Koen J.T.; Simpson, Julie H.; Bellen, Hugo J.

    2011-01-01

    Research in the fruit fly Drosophila melanogaster has lead to insights in neural development, axon guidance, ion channel function, synaptic transmission, learning and memory, diurnal rythmicity, and neural disease that have had broad implications for neuroscience. Drosophila is currently the eukaryotic model organism that permits the most sophisticated in vivo manipulations to address the function of neurons and neuronally expressed genes. Here, we summarize many of the techniques that help assess the role of specific neurons by labeling, removing, or altering their activity. We also survey genetic manipulations to identify and characterize neural genes by mutation, over-expression, and protein labeling. Here, we attempt to acquaint the reader with available options and contexts to apply these methods. PMID:22017985

  9. Genetics of Rapid and Extreme Size Evolution in Island Mice

    PubMed Central

    Gray, Melissa M.; Parmenter, Michelle D.; Hogan, Caley A.; Ford, Irene; Cuthbert, Richard J.; Ryan, Peter G.; Broman, Karl W.; Payseur, Bret A.

    2015-01-01

    Organisms on islands provide a revealing window into the process of adaptation. Populations that colonize islands often evolve substantial differences in body size from their mainland relatives. Although the ecological drivers of this phenomenon have received considerable attention, its genetic basis remains poorly understood. We use house mice (subspecies: Mus musculus domesticus) from remote Gough Island to provide a genetic portrait of rapid and extreme size evolution. In just a few hundred generations, Gough Island mice evolved the largest body size among wild house mice from around the world. Through comparisons with a smaller-bodied wild-derived strain from the same subspecies (WSB/EiJ), we demonstrate that Gough Island mice achieve their exceptional body weight primarily by growing faster during the 6 weeks after birth. We use genetic mapping in large F2 intercrosses between Gough Island mice and WSB/EiJ to identify 19 quantitative trait loci (QTL) responsible for the evolution of 16-week weight trajectories: 8 QTL for body weight and 11 QTL for growth rate. QTL exhibit modest effects that are mostly additive. We conclude that body size evolution on islands can be genetically complex, even when substantial size changes occur rapidly. In comparisons to published studies of laboratory strains of mice that were artificially selected for divergent body sizes, we discover that the overall genetic profile of size evolution in nature and in the laboratory is similar, but many contributing loci are distinct. Our results underscore the power of genetically characterizing the entire growth trajectory in wild populations and lay the foundation necessary for identifying the mutations responsible for extreme body size evolution in nature. PMID:26199233

  10. Common DNA sequences with potential for detection of genetically manipulated organisms in food.

    PubMed

    MacCormick, C A; Griffin, H G; Underwood, H M; Gasson, M J

    1998-06-01

    Foods produced by genetic engineering technology are now appearing on the market and many more are likely to emerge in the future. The safety aspects, regulation, and labelling of these foods are still contentious issues in most countries and recent surveys highlight consumer concerns about the safety and labelling of genetically modified food. In most countries it is necessary to have approval for the use of genetically manipulated organisms (GMOs) in the production of food. In order to police regulations, a technology to detect such foods is desirable. In addition, a requirement to label approved genetically modified food would necessitate a monitoring system. One solution is to 'tag' approved GMOs with some form of biological or genetic marker, permitting the surveillance of foods for the presence of approved products of genetic engineering. While non-approved GMOs would not be detected by such a surveillance, they might be detected by a screen for DNA sequences common to all or most GMOs. This review focuses on the potential of using common DNA sequences as detection probes for GMOs. The identification of vector sequences, plant transcription terminators, and marker genes by PCR and hybridization techniques is discussed.

  11. Novel PCR assay for determining the genetic sex of mice.

    PubMed

    McFarlane, L; Truong, V; Palmer, J S; Wilhelm, D

    2013-01-01

    A number of studies require the determination of the genetic sex of mouse embryos before sexual differentiation and/or of mutant mice that display partial or complete sex reversal. The majority of current methods for sexing by PCR involve multiplexing of 2 primer pairs. We have developed a novel sexing PCR using a single primer pair that amplifies fragments from the X and the Y chromosome with a clear size difference between the respective amplicons. This assay provides a rapid and reliable method to identify the genetic sex of mice across different mouse strains.

  12. Psychological aspects of human cloning and genetic manipulation: the identity and uniqueness of human beings.

    PubMed

    Morales, N M

    2009-01-01

    Human cloning has become one of the most controversial debates about reproduction in Western civilization. Human cloning represents asexual reproduction, but the critics of human cloning argue that the result of cloning is not a new individual who is genetically unique. There is also awareness in the scientific community, including the medical community, that human cloning and the creation of clones are inevitable. Psychology and other social sciences, together with the natural sciences, will need to find ways to help the healthcare system, to be prepared to face the new challenges introduced by the techniques of human cloning. One of those challenges is to help the healthcare system to find specific standards of behaviour that could be used to help potential parents to interact properly with cloned babies or children created through genetic manipulation. In this paper, the concepts of personality, identity and uniqueness are discussed in relationship to the contribution of twin studies in these areas. The author argues that an individual created by human cloning techniques or any other type of genetic manipulation will not show the donor's characteristics to the extent of compromising uniqueness. Therefore, claims to such an effect are needlessly alarmist.

  13. Public acceptance of human gene therapy and perceptions of human genetic manipulation.

    PubMed

    Macer, D R

    1992-10-01

    Clinical trials of gene therapy are underway in different countries, and further countries can be expected to use gene therapy soon. Little remains known, however, about public perceptions of gene therapy. Nationwide mail response opinion surveys were conducted in Japan in August-October, 1991. A total of 54% of the public, 65% of the high school biology teachers, and 54% of the scientists who responded said that they would be willing to use gene therapy, and 66%, 73%, and 62%, respectively, said that they would be willing to use gene therapy on their children. There appears to be growing acceptance of gene therapy in Japan, although about one-quarter of the population are against it. The underlying reasoning behind the acceptability of human genetic manipulation and perceived benefits and risks are presented, and these were found to be generally similar to reasoning expressed in a similar survey conducted in New Zealand in May, 1990. Public perceptions are also compared to those in Europe and the United States. People perceive both benefits and risks from genetic manipulation. There appears to be more teaching of ethical, social, and environmental issues associated with genetic engineering in senior high school biology classes in New Zealand than in Japan. In Japan and New Zealand, about 90% of the public would support including discussion of social issues associated with science and technology in the curriculum.

  14. Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei.

    PubMed

    Choi, Kyoung-Hee; Mima, Takehiko; Casart, Yveth; Rholl, Drew; Kumar, Ayush; Beacham, Ifor R; Schweizer, Herbert P

    2008-02-01

    Because of Burkholderia pseudomallei's classification as a select agent in the United States, genetic manipulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new tools were developed. A temperature-sensitive pRO1600 broad-host-range replicon was isolated and used to construct curable plasmids where the Flp and Cre recombinase genes are expressed from the rhamnose-regulated Escherichia coli P(BAD) promoter and kanamycin (nptI) and zeocin (ble) selection markers from the constitutive Burkholderia thailandensis ribosomal P(S12) or synthetic bacterial P(EM7) promoter. Flp and Cre site-specific recombination systems allow in vivo excision and recycling of nptII and ble selection markers contained on FRT or loxP cassettes. Finally, expression of Tn7 site-specific transposase from the constitutive P1 integron promoter allowed development of an efficient site-specific chromosomal integration system for B. pseudomallei. In conjunction with a natural transformation method, the utility of these new tools was demonstrated by isolating an unmarked delta(amrRAB-oprA) efflux pump mutant. Exploiting natural transformation, chromosomal DNA fragments carrying this mutation marked with zeocin resistance were transferred between the genomes of two different B. pseudomallei strains. Lastly, the deletion mutation was complemented by a chromosomally integrated mini-Tn7 element carrying the amrAB-oprA operon. The new tools allow routine select-agent-compliant genetic manipulations of B. pseudomallei and other Burkholderia species.

  15. ES cell technology: an introduction to genetic manipulation, differentiation and therapeutic cloning.

    PubMed

    Hook, Lilian; O'Brien, Carmel; Allsopp, Timothy

    2005-12-12

    ES cells are extraordinary cells, capable of proliferating in a pluripotent state indefinitely and of differentiating spontaneously into all cell types in vivo and many in vitro. However, the manipulation and modification of ES cells by processes such as directed differentiation and genetic modification have placed ES cells at the forefront of many biological studies and could lead to their application in biopharmaceutical areas such as cellular therapy and drug screening. Here we describe some of the ES cell based technologies that have lead to this realisation of ES cell potential.

  16. Advanced technologies for genetically manipulating the silkworm Bombyx mori, a model Lepidopteran insect

    PubMed Central

    Xu, Hanfu; O'Brochta, David A.

    2015-01-01

    Genetic technologies based on transposon-mediated transgenesis along with several recently developed genome-editing technologies have become the preferred methods of choice for genetically manipulating many organisms. The silkworm, Bombyx mori, is a Lepidopteran insect of great economic importance because of its use in silk production and because it is a valuable model insect that has greatly enhanced our understanding of the biology of insects, including many agricultural pests. In the past 10 years, great advances have been achieved in the development of genetic technologies in B. mori, including transposon-based technologies that rely on piggyBac-mediated transgenesis and genome-editing technologies that rely on protein- or RNA-guided modification of chromosomes. The successful development and application of these technologies has not only facilitated a better understanding of B. mori and its use as a silk production system, but also provided valuable experiences that have contributed to the development of similar technologies in non-model insects. This review summarizes the technologies currently available for use in B. mori, their application to the study of gene function and their use in genetically modifying B. mori for biotechnology applications. The challenges, solutions and future prospects associated with the development and application of genetic technologies in B. mori are also discussed. PMID:26108630

  17. Advanced technologies for genetically manipulating the silkworm Bombyx mori, a model Lepidopteran insect.

    PubMed

    Xu, Hanfu; O'Brochta, David A

    2015-07-07

    Genetic technologies based on transposon-mediated transgenesis along with several recently developed genome-editing technologies have become the preferred methods of choice for genetically manipulating many organisms. The silkworm, Bombyx mori, is a Lepidopteran insect of great economic importance because of its use in silk production and because it is a valuable model insect that has greatly enhanced our understanding of the biology of insects, including many agricultural pests. In the past 10 years, great advances have been achieved in the development of genetic technologies in B. mori, including transposon-based technologies that rely on piggyBac-mediated transgenesis and genome-editing technologies that rely on protein- or RNA-guided modification of chromosomes. The successful development and application of these technologies has not only facilitated a better understanding of B. mori and its use as a silk production system, but also provided valuable experiences that have contributed to the development of similar technologies in non-model insects. This review summarizes the technologies currently available for use in B. mori, their application to the study of gene function and their use in genetically modifying B. mori for biotechnology applications. The challenges, solutions and future prospects associated with the development and application of genetic technologies in B. mori are also discussed.

  18. A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

    PubMed Central

    Lipscomb, Gina L.; Conway, Jonathan M.; Blumer-Schuette, Sara E.; Kelly, Robert M.

    2016-01-01

    ABSTRACT Caldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in the pyrF genome region. The pyrF gene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand the C. bescii genetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs in C. bescii. These strains showed resistance to kanamycin at concentrations >50 μg · ml−1, whereas wild-type C. bescii was sensitive to kanamycin at 10 μg · ml−1. In addition, placement of the Cbhtk marker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of the pyrE gene in wild-type C. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. The pyrE gene functioned as a counterselectable marker, like pyrF, and was used together with Cbhtk in the ΔpyrE background strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme. IMPORTANCE Caldicellulosiruptor bescii is a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic

  19. A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii.

    PubMed

    Lipscomb, Gina L; Conway, Jonathan M; Blumer-Schuette, Sara E; Kelly, Robert M; Adams, Michael W W

    2016-07-15

    Caldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in the pyrF genome region. The pyrF gene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand the C. bescii genetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs in C. bescii These strains showed resistance to kanamycin at concentrations >50 μg · ml(-1), whereas wild-type C. bescii was sensitive to kanamycin at 10 μg · ml(-1) In addition, placement of the Cbhtk marker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of the pyrE gene in wild-type C. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. The pyrE gene functioned as a counterselectable marker, like pyrF, and was used together with Cbhtk in the ΔpyrE background strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme. Caldicellulosiruptor bescii is a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications

  20. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering.

    PubMed

    Yang, Hui; Wang, Haoyi; Jaenisch, Rudolf

    2014-08-01

    Mice with specific gene modifications are valuable tools for studying development and disease. Traditional gene targeting in mice using embryonic stem (ES) cells, although suitable for generating sophisticated genetic modifications in endogenous genes, is complex and time-consuming. We have recently described CRISPR/Cas-mediated genome engineering for the generation of mice carrying mutations in multiple genes, endogenous reporters, conditional alleles or defined deletions. Here we provide a detailed protocol for embryo manipulation by piezo-driven injection of nucleic acids into the cytoplasm to create gene-modified mice. Beginning with target design, the generation of gene-modified mice can be achieved in as little as 4 weeks. We also describe the application of the CRISPR/Cas technology for the simultaneous editing of multiple genes (five genes or more) after a single transfection of ES cells. The principles described in this protocol have already been applied in rats and primates, and they are applicable to sophisticated genome engineering in species in which ES cells are not available.

  1. Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium.

    PubMed

    Osanai, Takashi; Shirai, Tomokazu; Iijima, Hiroko; Nakaya, Yuka; Okamoto, Mami; Kondo, Akihiko; Hirai, Masami Y

    2015-01-01

    Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique.

  2. Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium

    PubMed Central

    Osanai, Takashi; Shirai, Tomokazu; Iijima, Hiroko; Nakaya, Yuka; Okamoto, Mami; Kondo, Akihiko; Hirai, Masami Y.

    2015-01-01

    Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique. PMID:26500619

  3. The genetic architecture of behavioural responses to novelty in mice.

    PubMed

    Crusio, W E; van Abeelen, J H

    1986-02-01

    The genetic architectures of 12 behavioural variables measured in adult male mice placed in a novel environment were analysed in a replicated 4 X 4 diallel cross. The results were combined with those obtained in a classical cross involving two of the four strains. Based on the hypothesis of an evolutionary history of stabilising selection for mouse exploratory behaviour, we expected additive genetic effects and ambidirectional dominance. Such genetic architectures were actually found for those exploratory behaviours where epistatic effects were of minor importance. Similar findings emerged for some non-exploratory phenotypes. All behaviours analysed appeared to be polygenically controlled.

  4. Genetically engineered mice in understanding the basis of neonatal lung disease.

    PubMed

    Glasser, Stephan W; Nogee, Lawrence M

    2006-12-01

    Advances in genetic engineering have allowed the creation of animals with additional or deleted genes. New genes may be inserted in mice, specific genes inactivated or "knocked out," and more complex animals created in which genes can be turned on or off at different times in development or in different tissues. These animal models allow for more detailed studies of the proteins encoded by the manipulated gene, an improved understanding of the pathophysiology of diseases resulting from the genetic alterations, and model organisms in which to study potential new therapies. Multiple mouse models involving genes important in surfactant production and regulation relevant to lung disease observed in human newborns have been created. This review will discuss the creation of such animals and illustrate their utility in understanding human disease.

  5. Using evolutionary costs to enhance the efficacy of malaria control via genetically manipulated mosquitoes.

    PubMed

    Koella, Jacob C; Zaghloul, Lamia

    2008-11-01

    An earlier mathematical model exploring the use of genetically manipulated mosquitoes for malaria control suggested that the prevalence of malaria is reduced significantly only if almost all mosquitoes become completely resistant to malaria. Central to the model was the 'cost of resistance': the reduction of a resistant mosquito's evolutionary fitness in comparison with a sensitive one's. Here, we consider the possibility of obtaining more optimistic outcomes by taking into account the epidemiological (in addition to the evolutionary) consequences of a cost of resistance that decreases the life-span of adult mosquitoes (the most relevant parameter for the parasite's epidemiology). There are two main results. First, if despite its cost, resistance is fixed in the population, increasing the cost of resistance decreases the intensity of transmission. However, this epidemiological effect is weak if resistance is effective enough to be considered relevant for control. Second, if the cost of resistance prevents its fixation, increasing it intensifies transmission. Thus, the epidemiological effect of the cost of resistance cannot compensate for the lower frequency of resistant mosquitoes in the population. Overall, our conclusion remains pessimistic: so that genetic manipulation can become a promising method of malaria control, we need techniques that enable almost all mosquitoes to be almost completely resistant to infection.

  6. In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation

    PubMed Central

    Kawasaki, Hiroshi; Toda, Tomohisa; Tanno, Kaori

    2013-01-01

    Summary Brain structures such as the outer subventricular zone (OSVZ) and the inner fiber layer (IFL) in the developing cerebral cortex are especially prominent in higher mammals. However, the molecular mechanisms underlying the formation of the OSVZ are still largely unknown, mainly because genetic manipulations that can be applied to the OSVZ in higher mammals had been poorly available. Here we developed and validated a rapid and efficient genetic manipulation technique for germinal zones including the OSVZ using in utero electroporation in developing gyrencephalic carnivore ferrets. We also determined the optimal conditions for using in utero electroporation to express transgenes in germinal zones. Using our electroporation procedure, the morphology of GFP-positive cells in the OSVZ was clearly visible even without immunostaining, and multiple genes were efficiently co-expressed in the same cells. Furthermore, we uncovered that fibers, which seemed to correspond to those in the IFL of monkeys, also existed in ferrets, and were derived from newly generated cortical neurons. Our technique promises to be a powerful tool for investigating the fundamental mechanisms underlying the formation and abnormalities of the cerebral cortex in higher mammals. PMID:23336081

  7. Facilitating Neuron-Specific Genetic Manipulations in Drosophila melanogaster Using a Split GAL4 Repressor.

    PubMed

    Dolan, Michael-John; Luan, Haojiang; Shropshire, William C; Sutcliffe, Ben; Cocanougher, Benjamin; Scott, Robert L; Frechter, Shahar; Zlatic, Marta; Jefferis, Gregory S X E; White, Benjamin H

    2017-06-01

    Efforts to map neural circuits have been galvanized by the development of genetic technologies that permit the manipulation of targeted sets of neurons in the brains of freely behaving animals. The success of these efforts relies on the experimenter's ability to target arbitrarily small subsets of neurons for manipulation, but such specificity of targeting cannot routinely be achieved using existing methods. In Drosophila melanogaster, a widely-used technique for refined cell type-specific manipulation is the Split GAL4 system, which augments the targeting specificity of the binary GAL4-UAS (Upstream Activating Sequence) system by making GAL4 transcriptional activity contingent upon two enhancers, rather than one. To permit more refined targeting, we introduce here the "Killer Zipper" (KZip(+)), a suppressor that makes Split GAL4 targeting contingent upon a third enhancer. KZip(+) acts by disrupting both the formation and activity of Split GAL4 heterodimers, and we show how this added layer of control can be used to selectively remove unwanted cells from a Split GAL4 expression pattern or to subtract neurons of interest from a pattern to determine their requirement in generating a given phenotype. To facilitate application of the KZip(+) technology, we have developed a versatile set of LexAop-KZip(+) fly lines that can be used directly with the large number of LexA driver lines with known expression patterns. KZip(+) significantly sharpens the precision of neuronal genetic control available in Drosophila and may be extended to other organisms where Split GAL4-like systems are used. Copyright © 2017 Dolan et al.

  8. Neurophenotyping genetically modified mice for social behavior.

    PubMed

    Rodriguiz, Ramona M; Colvin, Jennifer S; Wetsel, William C

    2011-01-01

    Sociability in mice is a multidimensional adaptive and functional response. Due to its complexity, it is important that researchers use well-defined behavioral assays that are easily replicated with clearly defined ethograms. In the Mouse Behavioral and Neuroendocrine Analysis Core Facility at Duke University, we have developed a broad series of tests that examine different components of neonatal and adult social behaviors that include sociability, sexual behavior, aggressive and territorial responses, and maternal behaviors. While the purpose of this chapter is not to provide an exhaustive description of all mouse social tests available, we provide investigators with a description of basic procedures and considerations necessary to develop a successful social behavior testing program within their laboratories.

  9. Genetic reassortment of mammalian reoviruses in mice.

    PubMed Central

    Wenske, E A; Chanock, S J; Krata, L; Fields, B N

    1985-01-01

    Reassortments between type 1 (Lang) and type 3 (Dearing) reoviruses were isolated from suckling mice infected perorally with an inoculum containing both type 1 and type 3 viruses. A total of five distinct reassortants (designated as E1 through E5) were isolated from animals during the course of the experiment. Two reassortants (E1 and E2) represented the majority of the reassortants isolated. The majority of genes of types E1 and E2 were derived from type 1 (Lang). However, E1 had an M2 gene and an S1 gene derived from type 3 (Dearing), while E2 had M2 and S2 genes derived from type 3 (Dearing). Thus, nonrandom reassortment between mammalian reoviruses can be demonstrated in vivo. PMID:4057359

  10. Advances in the application of genetic manipulation methods to apicomplexan parasites.

    PubMed

    Suarez, C E; Bishop, R P; Alzan, H F; Poole, W A; Cooke, B M

    2017-10-01

    Apicomplexan parasites such as Babesia, Theileria, Eimeria, Cryptosporidium and Toxoplasma greatly impact animal health globally, and improved, cost-effective measures to control them are urgently required. These parasites have complex multi-stage life cycles including obligate intracellular stages. Major gaps in our understanding of the biology of these relatively poorly characterised parasites and the diseases they cause severely limit options for designing novel control methods. Here we review potentially important shared aspects of the biology of these parasites, such as cell invasion, host cell modification, and asexual and sexual reproduction, and explore the potential of the application of relatively well-established or newly emerging genetic manipulation methods, such as classical transfection or gene editing, respectively, for closing important gaps in our knowledge of the function of specific genes and proteins, and the biology of these parasites. In addition, genetic manipulation methods impact the development of novel methods of control of the diseases caused by these economically important parasites. Transient and stable transfection methods, in conjunction with whole and deep genome sequencing, were initially instrumental in improving our understanding of the molecular biology of apicomplexan parasites and paved the way for the application of the more recently developed gene editing methods. The increasingly efficient and more recently developed gene editing methods, in particular those based on the CRISPR/Cas9 system and previous conceptually similar techniques, are already contributing to additional gene function discovery using reverse genetics and related approaches. However, gene editing methods are only possible due to the increasing availability of in vitro culture, transfection, and genome sequencing and analysis techniques. We envisage that rapid progress in the development of novel gene editing techniques applied to apicomplexan parasites of

  11. Unraveling the neurobiology of nicotine dependence using genetically engineered mice.

    PubMed

    Stoker, Astrid K; Markou, Athina

    2013-08-01

    This review article provides an overview of recent studies of nicotine dependence and withdrawal that used genetically engineered mice. Major progress has been made in recent years with mutant mice that have knockout and gain-of-function of specific neuronal nicotinic acetylcholine receptor (nAChR) subunit genes. Nicotine exerts its actions by binding to neuronal nAChRs that consist of five subunits. The different nAChR subunits that combine to compose a receptor determine the distinct pharmacological and kinetic properties of the specific nAChR. Recent findings in genetically engineered mice have indicated that while α4-containing and β2-containing nAChRs are involved in the acquisition of nicotine self-administration and initial stages of nicotine dependence, α7 homomeric nAChRs appear to be involved in the later stages of nicotine dependence. In the medial habenula, α5-containing, α3-containing, and β4-containing nAChRs were shown to be crucially important in the regulation of the aversive aspects of nicotine. Studies of the involvement of α6 nAChR subunits in nicotine dependence have only recently emerged. The use of genetically engineered mice continues to vastly improve our understanding of the neurobiology of nicotine dependence and withdrawal. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Genetic manipulation of periostin expression reveals a role in cardiac hypertrophy and ventricular remodeling

    PubMed Central

    Oka, Toru; Xu, Jian; Kaiser, Robert A.; Melendez, Jaime; Hambleton, Michael; Sargent, Michelle A.; Lorts, Angela; Brunskill, Eric W.; Dorn, Gerald W.; Conway, Simon J.; Aronow, Bruce J.; Robbins, Jeffrey; Molkentin, Jeffery D.

    2009-01-01

    The cardiac extracellular matrix is a dynamic structural support network that is both influenced by, and a regulator of, pathological remodeling and hypertrophic growth. In response to pathologic insults the adult heart re-expresses the secreted extracellular matrix protein periostin (Pn). Here we show that Pn is critically involved in regulating the cardiac hypertrophic response, interstitial fibrosis, and ventricular remodeling following long-term pressure overload stimulation and myocardial infarction. Mice lacking the gene encoding Pn (Postn) were more prone to ventricular rupture in the first 10 days after a myocardial infarction, but surviving mice showed less fibrosis and better ventricular performance. Pn−/− mice also showed less fibrosis and hypertrophy following long-term pressure overload, suggesting an intimate relationship between Pn and the regulation of cardiac remodeling. In contrast, inducible overexpression of Pn in the heart protected mice from rupture following myocardial infarction and induced spontaneous hypertrophy with aging. With respect to a mechanism underlying these alterations, Pn−/− hearts showed an altered molecular program in fibroblast function. Indeed, fibroblasts isolated from Pn−/− hearts were less effective in adherence to cardiac myocytes and were characterized by a dramatic alteration in global gene expression (7% of all genes). These are the first genetic data detailing the function of Pn in the adult heart as a regulator of cardiac remodeling and hypertrophy. PMID:17569887

  13. Of mice and men: molecular genetics of congenital heart disease.

    PubMed

    Andersen, Troels Askhøj; Troelsen, Karin de Linde Lind; Larsen, Lars Allan

    2014-04-01

    Congenital heart disease (CHD) affects nearly 1 % of the population. It is a complex disease, which may be caused by multiple genetic and environmental factors. Studies in human genetics have led to the identification of more than 50 human genes, involved in isolated CHD or genetic syndromes, where CHD is part of the phenotype. Furthermore, mapping of genomic copy number variants and exome sequencing of CHD patients have led to the identification of a large number of candidate disease genes. Experiments in animal models, particularly in mice, have been used to verify human disease genes and to gain further insight into the molecular pathology behind CHD. The picture emerging from these studies suggest that genetic lesions associated with CHD affect a broad range of cellular signaling components, from ligands and receptors, across down-stream effector molecules to transcription factors and co-factors, including chromatin modifiers.

  14. Analyses of glutathione reductase hypomorphic mice indicate a genetic knockout.

    PubMed

    Rogers, Lynette K; Tamura, Toshiya; Rogers, Bryan J; Welty, Stephen E; Hansen, Thomas N; Smith, Charles V

    2004-12-01

    A strain of mice (Gr1a1Neu) that exhibited tissue glutathione reductase (GR) activities that were substantially lower (less than 10% in liver) than the corresponding activities in control mice has been reported. The present report describes characterization of the mutation(s) in the GR gene of these mice. RT-PCR of mRNA from the Neu mice indicated a substantial deletion in the normal GR coding sequence. Southern blots revealed that the deletion involved a region spanning from intron 1 through intron 5. The exact breakpoints of the deletion were characterized by PCR and sequencing through the region encompassing the deletion. The deletion involves nucleotides 10840 through 23627 of the genomic GR gene and functionally deletes exons 2 through 5. In addition, the deletion produces a frame shift in exon 6 and introduces a stop codon in exon 7 that would prevent translation of the remainder of the protein. Consequently, the Neu mice are incapable of producing a functional GR protein and appear to be genetic knockouts for GR. The Neu mice offer live animal models with which to test hypotheses regarding oxidant mechanisms of tissue injury in vivo.

  15. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes.

    PubMed

    Xiao, Xiangwei; Guo, Ping; Prasadan, Krishna; Shiota, Chiyo; Peirish, Lauren; Fischbach, Shane; Song, Zewen; Gaffar, Iljana; Wiersch, John; El-Gohary, Yousef; Husain, Sohail Z; Gittes, George K

    2014-12-01

    Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type- and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.

  16. Increasing water-use efficiency directly through genetic manipulation of stomatal density.

    PubMed

    Franks, Peter J; W Doheny-Adams, Timothy; Britton-Harper, Zoe J; Gray, Julie E

    2015-07-01

    Improvement in crop water-use efficiency (WUE) is a critical priority for regions facing increased drought or diminished groundwater resources. Despite new tools for the manipulation of stomatal development, the engineering of plants with high WUE remains a challenge. We used Arabidopsis epidermal patterning factor (EPF) mutants exhibiting altered stomatal density to test whether WUE could be improved directly by manipulation of the genes controlling stomatal density. Specifically, we tested whether constitutive overexpression of EPF2 reduced stomatal density and maximum stomatal conductance (gw(max) ) sufficiently to increase WUE. We found that a reduction in gw(max) via reduced stomatal density in EPF2-overexpressing plants (EPF2OE) increased both instantaneous and long-term WUE without altering significantly the photosynthetic capacity. Conversely, plants lacking both EPF1 and EPF2 expression (epf1epf2) exhibited higher stomatal density, higher gw(max) and lower instantaneous WUE, as well as lower (but not significantly so) long-term WUE. Targeted genetic modification of stomatal conductance, such as in EPF2OE, is a viable approach for the engineering of higher WUE in crops, particularly in future high-carbon-dioxide (CO2 ) atmospheres.

  17. Screening and genetic manipulation of green organisms for establishment of biological life support systems in space

    PubMed Central

    Saei, Amir Ata; Omidi, Amir Ali; Barzegari, Abolfazl

    2013-01-01

    Curiosity has driven humankind to explore and conquer space. However, today, space research is not a means to relieve this curiosity anymore, but instead has turned into a need. To support the crew in distant expeditions, supplies should either be delivered from the Earth, or prepared for short durations through physiochemical methods aboard the space station. Thus, research continues to devise reliable regenerative systems. Biological life support systems may be the only answer to human autonomy in outposts beyond Earth. For construction of an artificial extraterrestrial ecosystem, it is necessary to search for highly adaptable super-organisms capable of growth in harsh space environments. Indeed, a number of organisms have been proposed for cultivation in space. Meanwhile, some manipulations can be done to increase their photosynthetic potential and stress tolerance. Genetic manipulation and screening of plants, microalgae and cyanobacteria is currently a fascinating topic in space bioengineering. In this commentary, we will provide a viewpoint on the realities, limitations and promises in designing biological life support system based on engineered and/or selected green organism. Special focus will be devoted to the engineering of key photosynthetic enzymes in pioneer green organisms and their potential use in establishment of transgenic photobioreactors in space. PMID:22992434

  18. Screening and genetic manipulation of green organisms for establishment of biological life support systems in space.

    PubMed

    Saei, Amir Ata; Omidi, Amir Ali; Barzegari, Abolfazl

    2013-01-01

    Curiosity has driven humankind to explore and conquer space. However, today, space research is not a means to relieve this curiosity anymore, but instead has turned into a need. To support the crew in distant expeditions, supplies should either be delivered from the Earth, or prepared for short durations through physiochemical methods aboard the space station. Thus, research continues to devise reliable regenerative systems. Biological life support systems may be the only answer to human autonomy in outposts beyond Earth. For construction of an artificial extraterrestrial ecosystem, it is necessary to search for highly adaptable super-organisms capable of growth in harsh space environments. Indeed, a number of organisms have been proposed for cultivation in space. Meanwhile, some manipulations can be done to increase their photosynthetic potential and stress tolerance. Genetic manipulation and screening of plants, microalgae and cyanobacteria is currently a fascinating topic in space bioengineering. In this commentary, we will provide a viewpoint on the realities, limitations and promises in designing biological life support system based on engineered and/or selected green organism. Special focus will be devoted to the engineering of key photosynthetic enzymes in pioneer green organisms and their potential use in establishment of transgenic photobioreactors in space.

  19. New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast.

    PubMed

    Wosika, Victoria; Durandau, Eric; Varidel, Clémence; Aymoz, Delphine; Schmitt, Marta; Pelet, Serge

    2016-12-01

    The tractability of the budding yeast genome has provided many insights into the fundamental mechanisms regulating cellular life. With the advent of synthetic biology and single-cell measurements, novel tools are required to manipulate the yeast genome in a more controlled manner. We present, here, a new family of yeast shuttle vectors called single integration vectors (pSIV). Upon transformation in yeast, these plasmids replace the entire deficient auxotrophy marker locus by a cassette containing an exogenous marker. As shown using flow cytometry, this complete replacement results in a unique integration of the desired DNA fragment at the marker locus. In addition, a second transcriptional unit can be inserted to achieve the simultaneous integration of two constructs. The selection marker cassettes, present in the pSIV, were also used to generate a complete set of gene tagging plasmids (pGT) encompassing a large palette of fluorescent proteins, from a cyan fluorescent protein to a near-infrared tandem dimer red fluorescent protein. These tagging cassettes are orthogonal to each other thanks to the use of different TEF promoter and terminator couples, thereby avoiding marker cassette switching and favoring integration in the desired locus. In summary, we have created two sets of robust molecular tools for the precise genetic manipulation of the budding yeast.

  20. Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.

    2013-03-01

    We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

  1. Considerations for importing live genetically modified mice from academic laboratories.

    PubMed

    Tyson, Jennifer A

    2012-06-01

    Genetically modified mice have been an invaluable tool for the study of gene function in an intact biological system. Researchers frequently obtain genetically modified mice from other academic institutions. This form of collaboration between laboratories comes with a unique set of challenges, and a clear set of guidelines for navigating the process has yet to be defined. The author provides suggestions for how to initiate an exchange of animal resources and steps for ensuring a successful collaboration. Both parties should be clear about their expectations. The importing lab should prepare in advance for potential animal health considerations and breeding and colony management strategies prior to importation. The number, gender, age and genotype of the imported animals should be confirmed as soon as possible by the importing lab. It is in the best interest of all parties to be courteous, forthright and thorough when sharing animal resources so that everyone can benefit from the resulting research.

  2. Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

    PubMed Central

    2014-01-01

    Glycosaminoglycans (GAGs) are constructed through the stepwise addition of respective monosaccharides by various glycosyltransferases and maturated by epimerases and sulfotransferases. The structural diversity of GAG polysaccharides, including their sulfation patterns and sequential arrangements, is essential for a wide range of biological activities such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Studies using knockout mice of enzymes responsible for the biosynthesis of the GAG side chains of proteoglycans have revealed their physiological functions. Furthermore, mutations in the human genes encoding glycosyltransferases, sulfotransferases, and related enzymes responsible for the biosynthesis of GAGs cause a number of genetic disorders including chondrodysplasia, spondyloepiphyseal dysplasia, and Ehlers-Danlos syndromes. This review focused on the increasing number of glycobiological studies on knockout mice and genetic diseases caused by disturbances in the biosynthetic enzymes for GAGs. PMID:25126564

  3. Genetics and evolution of hybrid male sterility in house mice.

    PubMed

    White, Michael A; Stubbings, Maria; Dumont, Beth L; Payseur, Bret A

    2012-07-01

    Comparative genetic mapping provides insights into the evolution of the reproductive barriers that separate closely related species. This approach has been used to document the accumulation of reproductive incompatibilities over time, but has only been applied to a few taxa. House mice offer a powerful system to reconstruct the evolution of reproductive isolation between multiple subspecies pairs. However, studies of the primary reproductive barrier in house mice-hybrid male sterility-have been restricted to a single subspecies pair: Mus musculus musculus and Mus musculus domesticus. To provide a more complete characterization of reproductive isolation in house mice, we conducted an F(2) intercross between wild-derived inbred strains from Mus musculus castaneus and M. m. domesticus. We identified autosomal and X-linked QTL associated with a range of hybrid male sterility phenotypes, including testis weight, sperm density, and sperm morphology. The pseudoautosomal region (PAR) was strongly associated with hybrid sterility phenotypes when heterozygous. We compared QTL found in this cross with QTL identified in a previous F(2) intercross between M. m. musculus and M. m. domesticus and found three shared autosomal QTL. Most QTL were not shared, demonstrating that the genetic basis of hybrid male sterility largely differs between these closely related subspecies pairs. These results lay the groundwork for identifying genes responsible for the early stages of speciation in house mice.

  4. Alleles that modulate late life hearing in genetically heterogeneous mice

    PubMed Central

    Schacht, Jochen; Altschuler, Richard; Burke, David T.; Chen, Shu; Dolan, David; Galecki, Andrzej T.; Kohrman, David; Miller, Richard A.

    2012-01-01

    A genetically heterogeneous population of mice was tested for hearing at 8, 18 and 22 months by auditory brainstem response (ABR), and genotyped at 128 markers to identify loci that modulate late life hearing loss. Half of the test mice were exposed to noise for 2 hr at age 20 months. Polymorphisms affecting hearing at 18 months were noted on chromosomes 2, 3, 7, 10, and 15. Most of these loci had effects only on responses to 48 kHz stimuli, but a subset also influenced the ABR at lower frequencies. Loci on chromosomes 4, 10, 12, and 14 had significant effects on hearing at 22 months in noise-exposed mice, and loci on chromosomes 10 and 11 had effects on mice not exposed to noise. Outer hair cell loss was modulated by polymorphisms on chromosomes 10, 11, 12, 17, and 19. Resistance to age-related hearing loss is thus modulated by a set of genetic effects, some age-specific, some frequency specific, some dependent on prior exposure to noise, and some of which compromise survival of cochlear hair cells. PMID:22305187

  5. Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells

    PubMed Central

    Pomeroy, Jordan E.; Nguyen, Hung X.; Hoffman, Brenton D.; Bursac, Nenad

    2017-01-01

    Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells. PMID:28912894

  6. Genetic manipulation of human embryonic stem cells in serum and feeder-free media.

    PubMed

    Braam, Stefan R; Denning, Chris; Mummery, Christine L

    2010-01-01

    Generic methods for genetic manipulation of human embryonic stem cells (hESCs) are important for both present research and future commercial applications. To date, differences in cell derivation and culture have required independent optimization of transfection and transduction protocols and some lines have remained refractile to all methods. Here we describe a culture protocol that has been extensively tested in 12 different hESC lines (1, 2) and shown to support efficient gene transfer independent of the method of gene delivery or history of the cell line. The system is based on Matrigel monolayer culture and conditioned medium from mouse embryonic feeder cells (MEFs) and entails transient high-density culture followed by rapid adaptation to low density for gene transfer. Under these conditions, plasmid transfection, virus infection, and siRNA transfection are highly effective. Stable genetically modified hESC lines can be generated with plasmid transfection, viral infection, or electroporation without loss of pluripotency or differentiation potential. The majority of lines generated in this system display a normal karyotype.

  7. Grafting of genetically manipulated cells into adult brain: toward graft-gene therapy.

    PubMed

    Uchida, K; Toya, S

    1996-06-01

    Accumulating evidence has shown that functional recoveries in various kinds of animal models of neurodegenerative diseases can be achieved by grafting fetal neurons into the brain. On the basis of these successful results, clinical trials are under way to determine whether human fetal mesencephalic tissue can ameliorate motor functions in patients with Parkinson's disease. Recent autopsy findings of parkinsonian patient implanted with human fetal mesencephalic tissue clearly revealed that the fetal neuronal graft can survive for extended period of time in the human brain and densely reinnervate the surrounding host striatal tissue. It is, however, still important to obtain more practical, effective and ethically justifiable donor material for the future clinical application of the procedures. Desirable properties for the donor cells include long-term survival in the host brain, neuronal cell type for the reconstruction of damaged neural circuits, and susceptibility to genetic manipulation for the practical use. With the development of molecular biology techniques, genetic modification and transplantation of the donor neuronal cells might be a feasible way to cure many kinds of central nervous system diseases toward a "graft-gene therapy".

  8. Tower of Babel: variation in ethical approaches, concepts of welfare and attitudes to genetic manipulation.

    PubMed

    Appleby, M C

    1999-01-01

    Attitudes to animal biotechnology are diverse, partly because people have different viewpoints and often do not recognize or acknowledge this to be so. First, people adopt different ethical approaches. If an opponent of genetic manipulation says 'I don't like the idea of altering animals' biology' and a proponent replies '...but it is useful', they are failing to communicate, because one is asking whether the action is right or wrong, whereas the other emphasizes the consequences. Another approach focuses on the person carrying out the action. Many people have hybrid views combining elements of these different approaches. Second, people's concepts of welfare vary, emphasizing animal minds, bodies or natures--or a combination of these. A proponent who argues that a particular genetic change will not cause suffering is unlikely to reassure an opponent who puts more emphasis on naturalness than on feelings or health. An improved dialogue, in which people attempt to understand one another's viewpoints, may enable common principles to be established and practical measures to be taken that enable more cooperation in attempts to improve both human and animal welfare.

  9. Genetic Basis of Atherosclerosis: Insights from Mice and Humans

    PubMed Central

    Stylianou, Ioannis M.; Bauer, Robert C.; Reilly, Muredach P.; Rader, Daniel J.

    2012-01-01

    Atherosclerosis is a complex and heritable disease involving multiple cell types and the interactions of many different molecular pathways. The genetic and molecular mechanisms of atherosclerosis have in part been elucidated by mouse models; at least 100 different genes have been shown to influence atherosclerosis in mice. Importantly, unbiased genome-wide association studies have recently identified a number of novel loci robustly associated with atherosclerotic coronary artery disease (CAD). Here we review the genetic data elucidated from mouse models of atherosclerosis, as well as significant associations for human CAD. Furthermore, we discuss in greater detail some of these novel human CAD loci. The combination of mouse and human genetics has the potential to identify and validate novel genes that influence atherosclerosis, some of which may be candidates for new therapeutic approaches. PMID:22267839

  10. Pharmacological and functional genetic assays to manipulate regeneration of the planarian Dugesia japonica.

    PubMed

    Chan, John D; Marchant, Jonathan S

    2011-08-31

    Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation. Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity. The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters, represent an important system for studying evolution of the central nervous system and have behavioral screening potential. Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs. Both

  11. Essentials of recombinase-based genetic fate mapping in mice.

    PubMed

    Jensen, Patricia; Dymecki, Susan M

    2014-01-01

    Fate maps, by defining the relationship between embryonic tissue organization and postnatal tissue structure, are one of the most important tools on hand to developmental biologists. In the past, generating such maps in mice was hindered by their in utero development limiting the physical access required for traditional methods involving tracer injection or cell transplantation. No longer is physical access a requirement. Innovations over the past decade have led to genetic techniques that offer means to "deliver" cell lineage tracers noninvasively. Such "genetic fate mapping" approaches employ transgenic strategies to express genetically encoded site-specific recombinases in a cell type-specific manner to switch on expression of a cell-heritable reporter transgene as lineage tracer. The behaviors and fate of marked cells and their progeny can then be explored and their contributions to different tissues examined. Here, we review the basic concepts of genetic fate mapping and consider the strengths and limitations for their application. We also explore two refinements of this approach that lend improved spatial and temporal resolution: (1) Intersectional and subtractive genetic fate mapping and (2) Genetic inducible fate mapping.

  12. Genetics and Evolution of Hybrid Male Sterility in House Mice

    PubMed Central

    White, Michael A.; Stubbings, Maria; Dumont, Beth L.; Payseur, Bret A.

    2012-01-01

    Comparative genetic mapping provides insights into the evolution of the reproductive barriers that separate closely related species. This approach has been used to document the accumulation of reproductive incompatibilities over time, but has only been applied to a few taxa. House mice offer a powerful system to reconstruct the evolution of reproductive isolation between multiple subspecies pairs. However, studies of the primary reproductive barrier in house mice—hybrid male sterility—have been restricted to a single subspecies pair: Mus musculus musculus and Mus musculus domesticus. To provide a more complete characterization of reproductive isolation in house mice, we conducted an F2 intercross between wild-derived inbred strains from Mus musculus castaneus and M. m. domesticus. We identified autosomal and X-linked QTL associated with a range of hybrid male sterility phenotypes, including testis weight, sperm density, and sperm morphology. The pseudoautosomal region (PAR) was strongly associated with hybrid sterility phenotypes when heterozygous. We compared QTL found in this cross with QTL identified in a previous F2 intercross between M. m. musculus and M. m. domesticus and found three shared autosomal QTL. Most QTL were not shared, demonstrating that the genetic basis of hybrid male sterility largely differs between these closely related subspecies pairs. These results lay the groundwork for identifying genes responsible for the early stages of speciation in house mice. PMID:22554891

  13. [Genetically engineered mice: mouse models for cancer research].

    PubMed

    Szymańska, Hanna

    2007-10-26

    Genetically engineered mice (GEM) have been extensively used to model human cancer. Mouse models mimic the morphology, histopathology, phenotype, and genotype of the corresponding cancer in humans. GEM mice are created by random integration of a transgene into the genome, which results in gene overexpression (transgenic mice); gene deletion (knock-out mice); or targeted insertion of the transgene in a selected locus (knock-in mice). Knock-out may be constitutive, i.e. total inactivation of the gene of interest in any cell, or conditional, i.e. tissue-specific inactivation of the gene. Gene knock-down (RNAi) and humanization of the mouse are more sophisticated models of GEM mice. RNA interference (RNAi) is a mechanism in which double-stranded RNAs inhibits the respective gene expression by inducing degradation of its mRNA. Humanization is based on replacing a mouse gene by its human counterpart. The alterations in genes in GEM have to be heritable. The opportunities provided by employing GEM cancer models are: analysis of the role of specific cancer genes and modifier genes, evaluation of conventional cancer therapies and new drugs, identification of cancer markers of tumor growth, analysis of the influence of the tumor's microenvironment on tumor formation, and the definition of the pre-clinical, discrete steps of tumorigenesis. The validation of mouse models of human cancer is the task of the MMHCC (Mouse Models of Human Cancer Consortium). The GEM models of breast, pancreatic, intestinal and colon, and prostate cancer are the most actively explored. In contrast, the models of brain tumors and ovary, cervical, and skin cancer are in the early stage of investigation.

  14. Genetic Analysis of Mice Skin Exposed by Hyper-Gravity

    NASA Astrophysics Data System (ADS)

    Takahashi, Rika; Terada, Masahiro; Seki, Masaya; Higashibata, Akira; Majima, Hideyuki J.; Ohira, Yoshinobu; Mukai, Chiaki; Ishioka, Noriaki

    2013-02-01

    In the space environment, physiological alterations, such as low bone density, muscle weakness and decreased immunity, are caused by microgravity and cosmic radiation. On the other hand, it is known that the leg muscles are hypertrophy by 2G-gravity. An understanding of the effects on human body from microgravity to hyper-gravity is very important. Recently, the Japan Aerospace Exploration Agency (JAXA) has started a project to detect the changes on gene expression and mineral metabolism caused by microgravity by analyzing the hair of astronauts who stay in the international Space Station (ISS) for a long time. From these results of human hair’s research, the genetic effects of human hair roots by microgravity will become clear. However, it is unclear how the gene expression of hair roots was effected by hypergravity. Therefore, in this experiment, we analyzed the effect on mice skin contained hair roots by comparing microgravity or hypergravity exposed mice. The purpose of this experiment is to evaluate the genetic effects on mice skin by microgravity or 2G-gravity. The samples were taken from mice exposed to space flight (FL) or hypergravity environment (2G) for 3-months, respectively. The extracted and amplified RNA from these mice skin was used to DNA microarray analysis. in this experiment, we analyzed the effect of gravity by using mice skin contained hair roots, which exposed space (FL) and hyper-gravity (2G) for 3 months and each control. By DNA microarray analysis, we found the common 98 genes changed in both FL and 2G. Among these 98 genes, the functions and pathways were identified by Gene Ontology (GO) analysis and Ingenuity Pathways Analysis (IPA) software. Next, we focused the one of the identified pathways and compared the effects on each molecules in this pathways by the different environments, such as FL and 2G. As the results, we could detect some interesting molecules, which might be depended on the gravity levels. In addition, to investigate

  15. Influence of genetic background on fluoride metabolism in mice.

    PubMed

    Carvalho, J G; Leite, A L; Yan, D; Everett, E T; Whitford, G M; Buzalaf, M A R

    2009-11-01

    A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis, due to their genetic backgrounds. This study tested whether these differences are due to variations in water intake and/or F metabolism. A/J (susceptible to dental fluorosis) and 129P3/J mice (resistant) received drinking water containing 0, 10, or 50 ppm F. Weekly F intake, excretion and retention, and terminal plasma and femur F levels were determined. Dental fluorosis was evaluated clinically and by quantitative fluorescence (QF). Data were tested by two-way ANOVA. Although F intakes by the strains were similar, excretion by A/J mice was significantly higher due to greater urinary F excretion, which resulted in lower plasma and femur F levels. Compared with 129P3/J mice given 50 ppm F, significantly higher QF scores were recorded for A/J mice. In conclusion, these strains differ with respect to several features of F metabolism, and amelogenesis in the 129P3/J strain seems to be unaffected by high F exposure.

  16. Influence of Genetic Background on Fluoride Metabolism in Mice

    PubMed Central

    Carvalho, J.G.; Leite, A.L.; Yan, D.; Everett, E.T.; Whitford, G.M.; Buzalaf, M.A.R.

    2009-01-01

    A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis, due to their genetic backgrounds. This study tested whether these differences are due to variations in water intake and/or F metabolism. A/J (susceptible to dental fluorosis) and 129P3/J mice (resistant) received drinking water containing 0, 10, or 50 ppm F. Weekly F intake, excretion and retention, and terminal plasma and femur F levels were determined. Dental fluorosis was evaluated clinically and by quantitative fluorescence (QF). Data were tested by two-way ANOVA. Although F intakes by the strains were similar, excretion by A/J mice was significantly higher due to greater urinary F excretion, which resulted in lower plasma and femur F levels. Compared with 129P3/J mice given 50 ppm F, significantly higher QF scores were recorded for A/J mice. In conclusion, these strains differ with respect to several features of F metabolism, and amelogenesis in the 129P3/J strain seems to be unaffected by high F exposure. PMID:19828896

  17. Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals

    PubMed Central

    Gompf, Heinrich S.; Budygin, Evgeny A.; Fuller, Patrick M.; Bass, Caroline E.

    2015-01-01

    Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to  the  rat  locus  coeruleus  (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from

  18. Ethanol-induced hypothermia and hyperglycemia in genetically obese mice

    SciTech Connect

    Haller, E.W.; Wittmers, L.E. Jr.

    1989-01-01

    Blood glucose and rectal temperatures were monitored in two strains of genetically obese mice (C57 BL/6J ob/ob) prior to and following intragastric ethanol administration in an attempt to relate the hypothermic response to ethanol to extracellular glucose concentration. In contrast to expectation, ethanol administration was typically associated with a hyperglycemia and a hypothermic response. In the ob/ob genotype, the hypothermic response was associated with pronounced hyperglycemia which was more emphatic in older animals. The data support the conclusion that ethanol-induced hypothermia is independent of blood glucose levels. In light of the known sensitivity of ob/ob mice to insulin, it is suggested further that the observed hypothermic response was not a function of the animals' ability to transport glucose into peripheral cells. The observed hyperglycemia of the obese animals was most likely stress-related

  19. Hybrid mice as genetic models of high alcohol consumption.

    PubMed

    Blednov, Y A; Ozburn, A R; Walker, D; Ahmed, S; Belknap, J K; Harris, R A

    2010-01-01

    We showed that F1 hybrid genotypes may provide a broader variety of ethanol drinking phenotypes than the inbred progenitor strains used to create the hybrids (Blednov et al. in Alcohol Clin Exp Res 29:1949-1958, 2005). To extend this work, we characterized alcohol consumption as well as intake of other tastants (saccharin, quinine and sodium chloride) in five inbred strains of mice (FVB, SJL, B6, BUB, NZB) and in their reciprocal F1 hybrids with B6 (FVBxB6; B6xFVB; NZBxB6; B6xNZB; BUBxB6; B6xBUB; SJLxB6; B6xSJL). We also compared ethanol intake in these mice for several concentrations before and after two periods of abstinence. F1 hybrid mice derived from the crosses of B6 and FVB and also B6 and SJL drank higher levels of ethanol than their progenitor strains, demonstrating overdominance for two-bottle choice drinking test. The B6 and NZB hybrid showed additivity in two-bottle choice drinking, whereas the hybrid of B6 and BUB demonstrated full or complete dominance. Genealogical origin, as well as non-alcohol taste preferences (sodium chloride), predicted ethanol consumption. Mice derived from the crosses of B6 and FVB showed high sustained alcohol preference and the B6 and NZB hybrids showed reduced alcohol preference after periods of abstinence. These new genetic models offer some advantages over inbred strains because they provide high, sustained, alcohol intake, and should allow mapping of loci important for the genetic architecture of these traits.

  20. Genetic Dissection of Learning and Memory in Mice

    PubMed Central

    Mineur, Yann S.; Crusio, Wim E.; Sluyter, Frans

    2004-01-01

    In this minireview, we discuss different strategies to dissect genetically the keystones of learning and memory. First, we broadly sketch the neurogenetic analysis of complex traits in mice. We then discuss two general strategies to find genes affecting learning and memory: candidate gene studies and whole genome searches. Next, we briefly review more recently developed techniques, such as microarrays and RNA interference. In addition, we focus on gene-environment interactions and endophenotypes. All sections are illustrated with examples from the learning and memory field, including a table summarizing the latest information about genes that have been shown to have effects on learning and memory. PMID:15656270

  1. Genetic dissection of learning and memory in mice.

    PubMed

    Mineur, Yann S; Crusio, Wim E; Sluyter, Frans

    2004-01-01

    In this minireview, we discuss different strategies to dissect genetically the keystones of learning and memory. First, we broadly sketch the neurogenetic analysis of complex traits in mice. We then discuss two general strategies to find genes affecting learning and memory: candidate gene studies and whole genome searches. Next, we briefly review more recently developed techniques, such as microarrays and RNA interference. In addition, we focus on gene-environment interactions and endophenotypes. All sections are illustrated with examples from the learning and memory field, including a table summarizing the latest information about genes that have been shown to have effects on learning and memory.

  2. Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

    PubMed Central

    Cue, D; Lam, H; Dillingham, R L; Hanson, R S; Flickinger, M C

    1997-01-01

    We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus. PMID:9097439

  3. The Genetic Basis of Baculum Size and Shape Variation in Mice

    PubMed Central

    Schultz, Nicholas G.; Ingels, Jesse; Hillhouse, Andrew; Wardwell, Keegan; Chang, Peter L.; Cheverud, James M.; Lutz, Cathleen; Lu, Lu; Williams, Robert W.; Dean, Matthew D.

    2016-01-01

    The rapid divergence of male genitalia is a preeminent evolutionary pattern. This rapid divergence is especially striking in the baculum, a bone that occurs in the penis of many mammalian species. Closely related species often display diverse baculum morphology where no other morphological differences can be discerned. While this fundamental pattern of evolution has been appreciated at the level of gross morphology, nearly nothing is known about the genetic basis of size and shape divergence. Quantifying the genetic basis of baculum size and shape variation has been difficult because these structures generally lack obvious landmarks, so comparing them in three dimensions is not straightforward. Here, we develop a novel morphometric approach to quantify size and shape variation from three-dimensional micro-CT scans taken from 369 bacula, representing 75 distinct strains of the BXD family of mice. We identify two quantitative trait loci (QTL) that explain ∼50% of the variance in baculum size, and a third QTL that explains more than 20% of the variance in shape. Together, our study demonstrates that baculum morphology may diverge relatively easily, with mutations at a few loci of large effect that independently modulate size and shape. Based on a combination of bioinformatic investigations and new data on RNA expression, we prioritized these QTL to 16 candidate genes, which have hypothesized roles in bone morphogenesis and may enable future genetic manipulation of baculum morphology. PMID:26935419

  4. Developing a genetic manipulation system for the Antarctic archaeon, Halorubrum lacusprofundi: investigating acetamidase gene function

    PubMed Central

    Liao, Y.; Williams, T. J.; Walsh, J. C.; Ji, M.; Poljak, A.; Curmi, P. M. G.; Duggin, I. G.; Cavicchioli, R.

    2016-01-01

    No systems have been reported for genetic manipulation of cold-adapted Archaea. Halorubrum lacusprofundi is an important member of Deep Lake, Antarctica (~10% of the population), and is amendable to laboratory cultivation. Here we report the development of a shuttle-vector and targeted gene-knockout system for this species. To investigate the function of acetamidase/formamidase genes, a class of genes not experimentally studied in Archaea, the acetamidase gene, amd3, was disrupted. The wild-type grew on acetamide as a sole source of carbon and nitrogen, but the mutant did not. Acetamidase/formamidase genes were found to form three distinct clades within a broad distribution of Archaea and Bacteria. Genes were present within lineages characterized by aerobic growth in low nutrient environments (e.g. haloarchaea, Starkeya) but absent from lineages containing anaerobes or facultative anaerobes (e.g. methanogens, Epsilonproteobacteria) or parasites of animals and plants (e.g. Chlamydiae). While acetamide is not a well characterized natural substrate, the build-up of plastic pollutants in the environment provides a potential source of introduced acetamide. In view of the extent and pattern of distribution of acetamidase/formamidase sequences within Archaea and Bacteria, we speculate that acetamide from plastics may promote the selection of amd/fmd genes in an increasing number of environmental microorganisms. PMID:27708407

  5. OptForce: An Optimization Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions

    PubMed Central

    Ranganathan, Sridhar; Suthers, Patrick F.; Maranas, Costas D.

    2010-01-01

    Computational procedures for predicting metabolic interventions leading to the overproduction of biochemicals in microbial strains are widely in use. However, these methods rely on surrogate biological objectives (e.g., maximize growth rate or minimize metabolic adjustments) and do not make use of flux measurements often available for the wild-type strain. In this work, we introduce the OptForce procedure that identifies all possible engineering interventions by classifying reactions in the metabolic model depending upon whether their flux values must increase, decrease or become equal to zero to meet a pre-specified overproduction target. We hierarchically apply this classification rule for pairs, triples, quadruples, etc. of reactions. This leads to the identification of a sufficient and non-redundant set of fluxes that must change (i.e., MUST set) to meet a pre-specified overproduction target. Starting with this set we subsequently extract a minimal set of fluxes that must actively be forced through genetic manipulations (i.e., FORCE set) to ensure that all fluxes in the network are consistent with the overproduction objective. We demonstrate our OptForce framework for succinate production in Escherichia coli using the most recent in silico E. coli model, iAF1260. The method not only recapitulates existing engineering strategies but also reveals non-intuitive ones that boost succinate production by performing coordinated changes on pathways distant from the last steps of succinate synthesis. PMID:20419153

  6. Manipulation of Ovarian Function Significantly Influenced Sarcopenia in Postreproductive-Age Mice

    PubMed Central

    Peterson, Rhett L.

    2016-01-01

    Previously, transplantation of ovaries from young cycling mice into old postreproductive-age mice increased life span. We anticipated that the same factors that increased life span could also influence health span. Female CBA/J mice received new (60 d) ovaries at 12 and 17 months of age and were evaluated at 16 and 25 months of age, respectively. There were no significant differences in body weight among any age or treatment group. The percentage of fat mass was significantly increased at 13 and 16 months of age but was reduced by ovarian transplantation in 16-month-old mice. The percentages of lean body mass and total body water were significantly reduced in 13-month-old control mice but were restored in 16- and 25-month-old recipient mice by ovarian transplantation to the levels found in six-month-old control mice. In summary, we have shown that skeletal muscle mass, which is negatively influenced by aging, can be positively influenced or restored by reestablishment of active ovarian function in aged female mice. These findings provide strong incentive for further investigation of the positive influence of young ovaries on restoration of health in postreproductive females. PMID:27747096

  7. CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.

    PubMed

    Yang, Fayu; Liu, Changbao; Chen, Ding; Tu, Mengjun; Xie, Haihua; Sun, Huihui; Ge, Xianglian; Tang, Lianchao; Li, Jin; Zheng, Jiayong; Song, Zongming; Qu, Jia; Gu, Feng

    2017-06-16

    Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%-68% at the human AAVS1 locus versus 4%-28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. An Allelic Exchange System for Compliant Genetic Manipulation of the Select Agents Burkholderia pseudomallei and Burkholderia mallei

    PubMed Central

    Hamad, Mohamad A.; Zajdowicz, Sheryl L.; Holmes, Randall K.; Voskuil, Martin I.

    2009-01-01

    Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei. PMID:19010402

  9. Genetic manipulation of cardiac Hsp72 levels does not alter substrate metabolism but reveals insights into high-fat feeding-induced cardiac insulin resistance.

    PubMed

    Henstridge, Darren C; Estevez, E; Allen, T L; Heywood, S E; Gardner, T; Yang, C; Mellett, N A; Kingwell, B A; Meikle, P J; Febbraio, M A

    2015-05-01

    Heat shock protein 72 (Hsp72) protects cells against a variety of stressors, and multiple studies have suggested that Hsp72 plays a cardioprotective role. As skeletal muscle Hsp72 overexpression can protect against high-fat diet (HFD)-induced insulin resistance, alterations in substrate metabolism may be a mechanism by which Hsp72 is cardioprotective. We investigated the impact of transgenically overexpressing (Hsp72 Tg) or deleting Hsp72 (Hsp72 KO) on various aspects of cardiac metabolism. Mice were fed a normal chow (NC) or HFD for 12 weeks from 8 weeks of age to examine the impact of diet-induced obesity on metabolic parameters in the heart. The HFD resulted in an increase in cardiac fatty acid oxidation and a decrease in cardiac glucose oxidation and insulin-stimulated cardiac glucose clearance; however, there was no difference in Hsp72 Tg or Hsp72 KO mice in these rates compared with their respective wild-type control mice. Although HFD-induced cardiac insulin resistance was not rescued in the Hsp72 Tg mice, it was preserved in the skeletal muscle, suggesting tissue-specific effects of Hsp72 overexpression on substrate metabolism. Comparison of two different strains of mice (BALB/c vs. C57BL/6J) also identified strain-specific differences in regard to HFD-induced cardiac lipid accumulation and insulin resistance. These strain differences suggest that cardiac lipid accumulation can be dissociated from cardiac insulin resistance. Our study finds that genetic manipulation of Hsp72 does not lead to alterations in metabolic processes in cardiac tissue under resting conditions, but identifies mouse strain-specific differences in cardiac lipid accumulation and insulin-stimulated glucose clearance.

  10. Genetic signature of histiocytic sarcoma revealed by a sleeping beauty transposon genetic screen in mice.

    PubMed

    Been, Raha A; Linden, Michael A; Hager, Courtney J; DeCoursin, Krista J; Abrahante, Juan E; Landman, Sean R; Steinbach, Michael; Sarver, Aaron L; Largaespada, David A; Starr, Timothy K

    2014-01-01

    Histiocytic sarcoma is a rare, aggressive neoplasm that responds poorly to therapy. Histiocytic sarcoma is thought to arise from macrophage precursor cells via genetic changes that are largely undefined. To improve our understanding of the etiology of histiocytic sarcoma we conducted a forward genetic screen in mice using the Sleeping Beauty transposon as a mutagen to identify genetic drivers of histiocytic sarcoma. Sleeping Beauty mutagenesis was targeted to myeloid lineage cells using the Lysozyme2 promoter. Mice with activated Sleeping Beauty mutagenesis had significantly shortened lifespan and the majority of these mice developed tumors resembling human histiocytic sarcoma. Analysis of transposon insertions identified 27 common insertion sites containing 28 candidate cancer genes. Several of these genes are known drivers of hematological neoplasms, like Raf1, Fli1, and Mitf, while others are well-known cancer genes, including Nf1, Myc, Jak2, and Pten. Importantly, several new potential drivers of histiocytic sarcoma were identified and could serve as targets for therapy for histiocytic sarcoma patients.

  11. Genetic Signature of Histiocytic Sarcoma Revealed by a Sleeping Beauty Transposon Genetic Screen in Mice

    PubMed Central

    Been, Raha A.; Linden, Michael A.; Hager, Courtney J.; DeCoursin, Krista J.; Abrahante, Juan E.; Landman, Sean R.; Steinbach, Michael; Sarver, Aaron L.; Largaespada, David A.; Starr, Timothy K.

    2014-01-01

    Histiocytic sarcoma is a rare, aggressive neoplasm that responds poorly to therapy. Histiocytic sarcoma is thought to arise from macrophage precursor cells via genetic changes that are largely undefined. To improve our understanding of the etiology of histiocytic sarcoma we conducted a forward genetic screen in mice using the Sleeping Beauty transposon as a mutagen to identify genetic drivers of histiocytic sarcoma. Sleeping Beauty mutagenesis was targeted to myeloid lineage cells using the Lysozyme2 promoter. Mice with activated Sleeping Beauty mutagenesis had significantly shortened lifespan and the majority of these mice developed tumors resembling human histiocytic sarcoma. Analysis of transposon insertions identified 27 common insertion sites containing 28 candidate cancer genes. Several of these genes are known drivers of hematological neoplasms, like Raf1, Fli1, and Mitf, while others are well-known cancer genes, including Nf1, Myc, Jak2, and Pten. Importantly, several new potential drivers of histiocytic sarcoma were identified and could serve as targets for therapy for histiocytic sarcoma patients. PMID:24827933

  12. Neuroinflammation in Parkinson's Disease and Related Disorders: A Lesson from Genetically Manipulated Mouse Models of α-Synucleinopathies

    PubMed Central

    Sekiyama, Kazunari; Sugama, Shuei; Fujita, Masayo; Sekigawa, Akio; Takamatsu, Yoshiki; Waragai, Masaaki; Takenouchi, Takato; Hashimoto, Makoto

    2012-01-01

    Neuroinflammation in Parkinson's disease (PD) is a chronic process that is associated with alteration of glial cells, including astrocytes and microglia. However, the precise mechanisms remain obscure. To better understand neuroinflammation in PD, we focused on glial activation in α-synuclein (αS) transgenic and related model mice. In the majority of αS transgenic mice, astrogliosis was observed concomitantly with accumulation of αS during the early stage of neurodegeneration. However, microglia were not extensively activated unless the mice were treated with lipopolysaccharides or through further genetic modification of other molecules, including familial PD risk factors. Thus, the results in αS transgenic mice and related model mice are consistent with the idea that neuroinflammation in PD is a double-edged sword that is protective in the early stage of neurodegeneration but becomes detrimental with disease progression. PMID:22550610

  13. Genetic architecture of testis and seminal vesicle weights in mice.

    PubMed Central

    Le Roy, I; Tordjman, S; Migliore-Samour, D; Degrelle, H; Roubertoux, P L

    2001-01-01

    Comparisons across 13 inbred strains of laboratory mice for reproductive organ (paired seminal vesicles and paired testes) weights indicated a very marked contrast between the C57BL/6By and NZB/BINJ mice. Subsequently these strains were selected to perform a quantitative genetic analysis and full genome scan for seminal vesicle and testis weights. An F(2) population was generated. The quantitative genetic analyses indicated that each was linked to several genes. Sixty-six short sequences for length polymorphism were used as markers in the wide genome scan strategy. For weight of paired testes, heritability was 82.3% of the total variance and five QTL contributed to 72.8% of the total variance. Three reached a highly significant threshold (>4.5) and were mapped on chromosome X (LOD score 9.11), chromosome 4 (LOD score 5.96), chromosome 10 (LOD score 5.81); two QTL were suggested: chromosome 13 (LOD score 3.10) and chromosome 18 (LOD score 2.80). Heritability for weight of seminal vesicles was 50.7%. One QTL was mapped on chromosome 4 (LOD score 9.21) and contributed to 24.2% of the total variance. The distance of this QTL to the centromere encompassed the distance of the QTL linked with testicular weight on chromosome 4, suggesting common genetic mechanisms as expected from correlations in the F(2). Both testis and seminal vesicle weights were associated with a reduction in the NZB/BINJ when this strain carried the Y(NPAR) from CBA/H whereas the Y(NPAR) from NZB/BINJ in the CBA/H strain did not modify reproductive organ weights, indicating that the Y(NPAR) interacts with the non-Y(NPAR) genes. The effects generated by this chromosomal region were significant but small in size. PMID:11333241

  14. Behavioural and physiological responses of wood mice (Apodemus sylvaticus) to experimental manipulations of predation and starvation risk.

    PubMed

    Monarca, Rita I; Mathias, Maria da Luz; Speakman, John R

    2015-10-01

    Body weight and the levels of stored body fat have fitness consequences. Greater levels of fat may provide protection against catastrophic failures in the food supply, but they may also increase the risk of predation. Animals may therefore regulate their fatness according to their perceived risks of predation and starvation: the starvation-predation trade-off model. We tested the predictions of this model in wood mice (Apodemus sylvaticus) by experimentally manipulating predation risk and starvation risk. We predicted that under increased predation risk individuals would lose weight and under increased starvation risk they would gain it. We simulated increased predation risk by playing the calls made by predatory birds (owls: Tyto alba and Bubo bubo) to the mice. Control groups included exposure to calls of a non-predatory bird (blackbird: Turdus merula) or silence. Mice exposed to owl calls at night lost weight relative to the silence group, mediated via reduced food intake, but exposure to owl calls in the day had no significant effect. Exposure to blackbird calls at night also resulted in weight loss, but blackbird calls in the day had no effect. Mice seemed to have a generalised response to bird calls at night irrespective of their actual source. This could be because in the wild any bird calling at night will be a predation risk, and any bird calling in the day would not be, because at that time the mice would normally be resting, and hence not exposed to avian predators. Consequently, mice have not evolved to distinguish different types of call but only to respond to the time of day that they occur. Mice exposed to stochastic 24h starvation events altered their behaviour (reduced activity) during the refeeding days that followed the deprivation periods to regain the lost mass. However, they only marginally elevated their food intake and consequently had reduced body weight/fat storage compared to that of the control unstarved group. This response may have

  15. Genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration.

    PubMed

    Holubowska, Anna; Mukherjee, Chaitali; Vadhvani, Mayur; Stegmüller, Judith

    2014-03-17

    Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

  16. Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens.

    PubMed

    Penno, Carlos Alberto; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

    2010-04-01

    We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors.

  17. Genetically manipulated progenitor cell sheet with diprotin A improves myocardial function and repair of infarcted hearts

    PubMed Central

    Zhang, Dongsheng; Huang, Wei; Dai, Bo; Zhao, Tiemin; Ashraf, Atif; Millard, Ronald W.; Ashraf, Muhammad

    2010-01-01

    We postulated that the combination of overexpression of CXCR4 in mesenchymal stem cells (MSC) with diprotin A would enhance MSC recruitment and penetration into ischemic myocardium, leading to an improvement in heart function after myocardial infarction (MI). Male rat MSC were genetically engineered with adenoviral vectors coexpressing CXCR4 and enhanced green fluorescent protein (EGFP) (MSCCXCR4), GFP alone (MSCNull, control), or siRNA-targeted CXCR4 (MSCsiRNA). Cell sheets were applied over the surface of infarcted left ventricle (LV) in female rats 7 days after ligation of the left anterior descending coronary artery (LAD) pretreated with either vehicle (VEH) or diprotin A (DIP). At 28 days after cell sheet implantation, echocardiography was performed. Hearts were harvested for histological analysis 7 days after LAD ligation or 28 days after cell sheet implantation. DPP-IV and stroma-derived factor-1α (SDF-1α) in the LV were analyzed. Efficacy of engraftment was determined by the presence of Y chromosome in nuclei (Ych+). LV blood vessel density and apoptosis were also analyzed. Myocardial SDF-1α was elevated before placement of the cell sheet in the DIP group compared with vehicle group on day 7 after LAD. On day 28 after cell sheet transplantation, the number of Ych+ was increased in the MSCCXCR4 + VEH group compared with the MSCNull + VEH group and further increased in the MSCCXCR4 + DIP treated group. This enhanced response was associated with increased angiogenesis in both sides of epicardium and improvement of LV function. Combination of gene-manipulated MSCCXCR4 patch with DIP pretreatment inhibits myocardial ischemia-induced apoptosis, promotes tissue angiogenesis, and enhances cell engraftment, leading to improved LV mechanical function after MI. PMID:20802132

  18. Genetically manipulated progenitor cell sheet with diprotin A improves myocardial function and repair of infarcted hearts.

    PubMed

    Zhang, Dongsheng; Huang, Wei; Dai, Bo; Zhao, Tiemin; Ashraf, Atif; Millard, Ronald W; Ashraf, Muhammad; Wang, Yigang

    2010-11-01

    We postulated that the combination of overexpression of CXCR4 in mesenchymal stem cells (MSC) with diprotin A would enhance MSC recruitment and penetration into ischemic myocardium, leading to an improvement in heart function after myocardial infarction (MI). Male rat MSC were genetically engineered with adenoviral vectors coexpressing CXCR4 and enhanced green fluorescent protein (EGFP) (MSC(CXCR4)), GFP alone (MSC(Null), control), or siRNA-targeted CXCR4 (MSC(siRNA)). Cell sheets were applied over the surface of infarcted left ventricle (LV) in female rats 7 days after ligation of the left anterior descending coronary artery (LAD) pretreated with either vehicle (VEH) or diprotin A (DIP). At 28 days after cell sheet implantation, echocardiography was performed. Hearts were harvested for histological analysis 7 days after LAD ligation or 28 days after cell sheet implantation. DPP-IV and stroma-derived factor-1α (SDF-1α) in the LV were analyzed. Efficacy of engraftment was determined by the presence of Y chromosome in nuclei (Y(ch+)). LV blood vessel density and apoptosis were also analyzed. Myocardial SDF-1α was elevated before placement of the cell sheet in the DIP group compared with vehicle group on day 7 after LAD. On day 28 after cell sheet transplantation, the number of Y(ch+) was increased in the MSC(CXCR4) + VEH group compared with the MSC(Null) + VEH group and further increased in the MSC(CXCR4) + DIP treated group. This enhanced response was associated with increased angiogenesis in both sides of epicardium and improvement of LV function. Combination of gene-manipulated MSC(CXCR4) patch with DIP pretreatment inhibits myocardial ischemia-induced apoptosis, promotes tissue angiogenesis, and enhances cell engraftment, leading to improved LV mechanical function after MI.

  19. Genetic manipulation of competition for nitrate between heterotrophic bacteria and diatoms

    SciTech Connect

    Diner, Rachel E.; Schwenck, Sarah M.; McCrow, John P.; Zheng, Hong; Allen, Andrew E.

    2016-06-09

    >A. macleodii to rescue nitrate reductase deficient P. tricomutum populations from nitrogen starvation, and RNA-seq transcriptomic evidence supports nitrogen-based interactions between diatoms and bacteria at the molecular level. As a result, this study provides key insights into the roles of carbon and nitrogen in phytoplankton-bacteria dynamics and lays the foundation for developing a mechanistic understanding of these interactions using co-culturing and genetic manipulation.

  20. Genetic manipulation of competition for nitrate between heterotrophic bacteria and diatoms

    DOE PAGES

    Diner, Rachel E.; Schwenck, Sarah M.; McCrow, John P.; ...

    2016-06-09

    from nitrogen starvation, and RNA-seq transcriptomic evidence supports nitrogen-based interactions between diatoms and bacteria at the molecular level. As a result, this study provides key insights into the roles of carbon and nitrogen in phytoplankton-bacteria dynamics and lays the foundation for developing a mechanistic understanding of these interactions using co-culturing and genetic manipulation.« less

  1. Genetic Manipulation of Competition for Nitrate between Heterotrophic Bacteria and Diatoms

    PubMed Central

    Diner, Rachel E.; Schwenck, Sarah M.; McCrow, John P.; Zheng, Hong; Allen, Andrew E.

    2016-01-01

    nitrogen starvation, and RNA-seq transcriptomic evidence supports nitrogen-based interactions between diatoms and bacteria at the molecular level. This study provides key insights into the roles of carbon and nitrogen in phytoplankton-bacteria dynamics and lays the foundation for developing a mechanistic understanding of these interactions using co-culturing and genetic manipulation. PMID:27375600

  2. Genetic Manipulation of Competition for Nitrate between Heterotrophic Bacteria and Diatoms

    NASA Astrophysics Data System (ADS)

    Diner, R. E.; Allen, A. E.

    2016-02-01

    with and without the addition of carbon. This study provides key insights into the roles of carbon and nitrogen in phytoplankton-bacteria dynamics and lays the foundation for developing a mechanistic understanding of these interactions using co-culturing and genetic manipulation.

  3. COMPETITIVE ABILITY IN MALE HOUSE MICE (Mus musculus): GENETIC INFLUENCES

    PubMed Central

    Cunningham, Christopher B.; Ruff, James S.; Chase, Kevin; Potts, Wayne K.; Carrier, David R.

    2013-01-01

    Conspecifics of many animal species physically compete to gain reproductive resources and thus fitness. Despite the importance of competitive ability across the animal kingdom, specific traits that influence or underpin competitive ability are poorly characterized. Here, we investigate whether there are genetic influences on competitive ability within male house mice. Additionally, we examined if litter demographics (litter size and litter sex ratio) influence competitive ability. We phenotyped two generations for a male s ability to possess a reproductive resource--a prime nesting site--using semi-natural enclosures with mixed sex groupings. We used the animal model coupled with an extensive pedigree to estimate several genetic parameters. Competitive ability was found to be highly heritable, but only displayed a moderate genetic correlation to body mass. Interestingly, litter sex ratio had a weak negative influence on competitive ability. Litter size had no significant influence on competitive ability. Our study also highlights how much remians unknown about the proximal causes of competitive ability. PMID:23291957

  4. Projections and interconnections of genetically defined serotonin neurons in mice

    PubMed Central

    Bang, Sun Jung; Jensen, Patricia; Dymecki, Susan M; Commons, Kathryn G.

    2012-01-01

    Brain serotonin neurons are heterogeneous and can be distinguished by several anatomical and physiological characteristics. Toward resolving this heterogeneity into classes of functional relevance, subtypes of mature serotonin neurons were previously identified based on gene expression differences initiated during development in different rhombomeric (r) segments of the hindbrain. This redefinition of mature serotonin neuron subtypes based on the criteria of genetic lineage, along with the enabling genetic fate mapping tools, now allows various functional properties, such as axonal projections, to be allocated onto these identified subtypes. Furthermore, our approach uniquely enables interconnections between the different serotonin neuron subtypes to be determined; this is especially relevant because serotonin neuron activity is regulated by several feedback mechanisms. We used intersectional and subtractive genetic fate mapping tools to generate three independent lines of mice in which serotonin neurons arising in different rhombomeric segments, either r1, r2 or both r3 and r5, were uniquely distinguished from all other serotonin neurons by their expression of enhanced green fluorescent protein. Each of these subgroups of serotonergic neurons had a unique combination of forebrain projection targets. Typically more than one subgroup innervated an individual target area. Unique patterns of interconnections between the different groups of serotonin neurons were also observed and these pathways could subserve feedback regulatory circuits. Overall, the current findings suggest that activation of subsets of serotonin neurons could result in topographic serotonin release in the forebrain coupled with feedback inhibition of serotonin neurons with alternative projection targets. PMID:22151329

  5. The genetic control of antibody affinity in mice.

    PubMed Central

    Katz, F E; Steward, M W

    1975-01-01

    Random-bred TO mice have been selectively bred into two lines on the basis of the relative affinity (KR) of antibody produced to protein antigens, one line producing high and the other low KR antibody. After four generations of selective breeding the difference in KR between the two lines was highly significant (P less than 0-001). The selection on the basis of KR did not result in a corresponding selection for antibody levels (Abt), which were not significantly different in the two lines. These results indicate that antibody affinity is a genetically controlled parameter of the immune response. Furthermore, this control appears to be expressed by a mechanism which is independent of the amount of antibody produced. PMID:1165110

  6. The genetic basis of parental care evolution in monogamous mice.

    PubMed

    Bendesky, Andres; Kwon, Young-Mi; Lassance, Jean-Marc; Lewarch, Caitlin L; Yao, Shenqin; Peterson, Brant K; He, Meng Xiao; Dulac, Catherine; Hoekstra, Hopi E

    2017-04-27

    Parental care is essential for the survival of mammals, yet the mechanisms underlying its evolution remain largely unknown. Here we show that two sister species of mice, Peromyscus polionotus and Peromyscus maniculatus, have large and heritable differences in parental behaviour. Using quantitative genetics, we identify 12 genomic regions that affect parental care, 8 of which have sex-specific effects, suggesting that parental care can evolve independently in males and females. Furthermore, some regions affect parental care broadly, whereas others affect specific behaviours, such as nest building. Of the genes linked to differences in nest-building behaviour, vasopressin is differentially expressed in the hypothalamus of the two species, with increased levels associated with less nest building. Using pharmacology in Peromyscus and chemogenetics in Mus, we show that vasopressin inhibits nest building but not other parental behaviours. Together, our results indicate that variation in an ancient neuropeptide contributes to interspecific differences in parental care.

  7. Genetically altered mice for evaluation of mode-of-action (MOA)

    EPA Science Inventory

    Genetically altered mice for evaluation of mode-of-action (MOA). Barbara D. Abbott, Cynthia J. Wolf, Kaberi P. Das, Christopher S. Lau. (Presented by B. Abbott). This presentation provides an example of the use of genetically modified mice to determine the mode-of-action of r...

  8. Genetically altered mice for evaluation of mode-of-action (MOA)

    EPA Science Inventory

    Genetically altered mice for evaluation of mode-of-action (MOA). Barbara D. Abbott, Cynthia J. Wolf, Kaberi P. Das, Christopher S. Lau. (Presented by B. Abbott). This presentation provides an example of the use of genetically modified mice to determine the mode-of-action of r...

  9. Acute genetic manipulation of neuronal activity for the functional dissection of neural circuits-a dream come true for the pioneers of behavioral genetics.

    PubMed

    Yoshihara, Moto; Ito, Kei

    2012-03-01

    Abstract: This review summarizes technical development of the functional manipulation of specific neural circuits through genetic techniques in Drosophila. Long after pioneers' efforts for the genetic dissection of behavior using this organism as a model, analyses with acute activation of specific neural circuits have finally become feasible using transgenic Drosophila that expresses light-, heat-, or cold-activatable cation channels by xxx/upstream activation sequence (Gal4/UAS)-based induction system. This methodology opened a new avenue to dissect functions of neural circuits to make dreams of the pioneers into reality.

  10. Automated, quantitative cognitive/behavioral screening of mice: for genetics, pharmacology, animal cognition and undergraduate instruction.

    PubMed

    Gallistel, C R; Balci, Fuat; Freestone, David; Kheifets, Aaron; King, Adam

    2014-02-26

    We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be

  11. Changes in cyst's nuclear chromatin resulting after experimental manipulation of Taenia crassiceps mice infections: biological implications.

    PubMed

    Fragoso, Gladis; Bobes, Raul J; Espinoza, Bertha; Martínez, María Luisa; Pérez-Morales, Deyanira; Rosas, Gabriela; Sciutto, Edda; Laclette, Juan Pedro

    2012-04-01

    During some estimations of the nuclear DNA content, based on determinations propidium iodide (PI) binding through fluorocytometry for Taenia crassiceps cysticerci, significant variation in the results were found. This initial observation led to a series of experiments designed to explain the variation. These changes could be induced by the diameter of the needles in the syringes used for the mouse to mouse transfer of the cysts. Nuclei from cysts transferred through 27-gauge needles showed 30% less PI staining than those transferred through 21 gauge needles, after 2 months infections. Reduction in PI capture induced by 27-gauge needle was reversible when the cysts were maintained in their mice hosts during 5 months. Moreover, variation in PI binding to cysticercal DNA was also found when comparing parasites grown in male versus female mice. The use of agents that homogenize the chromatin structure during PI staining, allowed demonstrating that variation were entirely due to differences in the chromatin relaxation/compaction. Additional experiments demonstrated that the higher compaction is accompanied by a reduced ability of cysts to grow in the peritoneal cavity of BALB/cAnN mice. Furthermore, proteomic analysis also showed that these changes in chromatin relaxation/compaction resulted in different levels and patterns of protein expression. Our results strongly suggest that chromatin is involved in several well characterized phenomena of the T. crassiceps murine model, and open new avenues for a detailed approach to understand such a complex host-parasite relationship.

  12. Genetic manipulation of periostin expression in the heart does not affect myocyte content, cell cycle activity or cardiac repair

    PubMed Central

    Lorts, Angela; Schwanekamp, Jennifer A.; Elrod, John W.; Sargent, Michelle A.; Molkentin, Jeffery D.

    2009-01-01

    Following a pathologic insult, the adult mammalian heart undergoes hypertrophic growth and remodeling of the extracellular matrix. While a small sub-population of cardiomyocytes can re-enter the cell cycle following cardiac injury, the myocardium is largely thought to be incapable of significant regeneration. Periostin, an extracellular matrix protein, has recently been proposed to induce re-entry of differentiated cardiomyocytes back into the cell cycle and promote meaningful repair following myocardial infarction. Here, we show that while periostin is induced in the heart following injury, it does not stimulate DNA synthesis, mitosis or cytokinesis of cardiomyocytes in vitro or in vivo. Mice lacking the gene encoding periostin and mice with inducible overexpression of full-length periostin were analyzed at baseline and after myocardial infarction. There was no difference in heart size or a change in cardiomyocyte number in either periostin transgenic or gene-targeted mice at baseline. Quantification of proliferating myocytes in the peri-infarct area showed no difference between periostin overexpressing and null mice compared with strain-matched controls. In support of these observations, neither overexpression of periostin in cell culture, via an adenoviral vector, nor stimulation with recombinant protein induced DNA synthesis, mitosis or cytokinesis. Periostin is a regulator of cardiac remodeling and hypertrophy and may be a reasonable pharmacological target to mitigate heart failure, but manipulation of this protein appears to have no obvious effect on myocardial regeneration. PMID:19038863

  13. Genetic Architecture of Atherosclerosis in Mice: A Systems Genetics Analysis of Common Inbred Strains

    PubMed Central

    Bennett, Brian J.; Davis, Richard C.; Civelek, Mete; Orozco, Luz; Wu, Judy; Qi, Hannah; Pan, Calvin; Packard, René R. Sevag; Eskin, Eleazar; Yan, Mujing; Kirchgessner, Todd; Wang, Zeneng; Li, Xinmin; Gregory, Jill C.; Hazen, Stanley L.; Gargalovic, Peter S.; Lusis, Aldons J.

    2015-01-01

    Common forms of atherosclerosis involve multiple genetic and environmental factors. While human genome-wide association studies have identified numerous loci contributing to coronary artery disease and its risk factors, these studies are unable to control environmental factors or examine detailed molecular traits in relevant tissues. We now report a study of natural variations contributing to atherosclerosis and related traits in over 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). The mice were made hyperlipidemic by transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). The mice were examined for lesion size and morphology as well as plasma lipid, insulin and glucose levels, and blood cell profiles. A subset of mice was studied for plasma levels of metabolites and cytokines. We also measured global transcript levels in aorta and liver. Finally, the uptake of acetylated LDL by macrophages from HMDP mice was quantitatively examined. Loci contributing to the traits were mapped using association analysis, and relationships among traits were examined using correlation and statistical modeling. A number of conclusions emerged. First, relationships among atherosclerosis and the risk factors in mice resemble those found in humans. Second, a number of trait-loci were identified, including some overlapping with previous human and mouse studies. Third, gene expression data enabled enrichment analysis of pathways contributing to atherosclerosis and prioritization of candidate genes at associated loci in both mice and humans. Fourth, the data provided a number of mechanistic inferences; for example, we detected no association between macrophage uptake of acetylated LDL and atherosclerosis. Fifth, broad sense heritability for atherosclerosis was much larger than narrow sense heritability, indicating an important role for gene-by-gene interactions. Sixth, stepwise linear regression

  14. Genetic Architecture of Atherosclerosis in Mice: A Systems Genetics Analysis of Common Inbred Strains.

    PubMed

    Bennett, Brian J; Davis, Richard C; Civelek, Mete; Orozco, Luz; Wu, Judy; Qi, Hannah; Pan, Calvin; Packard, René R Sevag; Eskin, Eleazar; Yan, Mujing; Kirchgessner, Todd; Wang, Zeneng; Li, Xinmin; Gregory, Jill C; Hazen, Stanley L; Gargalovic, Peter S; Lusis, Aldons J

    2015-12-01

    Common forms of atherosclerosis involve multiple genetic and environmental factors. While human genome-wide association studies have identified numerous loci contributing to coronary artery disease and its risk factors, these studies are unable to control environmental factors or examine detailed molecular traits in relevant tissues. We now report a study of natural variations contributing to atherosclerosis and related traits in over 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). The mice were made hyperlipidemic by transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). The mice were examined for lesion size and morphology as well as plasma lipid, insulin and glucose levels, and blood cell profiles. A subset of mice was studied for plasma levels of metabolites and cytokines. We also measured global transcript levels in aorta and liver. Finally, the uptake of acetylated LDL by macrophages from HMDP mice was quantitatively examined. Loci contributing to the traits were mapped using association analysis, and relationships among traits were examined using correlation and statistical modeling. A number of conclusions emerged. First, relationships among atherosclerosis and the risk factors in mice resemble those found in humans. Second, a number of trait-loci were identified, including some overlapping with previous human and mouse studies. Third, gene expression data enabled enrichment analysis of pathways contributing to atherosclerosis and prioritization of candidate genes at associated loci in both mice and humans. Fourth, the data provided a number of mechanistic inferences; for example, we detected no association between macrophage uptake of acetylated LDL and atherosclerosis. Fifth, broad sense heritability for atherosclerosis was much larger than narrow sense heritability, indicating an important role for gene-by-gene interactions. Sixth, stepwise linear regression

  15. Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo

    PubMed Central

    Kim, Ji-Yoen; Ash, Ryan T.; Ceballos-Diaz, Carolina; Levites, Yona; Golde, Todd E.; Smirnakis, Stelios M.; Jankowsky, Joanna L.

    2012-01-01

    Neonatal intraventricular injection of adeno-associated virus has been shown to transduce neurons widely throughout the brain, but its full potential for experimental neuroscience has not been adequately explored. We report a detailed analysis of the method’s versatility with an emphasis on experimental applications where tools for genetic manipulation are currently lacking. Viral injection into the neonatal mouse brain is fast, easy, and accesses regions of the brain including cerebellum and brain stem that have been difficult to target with other techniques such as electroporation. We show that viral transduction produces an inherently mosaic expression pattern that can be exploited by varying the titer to transduce isolated neurons or densely-packed populations. We demonstrate that expression of virally-encoded proteins is active much sooner than previously believed, allowing genetic perturbation during critical periods of neuronal plasticity, but is also long-lasting and stable, allowing chronic studies of aging. We harness these features to visualize and manipulate neurons in the hindbrain that have been recalcitrant to approaches commonly applied in the cortex. We show that viral labeling aids the analysis of postnatal dendritic maturation in cerebellar Purkinje neurons by allowing individual cells to be readily distinguished, and then demonstrate that the same sparse labeling allows live in vivo imaging of mature Purkinje neurons at resolution sufficient for complete analytical reconstruction. Given the rising availability of viral constructs, packaging services, and genetically modified animals, these techniques should facilitate a wide range of experiments into brain development, function, and degeneration. PMID:23347239

  16. Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture.

    PubMed

    Yi, Pei-Lu; Jou, Shuo-Bin; Wu, Yi-Jou; Chang, Fang-Chia

    2016-12-22

    Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

  17. Genetic Background Modulates Gene Expression Profile Induced by Skin Irradiation in Ptch1 Mice

    SciTech Connect

    Galvan, Antonella; Noci, Sara; Mancuso, Mariateresa; Pazzaglia, Simonetta; Saran, Anna; Dragani, Tommaso A.

    2008-12-01

    Purpose: Ptch1 germ-line mutations in mice predispose to radiation-induced basal cell carcinoma of the skin, with tumor incidence modulated by the genetic background. Here, we examined the possible mechanisms underlying skin response to radiation in F1 progeny of Ptch1{sup neo67/+} mice crossed with either skin tumor-susceptible (Car-S) or -resistant (Car-R) mice and X-irradiated (3 Gy) at 2 days of age or left untreated. Methods and Materials: We conducted a gene expression profile analysis in mRNA samples extracted from the skin of irradiated or control mice, using Affymetrix whole mouse genome expression array. Confirmation of the results was done using real-time reverse-transcriptase polymerase chain reaction. Results: Analysis of the gene expression profile of normal skin of F1 mice at 4 weeks of age revealed a similar basal profile in the nonirradiated mice, but alterations in levels of 71 transcripts in irradiated Ptch1{sup neo67/+} mice of the Car-R cross and modulation of only eight genes in irradiated Ptch1{sup neo67/+} mice of the Car-S cross. Conclusions: These results indicate that neonatal irradiation causes a persistent change in the gene expression profile of the skin. The tendency of mice genetically resistant to skin tumorigenesis to show a more complex pattern of transcriptional response to radiation than do genetically susceptible mice suggests a role for this response in genetic resistance to basal cell tumorigenesis.

  18. Amelioration of progressive renal injury by genetic manipulation of Klotho gene.

    PubMed

    Haruna, Yoshisuke; Kashihara, Naoki; Satoh, Minoru; Tomita, Naruya; Namikoshi, Tamehachi; Sasaki, Tamaki; Fujimori, Toshihiko; Xie, Ping; Kanwar, Yashpal S

    2007-02-13

    Klotho, an antiaging gene with restricted organ distribution, is mainly expressed in the kidney tubules; the mutant mice have shortened life span, arteriosclerosis, anemia, and osteoporesis, features common to patients with chronic renal failure. Conceivably, the reduction of the Klotho gene expression may contribute to the development of kidney failure; alternatively, its overexpression may lead to the amelioration of renal injury in an ICR-derived glomerulonephritis (ICGN) mouse model with subtle immune complex-mediated disease. To address this issue, four different strains of mice were generated by cross-breeding: ICGN mice without the Klotho transgene (ICGN), ICGN mice with the Klotho transgene (ICGN/klTG), wild-type mice with the Klotho transgene (klTG), and wild-type mice without the Klotho transgene (control). At 40 weeks old, the survival rate was approximately 30% in ICGN mice, and approximately 70% in the ICGN/klTG group. This improvement was associated with dramatic improvement in renal functions, morphological lesions, and cytochrome c oxidase activity but a reduction in beta-galactosidase activity (a senescence-associated protein), mitochondrial DNA fragmentation, superoxide anion generation, lipid peroxidation, and Bax protein expression and apoptosis. Interestingly, improvement was seen in both the tubular and glomerular compartments of the kidney, although Klotho is exclusively confined to the tubules, suggesting that its gene product has a remarkable renoprotective effect by potentially serving as a circulating hormone while mitigating the mitochondrial oxidative stress.

  19. Obesity-programmed mice are rescued by early genetic intervention.

    PubMed

    Bumaschny, Viviana F; Yamashita, Miho; Casas-Cordero, Rodrigo; Otero-Corchón, Verónica; de Souza, Flávio S J; Rubinstein, Marcelo; Low, Malcolm J

    2012-11-01

    Obesity is a chronic metabolic disorder affecting half a billion people worldwide. Major difficulties in managing obesity are the cessation of continued weight loss in patients after an initial period of responsiveness and rebound to pretreatment weight. It is conceivable that chronic weight gain unrelated to physiological needs induces an allostatic regulatory state that defends a supranormal adipose mass despite its maladaptive consequences. To challenge this hypothesis, we generated a reversible genetic mouse model of early-onset hyperphagia and severe obesity by selectively blocking the expression of the proopiomelanocortin gene (Pomc) in hypothalamic neurons. Eutopic reactivation of central POMC transmission at different stages of overweight progression normalized or greatly reduced food intake in these obesity-programmed mice. Hypothalamic Pomc rescue also attenuated comorbidities such as hyperglycemia, hyperinsulinemia, and hepatic steatosis and normalized locomotor activity. However, effectiveness of treatment to normalize body weight and adiposity declined progressively as the level of obesity at the time of Pomc induction increased. Thus, our study using a novel reversible monogenic obesity model reveals the critical importance of early intervention for the prevention of subsequent allostatic overload that auto-perpetuates obesity.

  20. Genetic and phenotypic influences on copulatory plug survival in mice

    PubMed Central

    Mangels, R; Young, B; Keeble, S; Ardekani, R; Meslin, C; Ferreira, Z; Clark, N L; Good, J M; Dean, M D

    2015-01-01

    Across a diversity of animals, male seminal fluid coagulates upon ejaculation to form a hardened structure known as a copulatory plug. Previous studies suggest that copulatory plugs evolved as a mechanism for males to impede remating by females, but detailed investigations into the time course over which plugs survive in the female's reproductive tract are lacking. Here, we cross males from eight inbred strains to females from two inbred strains of house mice (Mus musculus domesticus). Plug survival was significantly affected by male genotype. Against intuition, plug survival time was negatively correlated with plug size: long-lasting plugs were small and relatively more susceptible to proteolysis. Plug size was associated with divergence in major protein composition of seminal vesicle fluid, suggesting that changes in gene expression may play an important role in plug dynamics. In contrast, we found no correlation to genetic variation in the protein-coding regions of five genes thought to be important in copulatory plug formation (Tgm4, Svs1, Svs2, Svs4 and Svs5). Our study demonstrates a complex relationship between copulatory plug characteristics and survival. We discuss several models to explain unexpected variation in plug phenotypes. PMID:26103947

  1. Obesity-programmed mice are rescued by early genetic intervention

    PubMed Central

    Bumaschny, Viviana F.; Yamashita, Miho; Casas-Cordero, Rodrigo; Otero-Corchón, Verónica; de Souza, Flávio S.J.; Rubinstein, Marcelo; Low, Malcolm J.

    2012-01-01

    Obesity is a chronic metabolic disorder affecting half a billion people worldwide. Major difficulties in managing obesity are the cessation of continued weight loss in patients after an initial period of responsiveness and rebound to pretreatment weight. It is conceivable that chronic weight gain unrelated to physiological needs induces an allostatic regulatory state that defends a supranormal adipose mass despite its maladaptive consequences. To challenge this hypothesis, we generated a reversible genetic mouse model of early-onset hyperphagia and severe obesity by selectively blocking the expression of the proopiomelanocortin gene (Pomc) in hypothalamic neurons. Eutopic reactivation of central POMC transmission at different stages of overweight progression normalized or greatly reduced food intake in these obesity-programmed mice. Hypothalamic Pomc rescue also attenuated comorbidities such as hyperglycemia, hyperinsulinemia, and hepatic steatosis and normalized locomotor activity. However, effectiveness of treatment to normalize body weight and adiposity declined progressively as the level of obesity at the time of Pomc induction increased. Thus, our study using a novel reversible monogenic obesity model reveals the critical importance of early intervention for the prevention of subsequent allostatic overload that auto-perpetuates obesity. PMID:23093774

  2. Genetic Disruption of the Copulatory Plug in Mice Leads to Severely Reduced Fertility

    PubMed Central

    Dean, Matthew D.

    2013-01-01

    Seminal fluid proteins affect fertility at multiple stages in reproduction. In many species, a male's ejaculate coagulates to form a copulatory plug. Although taxonomically widespread, the molecular details of plug formation remain poorly understood, limiting our ability to manipulate the structure and understand its role in reproduction. Here I show that male mice knockouts for transglutaminase IV (Tgm4) fail to form a copulatory plug, demonstrating that this gene is necessary for plug formation and lending a powerful new genetic tool to begin characterizing plug function. Tgm4 knockout males show normal sperm count, sperm motility, and reproductive morphology. However, very little of their ejaculate migrates into the female's reproductive tract, suggesting the plug prevents ejaculate leakage. Poor ejaculate migration leads to a reduction in the proportion of oocytes fertilized. However, Tgm4 knockout males fertilized between 3–11 oocytes, which should be adequate for a normal litter. Nevertheless, females mated to Tgm4 knockout males for approximately 14 days were significantly less likely to give birth to a litter compared to females mated to wild-type males. Therefore, it appears that the plug also affects post-fertilization events such as implantation and/or gestation. This study shows that a gene influencing the viscosity of seminal fluid has a major influence on male fertility. PMID:23341775

  3. Impact of genetic variation on synaptic protein levels in genetically diverse mice.

    PubMed

    Loos, Maarten; Li, Ka Wan; van der Schors, Roel; Gouwenberg, Yvonne; van der Loo, Rolinka; Williams, Robert W; Smit, August B; Spijker, Sabine

    2016-04-01

    The relative abundance of synaptic proteins shapes protein complex formation and is essential for synapse function and behavioral fitness. Here, we have used a panel of highly diverse inbred strains of mice-NOD/LtJ, A/J, 129S1/SvImJ, FVB/NJ, C57BL/6J, WSB/EiJ, PWK/PhJ, and CAST/EiJ-to quantify the effects of genetic variation on the synaptic proteome between strains. Using iTRAQ-based quantitative proteome analyses, we detected significant differences in ∼20% of 400 core synaptic proteins. Surprisingly, the differentially abundant proteins showed a modest range of variation across strains, averaging about 1.3-fold. Analysis of protein abundance covariation across the eight strains identified known protein-protein relations (proteins of Arp2/3 complex), as well as novel relations (e.g. Dlg family, Fscn1). Moreover, covariation of synaptic proteins was substantially tighter (∼fourfold more dense than chance level) than corresponding networks of synaptic transcripts (∼twofold more dense than chance). The tight stoichiometry and coherent synaptic protein covariation networks suggest more intense evolutionary selection at this level of molecular organization. In conclusion, genetic diversity in the mouse genome differentially affects the transcriptome and proteome, and only partially penetrates the synaptic proteome. Protein abundance correlation analyses in genetically divergent strains can complement protein-protein interaction network analyses, to provide insight into protein interactomes.

  4. Cognitive components in mice and humans: combining genetics and touchscreens for medical translation.

    PubMed

    Nithianantharajah, Jess; Grant, Seth G N

    2013-10-01

    Human disorders of cognition arise from hundreds of gene mutations and mice serve as models for developing and testing therapeutic approaches. Recent advancements using touchscreen psychological tests that measure similar components of cognition in mice and humans can be combined with genetics. These experiments formally demonstrate that different components of cognition in humans and mice are not merely analogous, but homologous, sharing common descent and genetic constitution. They also show that it is possible to genetically dissect different behaviours and identify their underlying molecular mechanisms. Using these methods as standardised approaches offers the prospect of understanding the genetic architecture of the cognitive repertoire and the identification of new targets for drug development. Rigorously defining homologous mechanisms using genetics and touchscreen tests may also improve drug trial design. Recommendations for mouse clinical trial protocols combined with human genetics are proposed. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Genetic manipulation of reptilian embryos: toward an understanding of cortical development and evolution

    PubMed Central

    Nomura, Tadashi; Yamashita, Wataru; Gotoh, Hitoshi; Ono, Katsuhiko

    2015-01-01

    The mammalian neocortex is a remarkable structure that is characterized by tangential surface expansion and six-layered lamination. However, how the mammalian neocortex emerged during evolution remains elusive. Because all modern reptiles have a homolog of the neocortex at the dorsal pallium, developmental analyses of the reptilian cortex are valuable to explore the origin of the neocortex. However, reptilian cortical development and the underlying molecular mechanisms remain unclear, mainly due to technical difficulties with sample collection and embryonic manipulation. Here, we introduce a method of embryonic manipulations for the Madagascar ground gecko and Chinese softshell turtle. We established in ovo electroporation and an ex ovo culture system to address neural stem cell dynamics, neuronal differentiation and migration. Applications of these techniques illuminate the developmental mechanisms underlying reptilian corticogenesis, which provides significant insight into the evolutionary steps of different types of cortex and the origin of the mammalian neocortex. PMID:25759636

  6. Genetic manipulation of reptilian embryos: toward an understanding of cortical development and evolution.

    PubMed

    Nomura, Tadashi; Yamashita, Wataru; Gotoh, Hitoshi; Ono, Katsuhiko

    2015-01-01

    The mammalian neocortex is a remarkable structure that is characterized by tangential surface expansion and six-layered lamination. However, how the mammalian neocortex emerged during evolution remains elusive. Because all modern reptiles have a homolog of the neocortex at the dorsal pallium, developmental analyses of the reptilian cortex are valuable to explore the origin of the neocortex. However, reptilian cortical development and the underlying molecular mechanisms remain unclear, mainly due to technical difficulties with sample collection and embryonic manipulation. Here, we introduce a method of embryonic manipulations for the Madagascar ground gecko and Chinese softshell turtle. We established in ovo electroporation and an ex ovo culture system to address neural stem cell dynamics, neuronal differentiation and migration. Applications of these techniques illuminate the developmental mechanisms underlying reptilian corticogenesis, which provides significant insight into the evolutionary steps of different types of cortex and the origin of the mammalian neocortex.

  7. Real-Time Genetic Manipulations of the Cytokinin Pathway: A Tool for Laboratory and Field Studies.

    PubMed

    Schäfer, Martin; Meldau, Stefan

    2017-01-01

    Although many established tools for cytokinin (CK) pathway manipulations are well suitable for the analysis of molecular interactions, their use on a whole plant scale is often limited by the induction of severe developmental defects. To circumvent this problem, different methods were developed that allow for a more precise manipulation of the CK pathway. Here we present one of these systems, the pOp6/LhGR system for chemically inducible gene expression. This system allows regulation on a spatial, temporal, and quantitative scale and therefore provides a superior tool for analyzing the role of CKs in the interactions of plants with their environment. The pOp6/LhGR system was tested for RNAi-mediated gene silencing and heterologous gene expression and was successfully used for CK pathway manipulations in different model organisms (Arabidopsis thaliana, Nicotiana tabaccum, Nicotiana attenuata, Citrus sinensis × C. trifoliate). Here we describe specific aspects of the screening procedure and present an experimental setup that can not only be used in the laboratory but is also applicable under field conditions.

  8. Genome sequence of the highly weak-acid-tolerant Zygosaccharomyces bailii IST302, amenable to genetic manipulations and physiological studies.

    PubMed

    Palma, Margarida; Münsterkötter, Martin; Peça, João; Güldener, Ulrich; Sá-Correia, Isabel

    2017-06-01

    Zygosaccharomyces bailii is one of the most problematic spoilage yeast species found in the food and beverage industry particularly in acidic products, due to its exceptional resistance to weak acid stress. This article describes the annotation of the genome sequence of Z. bailii IST302, a strain recently proven to be amenable to genetic manipulations and physiological studies. The work was based on the annotated genomes of strain ISA1307, an interspecies hybrid between Z. bailii and a closely related species, and the Z. bailii reference strain CLIB 213T. The resulting genome sequence of Z. bailii IST302 is distributed through 105 scaffolds, comprising a total of 5142 genes and a size of 10.8 Mb. Contrasting with CLIB 213T, strain IST302 does not form cell aggregates, allowing its manipulation in the laboratory for genetic and physiological studies. Comparative cell cycle analysis with the haploid and diploid Saccharomyces cerevisiae strains BY4741 and BY4743, respectively, suggests that Z. bailii IST302 is haploid. This is an additional trait that makes this strain attractive for the functional analysis of non-essential genes envisaging the elucidation of mechanisms underlying its high tolerance to weak acid food preservatives, or the investigation and exploitation of the potential of this resilient yeast species as cell factory. © FEMS 2017.

  9. Therapeutic manipulation of peroxynitrite attenuates the development of opiate-induced antinociceptive tolerance in mice

    PubMed Central

    Muscoli, Carolina; Cuzzocrea, Salvatore; Ndengele, Michael M.; Mollace, Vincenzo; Porreca, Frank; Fabrizi, Francesca; Esposito, Emanuela; Masini, Emanuela; Matuschak, George M.; Salvemini, Daniela

    2007-01-01

    Severe pain syndromes reduce quality of life in patients with inflammatory and neoplastic diseases, often because chronic opiate therapy results in reduced analgesic effectiveness, or tolerance, leading to escalating doses and distressing side effects. The mechanisms leading to tolerance are poorly understood. Our studies revealed that development of antinociceptive tolerance to repeated doses of morphine in mice was consistently associated with the appearance of several tyrosine-nitrated proteins in the dorsal horn of the spinal cord, including the mitochondrial isoform of superoxide (O2–) dismutase, the glutamate transporter GLT-1, and the enzyme glutamine synthase. Furthermore, antinociceptive tolerance was associated with increased formation of several proinflammatory cytokines, oxidative DNA damage, and activation of the nuclear factor poly(ADP-ribose) polymerase. Inhibition of NO synthesis or removal of O2– blocked these biochemical changes and inhibited the development of tolerance, pointing to peroxynitrite (ONOO–), the product of the interaction between O2– and NO, as a signaling mediator in this setting. Indeed, coadministration of morphine with the ONOO– decomposition catalyst, Fe(III) 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, blocked protein nitration, attenuated the observed biochemical changes, and prevented the development of tolerance in a dose-dependent manner. Collectively, these data suggest a causal role for ONOO– in pathways culminating in antinociceptive tolerance to opiates. Peroxynitrite (ONOO–) decomposition catalysts may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain. PMID:17975673

  10. Generation of Genetically Modified Mice Using the CRISPR-Cas9 Genome-Editing System.

    PubMed

    Henao-Mejia, Jorge; Williams, Adam; Rongvaux, Anthony; Stein, Judith; Hughes, Cynthia; Flavell, Richard A

    2016-02-01

    Genetically modified mice are extremely valuable tools for studying gene function and human diseases. Although the generation of mice with specific genetic modifications through traditional methods using homologous recombination in embryonic stem cells has been invaluable in the last two decades, it is an extremely costly, time-consuming, and, in some cases, uncertain technology. The recently described CRISPR-Cas9 genome-editing technology significantly reduces the time and the cost that are required to generate genetically engineered mice, allowing scientists to test more precise and bold hypotheses in vivo. Using this revolutionary methodology we have generated more than 100 novel genetically engineered mouse strains. In the current protocol, we describe in detail the optimal conditions to generate mice carrying point mutations, chromosomal deletions, conditional alleles, fusion tags, or endogenous reporters.

  11. Genetic Differences in Hemoglobin as Markers for Bone Marrow Transplantation in Mice

    DTIC Science & Technology

    1959-03-01

    derived. Thus, genetic differences in hemoglobin can be used as markers for bone marrow transplantation in irradiated mice. Hemoglobin typing may be particularly useful where the H-2 markers cannot be used.

  12. Generation of Genetically Modified Mice using the CRISPR-Cas9 Genome-Editing System

    PubMed Central

    Rongvaux, Anthony; Stein, Judith; Hughes, Cynthia; Flavell, Richard A.

    2016-01-01

    Genetically modified mice are extremely valuable tools for studying gene function and human diseases. Although the generation of mice with specific genetic modifications through traditional methods using homologous recombination in embryonic stem (ES) has been invaluable in the last two decades, it is an extremely costly, time-consuming and in some cases uncertain technology. The recently described CRISPR/Cas9 genome-editing technology significantly reduces the time and the cost that are required to generate genetically engineered mice, allowing scientist to test more precise and bold hypothesis in vivo. Using this revolutionary methodology we have generated more than one hundred novel genetically engineered mouse strains. In the current protocol, we describe in detail the optimal conditions to generate mice carrying point mutations, chromosomal deletions, conditional alleles, fusion tags or endogenous reporters. PMID:26832688

  13. Probing the production of amidated peptides following genetic and dietary copper manipulations.

    PubMed

    Yin, Ping; Bousquet-Moore, Danielle; Annangudi, Suresh P; Southey, Bruce R; Mains, Richard E; Eipper, Betty A; Sweedler, Jonathan V

    2011-01-01

    Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.

  14. Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells

    PubMed Central

    Bressan, Raul Bardini; Dewari, Pooran Singh; Kalantzaki, Maria; Gangoso, Ester; Matjusaitis, Mantas; Garcia-Diaz, Claudia; Blin, Carla; Grant, Vivien; Bulstrode, Harry; Gogolok, Sabine; Skarnes, William C.

    2017-01-01

    Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable – experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis. PMID:28096221

  15. Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells.

    PubMed

    Bressan, Raul Bardini; Dewari, Pooran Singh; Kalantzaki, Maria; Gangoso, Ester; Matjusaitis, Mantas; Garcia-Diaz, Claudia; Blin, Carla; Grant, Vivien; Bulstrode, Harry; Gogolok, Sabine; Skarnes, William C; Pollard, Steven M

    2017-02-15

    Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis. © 2017. Published by The Company of Biologists Ltd.

  16. Hirschsprung's disease: genetic mutations in mice and men

    PubMed Central

    ROBERTSON, K; MASON, I; HALL, S

    1997-01-01

    MRC Brain Development Programme,Department of Developmental Neurobiology,UMDS Guy's Hospital,London SE1 9RT, UK S HALL Hirschsprung's disease is a neuronal dysplasia of the hindgut, characterised by a loss of neurones, which affects about 1in 5000 live births.1 Genetic factors have been implicated in the aetiology of this disease in about 20% of cases and a dominant pattern of inheritance has been revealed in several families. The pathogenesis of the aganglionosis is often attributed to a failure of migration of neural crest cells, although this has not been proven. 
Recently, mutations in a developmentally regulated receptor tyrosine kinase gene, ret, and mutations in the endothelin receptor-B gene (ENDR-B) have both been linked to familial Hirschsprung's disease in humans.4-6 Moreover, certain mutant mouse strains—namely piebald lethal and lethal spotted—exhibit striking similarities to the human condition. The mutation which gives rise to piebald lethal has now been found to be in the ENDR-B gene,7 and the mutation associated with lethal spotted occurs in the gene for endothelin-3 (ET-3), a ligand for ENDR-B.8 
Two transgenic mouse lines have been developed which also reflect the human disease: ret-k , which has a loss of function mutation of the ret gene,9 and ENDR-B null.10 In addition, the introduction of a Lac-Z reporter gene into neural crest cells of aganglionic mice has made it possible to study directly the fate of enteric neuroblasts which are affected by "Hirschsprung's-like" mutations.11 Here, we review the possible roles of RET and endothelin in the normal development of the enteric nervous system, and the significance of their mutated forms in the pathogenesis of familial aganglionosis. 
This review focuses on recent advances in our understanding of the genetic basis of the lesions which have been implicated in congenital forms of Hirschsprung's disease. Disruption of these genes in the mouse, either by transgenic "knockout" approaches or

  17. Genetic basis and biotechnological manipulation of sexual dimorphism and sex determination in fish.

    PubMed

    Mei, Jie; Gui, Jian-Fang

    2015-02-01

    Aquaculture has made an enormous contribution to the world food production, especially to the sustainable supply of animal proteins. The utility of diverse reproduction strategies in fish, such as the exploiting use of unisexual gynogenesis, has created a typical case of fish genetic breeding. A number of fish species show substantial sexual dimorphism that is closely linked to multiple economic traits including growth rate and body size, and the efficient development of sex-linked genetic markers and sex control biotechnologies has provided significant approaches to increase the production and value for commercial purposes. Along with the rapid development of genomics and molecular genetic techniques, the genetic basis of sexual dimorphism has been gradually deciphered, and great progress has been made in the mechanisms of fish sex determination and identification of sex-determining genes. This review summarizes the progress to provide some directive and objective thinking for further research in this field.

  18. Using Genetically Engineered Mice to Probe the Role of Bioactive Lipids in Prostate Carcinogenesis

    DTIC Science & Technology

    2006-07-01

    some of the mice generated perished unexpectedly and at random times . We analyzed the genotypes and life spans of these animals and found...AD_________________ Award Number: W81XWH-05-1-0135 TITLE: Using Genetically Engineered Mice to...0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing

  19. Control of chronic excessive alcohol drinking by genetic manipulation of the Edinger–Westphal nucleus urocortin-1 neuropeptide system

    PubMed Central

    Giardino, W J; Rodriguez, E D; Smith, M L; Ford, M M; Galili, D; Mitchell, S H; Chen, A; Ryabinin, A E

    2017-01-01

    Midbrain neurons of the centrally projecting Edinger–Westphal nucleus (EWcp) are activated by alcohol, and enriched with stress-responsive neuropeptide modulators (including the paralog of corticotropin-releasing factor, urocortin-1). Evidence suggests that EWcp neurons promote behavioral processes for alcohol-seeking and consumption, but a definitive role for these cells remains elusive. Here we combined targeted viral manipulations and gene array profiling of EWcp neurons with mass behavioral phenotyping in C57BL/6 J mice to directly define the links between EWcp-specific urocortin-1 expression and voluntary binge alcohol intake, demonstrating a specific importance for EWcp urocortin-1 activity in escalation of alcohol intake. PMID:28140406

  20. Humanized Androgen Receptor Mice: A Genetic Model for Differential Response to Prostate Cancer Therapy

    DTIC Science & Technology

    2010-05-01

    For each Q tract allele, we have currently obtained at least 30 experimental and 30 control mice . Some have reached their time points and tissues...overexpression of ETV1). Experimental mice have been generated and prostates are being microdissected as animals reach their time points. Initial...TITLE: Humanized Androgen Receptor Mice : A Genetic Model for Differential Response to Prostate Cancer Therapy PRINCIPAL INVESTIGATOR: Diane M

  1. The tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice.

    PubMed

    Cryan, John F; Mombereau, Cedric; Vassout, Annick

    2005-01-01

    Since its introduction almost 20 years ago, the tail suspension test has become one of the most widely used models for assessing antidepressant-like activity in mice. The test is based on the fact that animals subjected to the short-term, inescapable stress of being suspended by their tail, will develop an immobile posture. Various antidepressant medications reverse the immobility and promote the occurrence of escape-related behaviour. This review focuses on the utility this test as part of a research program aimed at understanding the mechanism of action of antidepressants. We discuss the inherent difficulties in modeling depression in rodents. We describe how the tail suspension differs from the closely related forced swim test. Further, we address some key issues associated with using the TST as a model of antidepressant action. We discuss issues regarding whether it satisfies criteria to be a valid model for assessing depression-related behavioural traits. We elaborate on the tests' ease of use, strain differences observed in the test and gender effects in the test. We focus on the utility of the test for genetic analysis. Furthermore, we discuss the concept of whether immobility maybe a behavioural trait relevant to depression. All of the available pharmacological data using the test in genetically modified mice is collated. Special attention is given to selective breeding programs such as the Rouen 'depressed' mice which have been bred for high and low immobility in the tail suspension test. We provide an extensive pooling of the pharmacological studies published to date using the test. Finally, we provide novel pharmacological validation of an automated system (Bioseb) for assessing immobility. Taken together, we conclude that the tail suspension test is a useful test for assessing the behavioural effects of antidepressant compounds and other pharmacological and genetic manipulations relevant to depression.

  2. Affiliative Behavior, Ultrasonic Communication and Social Reward Are Influenced by Genetic Variation in Adolescent Mice

    PubMed Central

    Panksepp, Jules B.; Jochman, Kimberly A.; Kim, Joseph U.; Koy, Jamie J.; Wilson, Ellie D.; Chen, QiLiang; Wilson, Clarinda R.; Lahvis, Garet P.

    2007-01-01

    Social approach is crucial for establishing relationships among individuals. In rodents, social approach has been studied primarily within the context of behavioral phenomena related to sexual reproduction, such as mating, territory defense and parental care. However, many forms of social interaction occur before the onset of reproductive maturity, which suggests that some processes underlying social approach among juvenile animals are probably distinct from those in adults. We conducted a longitudinal study of social investigation (SI) in mice from two inbred strains to assess the extent to which genetic factors influence the motivation for young mice to approach one another. Early-adolescent C57BL/6J (B6) mice, tested 4–6 days after weaning, investigated former cage mates to a greater degree than BALB/cJ (BALB) mice, irrespective of the sex composition within an interacting pair. This strain difference was not due to variation in maternal care, the phenotypic characteristics of stimulus mice or sensitivity to the length of isolation prior to testing, nor was it attributable to a general difference in appetitive motivation. Ultrasonic vocalization (USV) production was positively correlated with the SI responses of mice from both strains. Interestingly, several USV characteristics segregated with the genetic background of young mice, including a higher average frequency and shorter duration for the USVs emitted by B6 mice. An assessment of conditioned place preference responses indicated that there was a strain-dependent difference in the rewarding nature of social contact. As adolescent mice aged, SI responses gradually became less sensitive to genetic background and more responsive to the particular sex of individuals within an interacting pair. We have thus identified a specific, genetic influence on the motivation of early-adolescent mice to approach one another. Consistent with classical theories of motivation, which propose a functional relationship between

  3. Genetic Manipulation of NK Cells for Cancer Immunotherapy: Techniques and Clinical Implications

    PubMed Central

    Carlsten, Mattias; Childs, Richard W.

    2015-01-01

    Given their rapid and efficient capacity to recognize and kill tumor cells, natural killer (NK) cells represent a unique immune cell to genetically reprogram in an effort to improve the outcome of cell-based cancer immunotherapy. However, technical and biological challenges associated with gene delivery into NK cells have significantly tempered this approach. Recent advances in viral transduction and electroporation have now allowed detailed characterization of genetically modified NK cells and provided a better understanding for how these cells can be utilized in the clinic to optimize their capacity to induce tumor regression in vivo. Improving NK cell persistence in vivo via autocrine IL-2 and IL-15 stimulation, enhancing tumor targeting by silencing inhibitory NK cell receptors such as NKG2A, and redirecting tumor killing via chimeric antigen receptors, all represent approaches that hold promise in preclinical studies. This review focuses on available methods for genetic reprograming of NK cells and the advantages and challenges associated with each method. It also gives an overview of strategies for genetic reprograming of NK cells that have been evaluated to date and an outlook on how these strategies may be best utilized in clinical protocols. With the recent advances in our understanding of the complex biological networks that regulate the ability of NK cells to target and kill tumors in vivo, we foresee genetic engineering as an obligatory pathway required to exploit the full potential of NK-cell based immunotherapy in the clinic. PMID:26113846

  4. Tools for genetic manipulation of the plant growth-promoting bacterium Azospirillum amazonense

    PubMed Central

    2011-01-01

    Background Azospirillum amazonense has potential to be used as agricultural inoculant since it promotes plant growth without causing pollution, unlike industrial fertilizers. Owing to this fact, the study of this species has gained interest. However, a detailed understanding of its genetics and physiology is limited by the absence of appropriate genetic tools for the study of this species. Results Conjugation and electrotransformation methods were established utilizing vectors with broad host-replication origins (pVS1 and pBBR1). Two genes of interest - glnK and glnB, encoding PII regulatory proteins - were isolated. Furthermore, glnK-specific A. amazonense mutants were generated utilizing the pK19MOBSACB vector system. Finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium. Conclusion In this work, genetic tools that can support the study of A. amazonense were described. These methods could provide a better understanding of the genetic mechanisms of this species that underlie its plant growth promotion. PMID:21575234

  5. Tools for genetic manipulation of the plant growth-promoting bacterium Azospirillum amazonense.

    PubMed

    Sant'anna, Fernando H; Andrade, Dieime S; Trentini, Débora B; Weber, Shana S; Schrank, Irene S

    2011-05-16

    Azospirillum amazonense has potential to be used as agricultural inoculant since it promotes plant growth without causing pollution, unlike industrial fertilizers. Owing to this fact, the study of this species has gained interest. However, a detailed understanding of its genetics and physiology is limited by the absence of appropriate genetic tools for the study of this species. Conjugation and electrotransformation methods were established utilizing vectors with broad host-replication origins (pVS1 and pBBR1). Two genes of interest--glnK and glnB, encoding PII regulatory proteins--were isolated. Furthermore, glnK-specific A. amazonense mutants were generated utilizing the pK19MOBSACB vector system. Finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium. In this work, genetic tools that can support the study of A. amazonense were described. These methods could provide a better understanding of the genetic mechanisms of this species that underlie its plant growth promotion.

  6. Manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development.

    PubMed

    Blakely, Pennelope K; Delekta, Phillip C; Miller, David J; Irani, David N

    2015-02-01

    While alphaviruses spread naturally via mosquito vectors, some can also be transmitted as aerosols making them potential bioterrorism agents. One such pathogen, western equine encephalitis virus (WEEV), causes fatal human encephalitis via multiple routes of infection and thus presumably via multiple mechanisms. Although WEEV also produces acute encephalitis in non-human primates, a small animal model that recapitulates features of human disease would be useful for both pathogenesis studies and to evaluate candidate antiviral therapies. We have optimized conditions to infect mice with a low passage isolate of WEEV, thereby allowing detailed investigation of virus tropism, replication, neuroinvasion, and neurovirulence. We find that host factors strongly influence disease outcome, and in particular, that age, gender, and genetic background all have significant effects on disease susceptibility independent of virus tropism or replication within the central nervous system. Our data show that experimental variables can be adjusted in mice to recapitulate disease features known to occur in both non-human primates and humans, thus aiding further study of WEEV pathogenesis and providing a realistic therapeutic window for antiviral drug delivery.

  7. Overview of genetic tools and techniques to study Notch signaling in mice.

    PubMed

    Gridley, Thomas; Groves, Andrew K

    2014-01-01

    Aberrations of Notch signaling in humans cause both congenital and acquired defects and cancers. Genetically engineered mice provide the most efficient and cost-effective models to study Notch signaling in a mammalian system. Here, we review the various types of genetic models, tools, and strategies to study Notch signaling in mice, and provide examples of their use. We also provide advice on breeding strategies for conditional mutant mice, and a protocol for tamoxifen administration to mouse strains expressing inducible Cre recombinase-estrogen receptor fusion proteins.

  8. Anopheles arabiensis sperm production after genetic manipulation, dieldrin treatment, and irradiation.

    PubMed

    Damiens, D; Vreysen, M J B; Gilles, J R L

    2013-03-01

    The use of the sterile insect technique relies on the release of sterilized mass-reared male insects which, before field releases, endure several unnatural treatments. In the case of Anopheles arabiensis (Patton) sterile insect technique program in Sudan, the genetic background of the original strain was first changed to create a genetic sexing strain that is based on a dieldrin-resistant mutation. Secondly, the eggs of the genetic sexing strain require treatment with dieldrin to allow complete elimination of female L1 larvae to enable the release of males only. Finally, male mosquitoes receive an irradiation dose of 70 Gy as pupae for sterilization. The effects of these treatments on sperm production were tested separately and in combination. Irradiation alone significantly decreased the initial sperm number and prevented new sperm production. However, the dieldrin treatment, aimed at eliminating females, appears to have an unexpected radioprotectant effect.

  9. The genetic basis of adaptive melanism in pocket mice.

    PubMed

    Nachman, Michael W; Hoekstra, Hopi E; D'Agostino, Susan L

    2003-04-29

    Identifying the genes underlying adaptation is a major challenge in evolutionary biology. Here, we describe the molecular changes underlying adaptive coat color variation in a natural population of rock pocket mice, Chaetodipus intermedius. Rock pocket mice are generally light-colored and live on light-colored rocks. However, populations of dark (melanic) mice are found on dark lava, and this concealing coloration provides protection from avian and mammalian predators. We conducted association studies by using markers in candidate pigmentation genes and discovered four mutations in the melanocortin-1-receptor gene, Mc1r, that seem to be responsible for adaptive melanism in one population of lava-dwelling pocket mice. Interestingly, another melanic population of these mice on a different lava flow shows no association with Mc1r mutations, indicating that adaptive dark color has evolved independently in this species through changes at different genes.

  10. The genetic basis of adaptive melanism in pocket mice

    PubMed Central

    Nachman, Michael W.; Hoekstra, Hopi E.; D'Agostino, Susan L.

    2003-01-01

    Identifying the genes underlying adaptation is a major challenge in evolutionary biology. Here, we describe the molecular changes underlying adaptive coat color variation in a natural population of rock pocket mice, Chaetodipus intermedius. Rock pocket mice are generally light-colored and live on light-colored rocks. However, populations of dark (melanic) mice are found on dark lava, and this concealing coloration provides protection from avian and mammalian predators. We conducted association studies by using markers in candidate pigmentation genes and discovered four mutations in the melanocortin-1-receptor gene, Mc1r, that seem to be responsible for adaptive melanism in one population of lava-dwelling pocket mice. Interestingly, another melanic population of these mice on a different lava flow shows no association with Mc1r mutations, indicating that adaptive dark color has evolved independently in this species through changes at different genes. PMID:12704245

  11. Genetic Deletion of the NOS3 Gene in CAV1-/- Mice Restores Aqueous Humor Outflow Function.

    PubMed

    Song, Maomao; Wu, Jihong; Lei, Yuan; Sun, Xinghuai

    2017-10-01

    The purpose of this study was to investigate the impact of genetic deletion of NOS3 in CAV1-/- mice on aqueous humor outflow function using a mouse genetic double knockout model (DKO, NOS3-/- CAV1-/-). IOP was measured in DKO, NOS3 KO, CAV1 KO, and wild-type (WT) mice by rebound tonometry. Outflow facility was measured by perfusing enucleated mouse eyes at multiple pressure steps. Sodium nitroprusside (SNP) and L-NG-nitroarginine methyl ester (L-NAME) was administered topically, whereas the contralateral eyes served as vehicle controls. IOP was measured in both eyes before drug treatment and 1 hour after the last drug treatment. Mock aqueous humor ± the nitric oxide (NO) donor SNP or NOS inhibitor L-NAME was perfused into enucleated eyes. IOP was 11 ± 0.23 mm Hg in DKO mice, which was similar to WT mice and significantly lower than CAV1 KO mice (n = 18, P > 0.05). NOS3 deletion in CAV1-/- mice resulted in a 1.9-fold increase in conventional outflow facility (Ccon) compared with CAV1 KO mice (n = 7, P < 0.05). Topical application of NO donor SNP did not significantly change IOP (n = 18, P > 0.05) or Ccon in DKO mice (SNP, n = 20; vehicle, n = 11, P > 0.05). Topical application of L-NAME significantly increased IOP in WT, DKO, and CAV1 mice by reducing Ccon. Nitrotyrosine and PKG levels of DKO mice were similar to, whereas sGC was lower than, WT mice (P < 0.05). Genetic deletion of NOS3 in CAV1-deficient mice restored IOP and conventional aqueous humor drainage to WT level. NOS3 and CAV1 interaction is important to IOP regulation.

  12. IMPROVING PLANT GENETIC ENGINEERING BY MANIPULATING THE HOST. (R829479C001)

    EPA Science Inventory

    Agrobacterium-mediated transformation is a major technique for the genetic engineering of plants. However, there are many economically important crop and tree species that remain highly recalcitrant to Agrobacterium infection. Although attempts have been made to ...

  13. IMPROVING PLANT GENETIC ENGINEERING BY MANIPULATING THE HOST. (R829479C001)

    EPA Science Inventory

    Agrobacterium-mediated transformation is a major technique for the genetic engineering of plants. However, there are many economically important crop and tree species that remain highly recalcitrant to Agrobacterium infection. Although attempts have been made to ...

  14. Antisense-Mediated RNA Targeting: Versatile and Expedient Genetic Manipulation in the Brain

    PubMed Central

    Zalachoras, Ioannis; Evers, Melvin M.; van Roon-Mom, Willeke M. C.; Aartsma-Rus, Annemieke M.; Meijer, Onno C.

    2011-01-01

    A limiting factor in brain research still is the difficulty to evaluate in vivo the role of the increasing number of proteins implicated in neuronal processes. We discuss here the potential of antisense-mediated RNA targeting approaches. We mainly focus on those that manipulate splicing (exon skipping and exon inclusion), but will also briefly discuss mRNA targeting. Classic knockdown of expression by mRNA targeting is only one possible application of antisense oligonucleotides (AON) in the control of gene function. Exon skipping and inclusion are based on the interference of AONs with splicing of pre-mRNAs. These are powerful, specific and particularly versatile techniques, which can be used to circumvent pathogenic mutations, shift splice variant expression, knock down proteins, or to create molecular models using in-frame deletions. Pre-mRNA targeting is currently used both as a research tool, e.g., in models for motor neuron disease, and in clinical trials for Duchenne muscular dystrophy and amyotrophic lateral sclerosis. AONs are particularly promising in relation to brain research, as the modified AONs are taken up extremely fast in neurons and glial cells with a long residence, and without the need for viral vectors or other delivery tools, once inside the blood brain barrier. In this review we cover (1). The principles of antisense-mediated techniques, chemistry, and efficacy. (2) The pros and cons of AON approaches in the brain compared to other techniques of interfering with gene function, such as transgenesis and short hairpin RNAs, in terms of specificity of the manipulation, spatial, and temporal control over gene expression, toxicity, and delivery issues. (3) The potential applications for Neuroscience. We conclude that there is good evidence from animal studies that the central nervous system can be successfully targeted, but the potential of the diverse AON-based approaches appears to be under-recognized. PMID:21811437

  15. [Genetic sequelae of the accident at the Chernobyl nuclear power plant in house mice (Mus musculus)].

    PubMed

    Pomerantseva, M D; Ramaĭa, L K; Chekhovich, A V

    1996-02-01

    Genetic disorders were studied in house mice caught from 1986 to 1993 in areas contaminated by radionuclides after the Chernobyl disaster. Dose rates on soil surface ranged from 0.02 to 200 mR/h. Frequency of reciprocal translocations in spermatocytes of the mice studied was relatively low, but increased with dose rate. In populations, frequency of mice heterozygous for recessive lethal mutations decreased with time after the accident. The data obtained allow us to assume that induced mutations may lead to elimination of germ cells and decreased viability in mice heterozygous for the mutations. These processes result in removing excess mutations from the population.

  16. CRISPRi-Manipulation of Genetic Code Expansion via RF1 for Reassignment of Amber Codon in Bacteria

    PubMed Central

    Zhang, Bo; Yang, Qi; Chen, Jingxian; Wu, Ling; Yao, Tianzhuo; Wu, Yiming; Xu, Huan; Zhang, Lihe; Xia, Qing; Zhou, Demin

    2016-01-01

    The precise engineering of proteins in bacteria via the amber codon has been hampered by the poor incorporation of unnatural amino acid (UAA). Here we explored the amber assignment as a sense codon for UAA by CRISPRi targeting release factor 1 (RF1). Scanning of RF1 gene with sgRNAs identified target loci that differentiate RF1 repressions. Quantitation of RF1 repressions versus UAA incorporation indicated an increasing interrelation with the amber reassignment maximized upon RF1 knockdown to ~30%, disclosing the beneficial role of RF1 in amber assignment. However, further RF1 repression reversed this trend resulting from the detrimental effects on host cell growth, disclosing the harmful aspect of RF1 in reassignment of the amber codon. Our data indicate RF1 as a switch manipulating genetic code expansion and pave a direction via CRISPRi for precise engineering and efficient production of proteins in bacteria. PMID:26818534

  17. [Assisted reproduction and artificial insemination and genetic manipulation in the Criminal Code of the Federal District, Mexico].

    PubMed

    Brena Sesma, Ingrid

    2004-01-01

    The article that one presents has for purpose outline and comment on the recent modifications to the Penal Code for the Federal District of México which establish, for the first time, crimes related to the artificial procreation and to the genetic manipulation. Also one refers to the interaction of the new legal texts with the sanitary legislation of the country. Since it will be stated in some cases they present confrontations between the penal and the sanitary reglamentation and some points related to the legality or unlawfulness of a conduct that stayed without the enough development. These lacks will complicate the application of the new rules of the Penal Code of the Federal District.

  18. Genetic control of ATGL-mediated lipolysis modulates adipose triglyceride stores in leptin-deficient mice[S

    PubMed Central

    Marcelin, Genevieve; Liu, Shun-Mei; Li, Xiaosong; Schwartz, Gary J.; Chua, Streamson

    2012-01-01

    Dissecting the genetics of complex traits such as obesity allows the identification of causal genes for disease. Here, we show that the BALB/c mouse strain carries genetic variants that confer resistance to obesity induced by leptin-deficiency or a high-fat diet (HFD). We set out to identify the physiological and genetic bases underlying this phenotype. When compared with C57BL6/J ob/ob mice (B6), BALB/c ob/ob mice exhibited decreased food intake, increased thermogenic capacity, and improved fat catabolism, each of which can potentially modify obesity. Interestingly, analysis of F1 ob/ob (progeny of B6 ob/+ × BALB/c ob+) mice revealed that obesity resistance in BALB/c ob/ob mice principally relied upon improved fat mobilization. This was mechanistically explained by increased adipose triglyceride lipase (ATGL) content in adipocytes, along with increased lipolysis and fatty acid oxidation. We conducted a genome-wide scan and defined a quantitative trait locus (QTL) on chromosome 2. BALB/c alleles on chromosome 2 not only associated with the obesity resistance phenotype but also supported increased ATGL content in adipose tissue. In summary, our study provides evidence that leptin-independent control of adipocyte lipolysis rates directly modifies the balance of macronutrient handling and is sufficient to regulate fat mass in the absence of alterations in food intake and energy expenditure.—Marcelin, G., S-M. Liu, X. Li, G. J. Schwartz, and S. Chua. PMID:22383686

  19. Glutamate taste and appetite in laboratory mice: physiologic and genetic analyses.

    PubMed

    Bachmanov, Alexander A; Inoue, Masashi; Ji, Hong; Murata, Yuko; Tordoff, Michael G; Beauchamp, Gary K

    2009-09-01

    This article provides an overview of our studies of variation in voluntary glutamate consumption in mice. In 2-bottle preference tests, mice from the C57BL/6ByJ (B6) strain consume more monosodium l-glutamate (MSG) than do mice from the 129P3/J (129) strain. We used these mice to study physiologic and genetic mechanisms that underlie the strain differences in glutamate intake. Our genetic analyses showed that differences between B6 mice and 129 mice in MSG consumption are unrelated to strain variation in consumption of sodium or sweeteners and therefore are attributed to mechanisms specific for glutamate. These strain differences could be due to variation in responses to either taste or postingestive effects of glutamate. To examine the role of taste responsiveness, we measured MSG-evoked activity in gustatory nerves and showed that it is similar in B6 and 129 mice. On the other hand, strain-specific postingestive effects of glutamate were evident from our finding that exposure to MSG increases its consumption in B6 mice and decreases its consumption in 129 mice. We therefore examined whether B6 mice and 129 mice differ in postingestive metabolism of glutamate. We showed that, after intragastric administration of MSG, the MSG is preferentially metabolized through gluconeogenesis in B6 mice, whereas thermogenesis is the predominant process for 129 mice. We hypothesize that a process related to gluconeogenesis of the ingested glutamate generates the rewarding stimulus, which probably occurs in the liver before glucose enters the general circulation, and that the glutamate-induced postingestive thermogenesis generates an aversive stimulus. Our animal model studies raise the question of whether humans also vary in glutamate metabolism in a manner that influences their glutamate preference, consumption, and postingestive processing.

  20. Enhancement in Motor Learning through Genetic Manipulation of the Lynx1 Gene

    PubMed Central

    Miwa, Julie M.; Walz, Andreas

    2012-01-01

    The cholinergic system is a neuromodulatory neurotransmitter system involved in a variety of brain processes, including learning and memory, attention, and motor processes, among others. The influence of nicotinic acetylcholine receptors of the cholinergic system are moderated by lynx proteins, which are GPI-anchored membrane proteins forming tight associations with nicotinic receptors. Previous studies indicate lynx1 inhibits nicotinic receptor function and limits neuronal plasticity. We sought to investigate the mechanism of action of lynx1 on nicotinic receptor function, through the generation of lynx mouse models, expressing a soluble version of lynx and comparing results to the full length overexpression. Using rotarod as a test for motor learning, we found that expressing a secreted variant of lynx leads to motor learning enhancements whereas overexpression of full-length lynx had no effect. Further, adult lynx1KO mice demonstrated comparable motor learning enhancements as the soluble transgenic lines, whereas previously, aged lynx1KO mice showed performance augmentation only with nicotine treatment. From this we conclude the motor learning is more sensitive to loss of lynx function, and that the GPI anchor plays a role in the normal function of the lynx protein. In addition, our data suggests that the lynx gene plays a modulatory role in the brain during aging, and that a soluble version of lynx has potential as a tool for adjusting cholinergic-dependent plasticity and learning mechanisms in the brain. PMID:23139735

  1. Enhancement in motor learning through genetic manipulation of the Lynx1 gene.

    PubMed

    Miwa, Julie M; Walz, Andreas

    2012-01-01

    The cholinergic system is a neuromodulatory neurotransmitter system involved in a variety of brain processes, including learning and memory, attention, and motor processes, among others. The influence of nicotinic acetylcholine receptors of the cholinergic system are moderated by lynx proteins, which are GPI-anchored membrane proteins forming tight associations with nicotinic receptors. Previous studies indicate lynx1 inhibits nicotinic receptor function and limits neuronal plasticity. We sought to investigate the mechanism of action of lynx1 on nicotinic receptor function, through the generation of lynx mouse models, expressing a soluble version of lynx and comparing results to the full length overexpression. Using rotarod as a test for motor learning, we found that expressing a secreted variant of lynx leads to motor learning enhancements whereas overexpression of full-length lynx had no effect. Further, adult lynx1KO mice demonstrated comparable motor learning enhancements as the soluble transgenic lines, whereas previously, aged lynx1KO mice showed performance augmentation only with nicotine treatment. From this we conclude the motor learning is more sensitive to loss of lynx function, and that the GPI anchor plays a role in the normal function of the lynx protein. In addition, our data suggests that the lynx gene plays a modulatory role in the brain during aging, and that a soluble version of lynx has potential as a tool for adjusting cholinergic-dependent plasticity and learning mechanisms in the brain.

  2. A single injection of gain-of-function mutant PCSK9 adeno-associated virus vector induces cardiovascular calcification in mice with no genetic modification

    PubMed Central

    Goettsch, Claudia; Hutcheson, Joshua D.; Hagita, Sumihiko; Rogers, Maximillian A.; Creager, Michael D.; Pham, Tan; Choi, Jung; Mlynarchik, Andrew K; Pieper, Brett; Kjolby, Mads; Aikawa, Masanori; Aikawa, Elena

    2016-01-01

    Background Studying atherosclerotic calcification in vivo requires mouse models with genetic modifications. Previous studies showed that injection of recombinant adeno-associated virus vector (AAV) encoding a gain-of-function mutant PCSK9 into mice promotes atherosclerosis. Aim We aim to study cardiovascular calcification induced by PCSK9 AAV in C57BL/6J mice. Methods 10 week-old C57BL/6J mice received a single injection of AAV encoding mutant mPCSK9 (rAAV8/D377Y-mPCSK9). Ldlr−/− mice served as positive controls. Mice consumed a high-fat, high-cholesterol diet for 15 or 20 weeks. Aortic calcification was assessed by fluorescence reflectance imaging (FRI) of a near-infrared calcium tracer. Results Serum levels of PCSK9 (0.14 µg/ml to 20 µg/ml, p < 0.01) and total cholesterol (82 mg/dL to 820 mg/dL, p < 0.01) increased within one week after injection and remained elevated for 20 weeks. Atherosclerotic lesion size was similar between PCSK9 AAV and Ldlr−/− mice. Aortic calcification was 0.01%±0.01 in PCSK9 AAV mice and 15.3%±6.1 in Ldlr−/− mice at 15 weeks (p < 0.01); by 20 weeks, the PCSK9 AAV mice aortic calcification grew to 12.4%±4.9. Tissue non-specific alkaline phosphatase activity was similar in PCSK9 AAV mice and Ldlr−/− mice at 15 and 20 weeks, respectively. As example of the utility of this model in testing modulators of calcification in vivo, PCSK9 AAV injection to sortilin-deficient mice demonstrated reduced aortic calcification by 46.3% (p < 0.05) compared to littermate controls. Conclusion A single injection of gain-of-function PCSK9 AAV into C57BL/6J mice is a useful tool to study cardiovascular calcification in mice with no genetic manipulation. PMID:27318830

  3. A modified recombineering protocol for the genetic manipulation of gene clusters in Aspergillus fumigatus.

    PubMed

    Alcazar-Fuoli, Laura; Cairns, Timothy; Lopez, Jordi F; Zonja, Bozo; Pérez, Sandra; Barceló, Damià; Igarashi, Yasuhiro; Bowyer, Paul; Bignell, Elaine

    2014-01-01

    Genomic analyses of fungal genome structure have revealed the presence of physically-linked groups of genes, termed gene clusters, where collective functionality of encoded gene products serves a common biosynthetic purpose. In multiple fungal pathogens of humans and plants gene clusters have been shown to encode pathways for biosynthesis of secondary metabolites including metabolites required for pathogenicity. In the major mould pathogen of humans Aspergillus fumigatus, multiple clusters of co-ordinately upregulated genes were identified as having heightened transcript abundances, relative to laboratory cultured equivalents, during the early stages of murine infection. The aim of this study was to develop and optimise a methodology for manipulation of gene cluster architecture, thereby providing the means to assess their relevance to fungal pathogenicity. To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes. By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates. We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge.

  4. A Modified Recombineering Protocol for the Genetic Manipulation of Gene Clusters in Aspergillus fumigatus

    PubMed Central

    Alcazar-Fuoli, Laura; Cairns, Timothy; Lopez, Jordi F.; Zonja, Bozo; Pérez, Sandra; Barceló, Damià; Igarashi, Yasuhiro; Bowyer, Paul; Bignell, Elaine

    2014-01-01

    Genomic analyses of fungal genome structure have revealed the presence of physically-linked groups of genes, termed gene clusters, where collective functionality of encoded gene products serves a common biosynthetic purpose. In multiple fungal pathogens of humans and plants gene clusters have been shown to encode pathways for biosynthesis of secondary metabolites including metabolites required for pathogenicity. In the major mould pathogen of humans Aspergillus fumigatus, multiple clusters of co-ordinately upregulated genes were identified as having heightened transcript abundances, relative to laboratory cultured equivalents, during the early stages of murine infection. The aim of this study was to develop and optimise a methodology for manipulation of gene cluster architecture, thereby providing the means to assess their relevance to fungal pathogenicity. To this end we adapted a recombineering methodology which exploits lambda phage-mediated recombination of DNA in bacteria, for the generation of gene cluster deletion cassettes. By exploiting a pre-existing bacterial artificial chromosome (BAC) library of A. fumigatus genomic clones we were able to implement single or multiple intra-cluster gene replacement events at both subtelomeric and telomere distal chromosomal locations, in both wild type and highly recombinogenic A. fumigatus isolates. We then applied the methodology to address the boundaries of a gene cluster producing a nematocidal secondary metabolite, pseurotin A, and to address the role of this secondary metabolite in insect and mammalian responses to A. fumigatus challenge. PMID:25372385

  5. Genetic manipulation of starch properties in plants: patents 2001-2006.

    PubMed

    Waters, Daniel L E; Henry, Robert J

    2007-01-01

    Starch is the major energy store for many plants and has been extensively exploited by humans for millennia, first as a food source and more recently in a wide variety of non-food applications. The starch properties of the plant species which were first selected by humans have been improved for some time, first by the identification and selection of genotypes with favourable natural variation, then by a process of controlled crosses and selection of offspring with desirable traits and more recently by the generation of synthetic mutants and transgenic plants. Many genes involved in starch synthesis have been identified at the molecular level, providing the means of both manipulating starch properties in novel ways and defining how useful changes to the starch have been made. The commercial value of this understanding is reflected in the expansion of the patent literature where knowledge surrounding each of the genes of starch biosynthesis has been protected in the patent literature. Changes to starch structure have been made using transgenic and conventional breeding methods and these methods are described in the patent literature. Transgenic approaches usually either reduce the activity of an endogenous protein or replace an endogenous protein with a protein from another species. Conventional breeding still plays a role in the recent patent literature and has been used to create new combinations of alleles which in turn generates starch with novel properties.

  6. Modulation of a Circulating Uremic Solute via Rational Genetic Manipulation of the Gut Microbiota.

    PubMed

    Devlin, A Sloan; Marcobal, Angela; Dodd, Dylan; Nayfach, Stephen; Plummer, Natalie; Meyer, Tim; Pollard, Katherine S; Sonnenburg, Justin L; Fischbach, Michael A

    2016-12-14

    Renal disease is growing in prevalence and has striking co-morbidities with metabolic and cardiovascular disease. Indoxyl sulfate (IS) is a toxin that accumulates in plasma when kidney function declines and contributes to the progression of chronic kidney disease. IS derives exclusively from the gut microbiota. Bacterial tryptophanases convert tryptophan to indole, which is absorbed and modified by the host to produce IS. Here, we identify a widely distributed family of tryptophanases in the gut commensal Bacteroides and find that deleting this gene eliminates the production of indole in vitro. By altering the status or abundance of the Bacteroides tryptophanase, we can modulate IS levels in gnotobiotic mice and in the background of a conventional murine gut community. Our results demonstrate that it is possible to control host IS levels by targeting the microbiota and suggest a possible strategy for treating renal disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Genetic and Pharmacologic Manipulation of TLR4 Has Minimal Impact on Ethanol Consumption in Rodents.

    PubMed

    Harris, R Adron; Bajo, Michal; Bell, Richard L; Blednov, Yuri A; Varodayan, Florence P; Truitt, Jay M; de Guglielmo, Giordano; Lasek, Amy W; Logrip, Marian L; Vendruscolo, Leandro F; Roberts, Amanda J; Roberts, Edward; George, Olivier; Mayfield, Jody; Billiar, Timothy R; Hackam, David J; Mayfield, R Dayne; Koob, George F; Roberto, Marisa; Homanics, Gregg E

    2017-02-01

    Toll-like receptor 4 (TLR4) is a critical component of innate immune signaling and has been implicated in alcohol responses in preclinical and clinical models. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium tested the hypothesis that TLR4 mediates excessive ethanol drinking using the following models: (1) Tlr4 knock-out (KO) rats, (2) selective knockdown of Tlr4 mRNA in mouse nucleus accumbens (NAc), and (3) injection of the TLR4 antagonist (+)-naloxone in mice. Lipopolysaccharide (LPS) decreased food/water intake and body weight in ethanol-naive and ethanol-trained wild-type (WT), but not Tlr4 KO rats. There were no consistent genotypic differences in two-bottle choice chronic ethanol intake or operant self-administration in rats before or after dependence. In mice, (+)-naloxone did not decrease drinking-in-the-dark and only modestly inhibited dependence-driven consumption at the highest dose. Tlr4 knockdown in mouse NAc did not decrease drinking in the two-bottle choice continuous or intermittent access tests. However, the latency to ethanol-induced loss of righting reflex increased and the duration decreased in KO versus WT rats. In rat central amygdala neurons, deletion of Tlr4 altered GABAA receptor function, but not GABA release. Although there were no genotype differences in acute ethanol effects before or after chronic intermittent ethanol exposure, genotype differences were observed after LPS exposure. Using different species and sexes, different methods to inhibit TLR4 signaling, and different ethanol consumption tests, our comprehensive studies indicate that TLR4 may play a role in ethanol-induced sedation and GABAA receptor function, but does not regulate excessive drinking directly and would not be an effective therapeutic target.

  8. “Real time” genetic manipulation: a new tool for ecological field studies

    PubMed Central

    Schäfer, Martin; Brütting, Christoph; Gase, Klaus; Reichelt, Michael; Baldwin, Ian; Meldau, Stefan

    2014-01-01

    Summary Field experiments with transgenic plants often reveal the functional significance of genetic traits important for plant performance in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene-related phenotypes in natural habitats. These methods do not allow sufficient control over gene expression to study ecological interactions in real-time, genetic traits playing essential roles in development, or dose-dependent effects. We applied the sensitive dexamethasone (DEX)-inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin-based DEX application method to facilitate ectopic gene expression and RNAi mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field-grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CK’s in both the glasshouse and field to understand resistance to the native herbivore Tupiocoris notatus, which attack plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, T. notatus damage increased, demonstrating CK’s role in this plant-herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature. PMID:23906159

  9. 'Real time' genetic manipulation: a new tool for ecological field studies.

    PubMed

    Schäfer, Martin; Brütting, Christoph; Gase, Klaus; Reichelt, Michael; Baldwin, Ian; Meldau, Stefan

    2013-11-01

    Field experiments with transgenic plants often reveal the functional significance of genetic traits that are important for the performance of the plants in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene-related phenotypes in natural habitats. These methods do not allow sufficient control over gene expression for the study of ecological interactions in real time, of genetic traits that play essential roles in development, or of dose-dependent effects. We applied the sensitive dexamethasone (DEX)-inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin-based DEX application method to facilitate ectopic gene expression and RNA interference-mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field-grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CKs in both the glasshouse and the field to understand resistance to the native herbivore Tupiocoris notatus, which attacks plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, damage by T. notatus increased, demonstrating the role of CKs in this plant-herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature.

  10. Generating high-yielding varieties by genetic manipulation of plant architecture.

    PubMed

    Sakamoto, Tomoaki; Matsuoka, Makoto

    2004-04-01

    Despite a huge population increase since the 1960s, the green revolution more than doubled world grain production and averted large-scale famine. Food crop productivity will have to be further raised, however, because the world population is still increasing rapidly. Among several parameters associated with the increase in yield potential, genes that control plant height and tiller number (in cereal crops) have recently been identified. In addition, a promising strategy to generate semi-dwarf varieties has been developed. Recent advances in plant genome analyses and plant biotechnology will realize a second green revolution through the genetic engineering of food crops.

  11. Characterization of organophosphorus hydrolases and the genetic manipulation of the phosphotriesterase from pseudomonas diminuta

    SciTech Connect

    Dave, K.I.; Miller, C.E.; Wild, J.R.

    1993-12-31

    There are a variety of enzymes which are specifically capable of hydrolyzing organophosphorus esters with different phosphoryl bonds from the typical phosphotriester bonds of common insecticidal neurotoxins (e.g. paraoxon or coumaphos) to the phosphonate-fluoride bonds of chemical warfare agents (e.g. soman or sarin). These enzymes comprise a diverse set of enzymes whose basic architecture and substrate specificities vary dramatically, yet they appear to be ubiquitous throughout nature. The most thoroughly studied of these enzymes is the organophosphate hydrolase (opd gene product) of Pseudomonas diminuta and Ftavobacterium sp. ATCC 27551, and the heterologous expression, post-translational modification, and genetic engineering studies undertaken with this enzyme are described.

  12. Characterization of organophosphorus hydrolases and the genetic manipulation of the phosphotriesterase from Pseudomonas diminuta.

    PubMed

    Dave, K I; Miller, C E; Wild, J R

    1993-06-01

    There are a variety of enzymes which are specifically capable of hydrolyzing organophosphorus esters with different phosphoryl bonds from the typical phosphotriester bonds of common insecticidal neurotoxins (e.g. paraoxon or coumaphos) to the phosphonate-fluoride bonds of chemical warfare agents (e.g. soman or sarin). These enzymes comprise a diverse set of enzymes whose basic architecture and substrate specificities vary dramatically, yet they appear to be ubiquitous throughout nature. The most thoroughly studied of these enzymes is the organophosphate hydrolase (opd gene product) of Pseudomonas diminuta and Flavobacterium sp. ATCC 27551, and the heterologous expression, post-translational modification, and genetic engineering studies undertaken with this enzyme are described.

  13. Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1

    PubMed Central

    Ulhaq, Amber; Yan, Jun; Pantapalangkoor, Paul; Nielsen, Travis B.; Davies, Bryan W.; Actis, Luis A.; Spellberg, Brad

    2017-01-01

    ABSTRACT Acinetobacter baumannii is one of the most antibiotic-resistant pathogens in clinical medicine, and extensively drug-resistant (XDR) strains are commonly isolated from infected patients. Such XDR strains are already resistant to traditional selectable genetic markers, limiting the ability to conduct pathogenesis research by genetic disruption. Optimization of selectable markers is therefore critical for the advancement of fundamental molecular biology techniques to use in these strains. We screened 23 drugs that constitute a broad array of antibiotics spanning multiple drug classes against HUMC1, a highly virulent and XDR A. baumannii clinical blood and lung isolate. HUMC1 is resistant to all clinically useful antibiotics that are reported by the clinical microbiology laboratory, except for colistin. Ethical concerns about intentionally establishing pan-resistance, including to the last-line agent, colistin, in a clinical isolate made identification of other markers desirable. We screened additional antibiotics that are in clinical use and those that are useful only in a lab setting to identify selectable markers that were effective at selecting for transformants in vitro. We show that supraphysiological levels of tetracycline can overcome innate drug resistance displayed by this XDR strain. Last, we demonstrate that transformation of the tetA (tetracycline resistance) and Sh ble (zeocin resistance), but not pac (puromycin resistance), resistance cassettes allow for selection of drug-resistant transformants. These results make the genetic manipulation of XDR A. baumannii strains easily achieved. IMPORTANCE Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report

  14. Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1.

    PubMed

    Luna, Brian M; Ulhaq, Amber; Yan, Jun; Pantapalangkoor, Paul; Nielsen, Travis B; Davies, Bryan W; Actis, Luis A; Spellberg, Brad

    2017-01-01

    Acinetobacter baumannii is one of the most antibiotic-resistant pathogens in clinical medicine, and extensively drug-resistant (XDR) strains are commonly isolated from infected patients. Such XDR strains are already resistant to traditional selectable genetic markers, limiting the ability to conduct pathogenesis research by genetic disruption. Optimization of selectable markers is therefore critical for the advancement of fundamental molecular biology techniques to use in these strains. We screened 23 drugs that constitute a broad array of antibiotics spanning multiple drug classes against HUMC1, a highly virulent and XDR A. baumannii clinical blood and lung isolate. HUMC1 is resistant to all clinically useful antibiotics that are reported by the clinical microbiology laboratory, except for colistin. Ethical concerns about intentionally establishing pan-resistance, including to the last-line agent, colistin, in a clinical isolate made identification of other markers desirable. We screened additional antibiotics that are in clinical use and those that are useful only in a lab setting to identify selectable markers that were effective at selecting for transformants in vitro. We show that supraphysiological levels of tetracycline can overcome innate drug resistance displayed by this XDR strain. Last, we demonstrate that transformation of the tetA (tetracycline resistance) and Sh ble (zeocin resistance), but not pac (puromycin resistance), resistance cassettes allow for selection of drug-resistant transformants. These results make the genetic manipulation of XDR A. baumannii strains easily achieved. IMPORTANCE Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable

  15. Characterization of pNC1, a small and mobilizable plasmid for use in genetic manipulation of Desulfovibrio africanus.

    PubMed

    Castañeda-Carrión, I Nydia; Whiteley, Marvin; Krumholz, Lee R

    2009-10-01

    To develop a vector system that facilitates genetic manipulation in Desulfovibrio species, we screened native sulfate-reducing bacteria for small plasmids. A self-replicating plasmid was discovered in Desulfovibrio africanus SR-1. Sequence analysis of this 8568-bp plasmid (pNC1) revealed a G+C content of 47.2% and nine open reading frames. This plasmid has a copy number of six. Compatible hosts include D. africanus and Pseudomonas aeruginosa PA14. Genetic characterization of pNC1 revealed that 53.6% of the plasmid contains genes associated with replication, mobilization, and partitioning. The 1123-bp replicon is composed of a rep gene and four 22-bp iterons. The mobilization operon is composed of three genes with a putative 144-bp oriT. The partitioning operon is composed of parA and parB with a downstream parS. We report the construction of a small pNC1-based cloning vector which transforms D. africanus at high frequencies (approximately 1.5 x 10(3) CFU/microg DNA), is mobilizable at high transfer frequency (4.8 x 10(-4) transconjugants/donor), and is stably maintained under non-selective pressure. This study provides a potential host-vector system for Desulfovibrio gene functional analyses.

  16. Energy crops for biofuel feedstocks: facts and recent patents on genetic manipulation to improve biofuel crops.

    PubMed

    Kumar, Suresh

    2013-12-01

    Burning fossil-fuels to meet the global energy requirements by human being has intensified the concerns of increasing concentrations of greenhouse gases. Therefore, serious efforts are required to develop nonfossil-based renewable energy sources. Plants are more efficient in utilizing solar energy to convert it into biomass which can be used as feedstocks for biofuel production. Hence with the increasing demands of energy and the needs of cost-effective, sustainable production of fuels, it has become necessary to switch over to plant biomass as a renewable source of energy. Biofuels derived from more sustainable biological materials such as lignocellulosic plant residues, considered as second generation biofuels, are more dependable. However, there are technical challenges such as pretreatment and hydrolysis of lignocellulosic biomass to convert it into fermentable sugars. Plant genetic engineering has already proven its potential in modifying cell wall composition of plants for enhancing the efficiency of biofuel production. Interest and potential in the area are very much evident from the growing number of patents in the recent years on the subject. In this review, recent trends in genetic engineering of energy crops for biofuel production have been introduced, and strategies for the future developments have been discussed.

  17. Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine

    PubMed Central

    Nobrega, Franklin L.; Costa, Ana Rita; Santos, José F.; Siliakus, Melvin F.; van Lent, Jan W. M.; Kengen, Servé W. M.; Azeredo, Joana; Kluskens, Leon D.

    2016-01-01

    Orally administered phages to control zoonotic pathogens face important challenges, mainly related to the hostile conditions found in the gastrointestinal tract (GIT). These include temperature, salinity and primarily pH, which is exceptionally low in certain compartments. Phage survival under these conditions can be jeopardized and undermine treatment. Strategies like encapsulation have been attempted with relative success, but are typically complex and require several optimization steps. Here we report a simple and efficient alternative, consisting in the genetic engineering of phages to display lipids on their surfaces. Escherichia coli phage T7 was used as a model and the E. coli PhoE signal peptide was genetically fused to its major capsid protein (10 A), enabling phospholipid attachment to the phage capsid. The presence of phospholipids on the mutant phages was confirmed by High Performance Thin Layer Chromatography, Dynamic Light Scattering and phospholipase assays. The stability of phages was analysed in simulated GIT conditions, demonstrating improved stability of the mutant phages with survival rates 102–107 pfu.mL−1 higher than wild-type phages. Our work demonstrates that phage engineering can be a good strategy to improve phage tolerance to GIT conditions, having promising application for oral administration in veterinary medicine. PMID:27976713

  18. Inducible and Selective Erasure of Memories in the Mouse Brain via Chemical-Genetic Manipulation

    PubMed Central

    Cao, Xiaohua; Wang, Huimin; Mei, Bing; An, Shuming; Yin, Liang; Wang, L. Phillip; Tsien, Joe Z.

    2010-01-01

    SUMMARY Rapid and selective erasures of certain types of memories in the brain would be desirable under certain clinical circumstances. By employing an inducible and reversible chemical-genetic technique, we find that transient αCaMKII overexpression at the time of recall impairs the retrieval of both newly formed one-hour object recognition memory and fear memories, as well as 1-month-old fear memories. Systematic analyses suggest that excessive αCaMKII activity-induced recall deficits are not caused by disrupting the retrieval access to the stored information but are, rather, due to the active erasure of the stored memories. Further experiments show that the recall-induced erasure of fear memories is highly restricted to the memory being retrieved while leaving other memories intact. Therefore, our study reveals a molecular genetic paradigm through which a given memory, such as new or old fear memory, can be rapidly and specifically erased in a controlled and inducible manner in the brain. PMID:18957226

  19. Mosquitocidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes.

    PubMed Central

    Porter, A G; Davidson, E W; Liu, J W

    1993-01-01

    The identification, cloning, and characterization of protein toxins from various species of bacilli have demonstrated the existence of mosquitocidal toxins with different structures, mechanisms of action, and host ranges. A start has been made in understanding the polypeptide determinants of toxicity and insecticidal activity, and the purification of toxins from recombinant organisms may lead to the elucidation of their X-ray crystal structures and the cloning of brush border membrane receptors. The results of cloning mosquitocidal toxins in heterologous microorganisms show the potential of expanding the range of susceptible mosquito species by combining several toxins of different host specificity in one cell. Toxins have been expressed in new microorganisms with the potential for increasing potency by persisting at the larval feeding zone. The powerful tools of bacterial genetics are being applied to engineer genetically stable, persistent toxin expression and expand the insecticidal host ranges of Bacillus sphaericus and Bacillus thuringiensis strains. These techniques, together with modern formulation technology, should eventually lead to the construction of mosquitocidal microorganisms which are effective enough to have a real impact on mosquito-borne diseases. Images PMID:7905597

  20. Genetic and biochemical manipulation of a broad-spectrum organophosphate degrading system. Final report

    SciTech Connect

    Wild, J.R.

    1994-08-01

    Recent studies on the plasmid-borne organophosphorus-degrading gene of Pseudomonas diminuta and its enzyme have sought to define both the genetic organization and the protein chemistry involved in this system. The bacterial gene encodes a single, unique enzyme, a phosphotriesterase (organophosphorus anhydrase), which is capable of hydrolyzing a wide spectrum of organophosphorus neurotoxins ranging from insecticides such a parathion, orthene, coumaphos and diazinon to mammalian neurotoxins such as diisopropylfluorophosphate (DFP), sarin, soman and mipafox. The organophosphorus degrading genes (opd) from two different plasmids in the soil bacteria P. diminuta and Flavobacterium have been sequenced andtheir structural organizations are being characterized. The cloned geneshave been expressed in a number of biological systems from bacteria to insect tissue culture, and the enzyme has been purified and characterized from several different sources. The catalytic reaction hasbeen determined to involve a stereospecific mechanism which proceeds by the direct nucleophilic attack of an activated water at the reaction center. The reaction rate approaches a diffusion limited catalysis at 2100/M/s and the enzyme is actively adsorbed to various column and particular matrices. This proposal will define the structure of the active site of the phosphotriesterase, evaluate its membrane signal sequence, and develop new genetic constructions to evaluate the heterologous expression/processing of the apoprotein.

  1. Genetic and Pharmacologic Manipulation of TLR4 Has Minimal Impact on Ethanol Consumption in Rodents

    PubMed Central

    Blednov, Yuri A.; Varodayan, Florence P.; Truitt, Jay M.; Lasek, Amy W.; Logrip, Marian L.; Roberts, Amanda J.; Roberts, Edward; Mayfield, Jody; Billiar, Timothy R.; Hackam, David J.; Koob, George F.; Roberto, Marisa

    2017-01-01

    Toll-like receptor 4 (TLR4) is a critical component of innate immune signaling and has been implicated in alcohol responses in preclinical and clinical models. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium tested the hypothesis that TLR4 mediates excessive ethanol drinking using the following models: (1) Tlr4 knock-out (KO) rats, (2) selective knockdown of Tlr4 mRNA in mouse nucleus accumbens (NAc), and (3) injection of the TLR4 antagonist (+)-naloxone in mice. Lipopolysaccharide (LPS) decreased food/water intake and body weight in ethanol-naive and ethanol-trained wild-type (WT), but not Tlr4 KO rats. There were no consistent genotypic differences in two-bottle choice chronic ethanol intake or operant self-administration in rats before or after dependence. In mice, (+)-naloxone did not decrease drinking-in-the-dark and only modestly inhibited dependence-driven consumption at the highest dose. Tlr4 knockdown in mouse NAc did not decrease drinking in the two-bottle choice continuous or intermittent access tests. However, the latency to ethanol-induced loss of righting reflex increased and the duration decreased in KO versus WT rats. In rat central amygdala neurons, deletion of Tlr4 altered GABAA receptor function, but not GABA release. Although there were no genotype differences in acute ethanol effects before or after chronic intermittent ethanol exposure, genotype differences were observed after LPS exposure. Using different species and sexes, different methods to inhibit TLR4 signaling, and different ethanol consumption tests, our comprehensive studies indicate that TLR4 may play a role in ethanol-induced sedation and GABAA receptor function, but does not regulate excessive drinking directly and would not be an effective therapeutic target. SIGNIFICANCE STATEMENT Toll-like receptor 4 (TLR4) is a key mediator of innate immune signaling and has been implicated in alcohol responses in animal models and human

  2. Genetic manipulation of adult-born hippocampal neurons rescues memory in a mouse model of Alzheimer's disease.

    PubMed

    Richetin, Kevin; Leclerc, Clémence; Toni, Nicolas; Gallopin, Thierry; Pech, Stéphane; Roybon, Laurent; Rampon, Claire

    2015-02-01

    In adult mammals, neural progenitors located in the dentate gyrus retain their ability to generate neurons and glia throughout lifetime. In rodents, increased production of new granule neurons is associated with improved memory capacities, while decreased hippocampal neurogenesis results in impaired memory performance in several memory tasks. In mouse models of Alzheimer's disease, neurogenesis is impaired and the granule neurons that are generated fail to integrate existing networks. Thus, enhancing neurogenesis should improve functional plasticity in the hippocampus and restore cognitive deficits in these mice. Here, we performed a screen of transcription factors that could potentially enhance adult hippocampal neurogenesis. We identified Neurod1 as a robust neuronal determinant with the capability to direct hippocampal progenitors towards an exclusive granule neuron fate. Importantly, Neurod1 also accelerated neuronal maturation and functional integration of new neurons during the period of their maturation when they contribute to memory processes. When tested in an APPxPS1 mouse model of Alzheimer's disease, directed expression of Neurod1 in cycling hippocampal progenitors conspicuously reduced dendritic spine density deficits on new hippocampal neurons, to the same level as that observed in healthy age-matched control animals. Remarkably, this population of highly connected new neurons was sufficient to restore spatial memory in these diseased mice. Collectively our findings demonstrate that endogenous neural stem cells of the diseased brain can be manipulated to become new neurons that could allow cognitive improvement. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  3. Non-human Primate Models for Brain Disorders - Towards Genetic Manipulations via Innovative Technology.

    PubMed

    Qiu, Zilong; Li, Xiao

    2017-04-01

    Modeling brain disorders has always been one of the key tasks in neurobiological studies. A wide range of organisms including worms, fruit flies, zebrafish, and rodents have been used for modeling brain disorders. However, whether complicated neurological and psychiatric symptoms can be faithfully mimicked in animals is still debatable. In this review, we discuss key findings using non-human primates to address the neural mechanisms underlying stress and anxiety behaviors, as well as technical advances for establishing genetically-engineered non-human primate models of autism spectrum disorders and other disorders. Considering the close evolutionary connections and similarity of brain structures between non-human primates and humans, together with the rapid progress in genome-editing technology, non-human primates will be indispensable for pathophysiological studies and exploring potential therapeutic methods for treating brain disorders.

  4. Genetic background influences nicotine-induced conditioned place preference and place aversion in mice.

    PubMed

    Ise, Yuya; Mori, Tomohisa; Katayama, Shirou; Suzuki, Tsutomu; Wang, Tzu-Chueh

    2014-01-01

    This study was designed to determine whether genetic differences influence the rewarding effects of nicotine in 4 inbred strains of mice (DBA/2, BALB/c, C3H, and C57BL/6). Nicotine (subcutaneous) induced a place preference in DBA/2 and BALB/c mice but a place aversion in C57BL/6 mice. A low dose of nicotine produced a significant place preference, whereas a high dose of nicotine produced place aversion in C3H mice. These effects were completely reversed by the nicotinic receptor antagonist mecamylamine. These results strongly suggest that a conditioned state, such as rewarding effects or aversive effects, can be influenced by genetic background.

  5. Genetic Manipulation of Carotenoid Biosynthesis in the Green Sulfur Bacterium Chlorobium tepidum†

    PubMed Central

    Frigaard, Niels-Ulrik; Maresca, Julia A.; Yunker, Colleen E.; Jones, A. Daniel; Bryant, Donald A.

    2004-01-01

    The green sulfur bacterium Chlorobium tepidum is a strict anaerobe and an obligate photoautotroph. On the basis of sequence similarity with known enzymes or sequence motifs, nine open reading frames encoding putative enzymes of carotenoid biosynthesis were identified in the genome sequence of C. tepidum, and all nine genes were inactivated. Analysis of the carotenoid composition in the resulting mutants allowed the genes encoding the following six enzymes to be identified: phytoene synthase (crtB/CT1386), phytoene desaturase (crtP/CT0807), ζ-carotene desaturase (crtQ/CT1414), γ-carotene desaturase (crtU/CT0323), carotenoid 1′,2′-hydratase (crtC/CT0301), and carotenoid cis-trans isomerase (crtH/CT0649). Three mutants (CT0180, CT1357, and CT1416 mutants) did not exhibit a discernible phenotype. The carotenoid biosynthetic pathway in C. tepidum is similar to that in cyanobacteria and plants by converting phytoene into lycopene using two plant-like desaturases (CrtP and CrtQ) and a plant-like cis-trans isomerase (CrtH) and thus differs from the pathway known in all other bacteria. In contrast to the situation in cyanobacteria and plants, the construction of a crtB mutant completely lacking carotenoids demonstrates that carotenoids are not essential for photosynthetic growth of green sulfur bacteria. However, the bacteriochlorophyll a contents of mutants lacking colored carotenoids (crtB, crtP, and crtQ mutants) were decreased from that of the wild type, and these mutants exhibited a significant growth rate defect under all light intensities tested. Therefore, colored carotenoids may have both structural and photoprotection roles in green sulfur bacteria. The ability to manipulate the carotenoid composition so dramatically in C. tepidum offers excellent possibilities for studying the roles of carotenoids in the light-harvesting chlorosome antenna and iron-sulfur-type (photosystem I-like) reaction center. The phylogeny of carotenogenic enzymes in green sulfur

  6. Genetic inhibition of Anaplastic Lymphoma Kinase rescues cognitive impairments in Neurofibromatosis 1 mutant mice.

    PubMed

    Weiss, Joseph B; Weber, Sydney J; Torres, Eileen Ruth S; Marzulla, Tessa; Raber, Jacob

    2017-03-15

    Heterozygous Neurofibromatosis 1 (NF1) loss of function mutations occur in approximately 90% of patients with neurofibromatosis. A major, disabling phenotypic consequence of reduced NF1 function is cognitive impairment; a possibly related behavioral phenotype is impaired sleep. Recent results in Drosophila have demonstrated a genetic interaction between Anaplastic Lymphoma Kinase (Alk) and NF1 for both associative learning and sleep. Inhibition of Alk improves associative learning and sleep in heterozygous NF1 mutant flies. The results in Drosophila provide a strong motivation to investigate NF1/Alk genetic interactions in mice. In Drosophila, activation of Alk by its ligand, Jelly belly (Jeb), is the physiologically relevant target of negative regulation by NF1. Therefore, we tested whether genetic inhibition of Alk in heterozygous NF1 mutant mice attenuates or rescues cognitive impairments in mice. Our results are consistent with the hypothesis that NF1 functions in mice biochemically to inhibit signaling from Alk through Ras. The cognitive phenotypes observed in heterozygous NF1 mutant mice are rescued or ameliorated by genetic inhibition of Alk activity. In two tests of hippocampus-dependent learning, the Morris water maze and extinction of contextual fear, mutation of one or both alleles of Alk was sufficient to improve performance to wild type or near wild type levels in NF1-/+ mice. In addition, in NF1 mice genetic inhibition of Alk improves circadian activity levels. These data are intriguing in light of the circadian alterations seen in NF1 patients and indicate that inhibition of Alk activity may cognitively benefit patients with Neurofibromatosis 1.

  7. The effects of selection for gain in mice on the direct-maternal genetic correlation.

    PubMed

    Swartz, A R; Famula, T R

    1994-10-01

    Components of genetic variation for postweaning growth traits were estimated for both control and growth stocks of mice. The effect of phenotypic selection for gain, which genetically combines selection for additive direct and maternal effects, on additive genetic variance components, heritability, and additive genetic correlationsis discussed. Quantitative genetic theory predicts that simultaneous selection for two metric traits in the same direction will cause the genetic correlation between the two traits to become more negative. The results presented in this paper conflict with this theory. The direct-maternal additive genetic correlation was more negative in the control line (with 356 mice) than in the growth-selected line (with 320 mice) for the three traits analyzed (0.310 vs 0.999 for 21-day weight, 0.316 vs 1.000 for 42-day weight, and 0.506 vs 1.000 for gain from 21-42 days). Estimates were obtained by restricted maximum likelihood (REML) computed under a derivative free algorithm (DFREML).

  8. Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management

    PubMed Central

    Sardar, Abul Hasan; Jardim, Armando; Ghosh, Ayan Kumar; Mandal, Abhishek; Das, Sushmita; Saini, Savita; Abhishek, Kumar; Singh, Ruby; Verma, Sudha; Kumar, Ajay; Das, Pradeep

    2016-01-01

    Reactive oxygen and nitrogen species (ROS and RNS) produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS) plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL). There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed environments

  9. Genetic and Proteomics Analyses of Space Flown Mice Skin

    NASA Astrophysics Data System (ADS)

    Terada, Masahiro; Takahashi, Rika; Yamada, Shin; Masaya, Seki; Higashibata, Akira; Majima, Hideyuki J.; Ohira, Yoshinobu; Mukai, Chiaki; Ishioka, Noriaki

    2013-02-01

    Many astronauts stay in the International Space Station (ISS) for a long period of time. Therefore, the development of astronaut health care technologies is very important. Especially, an understanding of the effects of the space environment, such as microgravity and radiation, on protein, gene, and mineral metabolism is important for developing countermeasures against the adverse effects experienced by astronauts who are in space for long periods of time. Since December 2009, the Japan Aerospace Exploration Agency (JAXA) has initiated a human research study to investigate the effects of long-term space flight on gene expression and mineral metabolism by analyzing hair samples from ISS crew members who have been in space (experiment nicknamed “HAIR”). As animal control experiments, we could have an opportunity to analyze rodents samples by participating the tissue sharing program of space-flown mice organized by Italian Space Agency (AGI) and National Aeronautics and Space Administration (NASA). It will reasonably complement human hair experiment because we able to conduct more detailed skin analysis which is enable in human experiment. The purpose of this flown-mice experiment is to study the effects of long-term exposure to space environment. In this experiment, we analyzed mice skin contained hair roots. The samples were taken from space-flown (3-month and 2-week) and 3-month hindlimb suspensioned and 3-month 2G exposed mice, and ground-control mice. For the skin contained hair roots, the extracted and amplified RNA was used to DNA microarray analysis, and was further analyzed with expression on the interesting genes by real time Reverse Transcription Polymerase Chain Reaction (RT-PCR) method. And the extracted protein was used to Mass Spectrometer analysis. Data analysis on the specimen are in progress.

  10. Molecular Toolbox for Genetic Manipulation of the Stalked Budding Bacterium Hyphomonas neptunium

    PubMed Central

    Jung, Alexandra; Eisheuer, Sabrina; Cserti, Emöke; Leicht, Oliver; Strobel, Wolfgang; Möll, Andrea; Schlimpert, Susan; Kühn, Juliane

    2014-01-01

    The alphaproteobacterium Hyphomonas neptunium proliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies two H. neptunium promoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions in H. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species. PMID:25398860

  11. Molecular toolbox for genetic manipulation of the stalked budding bacterium Hyphomonas neptunium.

    PubMed

    Jung, Alexandra; Eisheuer, Sabrina; Cserti, Emöke; Leicht, Oliver; Strobel, Wolfgang; Möll, Andrea; Schlimpert, Susan; Kühn, Juliane; Thanbichler, Martin

    2015-01-01

    The alphaproteobacterium Hyphomonas neptunium proliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies two H. neptunium promoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions in H. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Improved vectors for Agrobacterium mediated genetic manipulation of Hypholoma spp. and other homobasidiomycetes.

    PubMed

    Al-Salihi, Suhad A A; Scott, Thomas A; Bailey, Andy M; Foster, Gary D

    2017-08-24

    The basidiomycete fungi Hypholoma fasciculare and H. sublateritium are both prolific producers of sesquiterpenes and triterpenes, some of which have relevant pharmaceutical properties. Although H. sublateritium has been transformed in the past, the low reported efficiencies highlighted the need for establishing an effective simple transformation system for these valuable species. We have optimized Agrobacterium tumefaciens-mediated transformation through testing various parameters in these two Hypholoma species, showing that a mixture of homogenized mycelia and Agrobacterium (strain LBA4404) co-cultivated for 84h at 25°C is optimal for efficient transformation in these basidiomycetes. This study also reveals the requirements for transgene expression, with the first report of GFP expression in these Hypholoma, the need for an intron for such transgene expression, and further demonstrates the functionality of the expression vector by its use in Clitopilus passeckerianus. This development of transformation system and expression constructs, can facilitate further genetic investigation such as gene functionality in these fungi. Copyright © 2017. Published by Elsevier B.V.

  13. Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass

    PubMed Central

    Fu, Chunxiang; Mielenz, Jonathan R.; Xiao, Xirong; Ge, Yaxin; Hamilton, Choo Y.; Rodriguez, Miguel; Chen, Fang; Foston, Marcus; Ragauskas, Arthur; Bouton, Joseph; Dixon, Richard A.; Wang, Zeng-Yu

    2011-01-01

    Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300–400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions. PMID:21321194

  14. Lentiviral vectors and cardiovascular diseases: a genetic tool for manipulating cardiomyocyte differentiation and function.

    PubMed

    Di Pasquale, E; Latronico, M V G; Jotti, G S; Condorelli, G

    2012-06-01

    Engineered recombinant viral vectors are a powerful tool for vehiculating genetic information into mammalian cells. Because of their ability to infect both dividing and non-dividing cells with high efficiency, lentiviral vectors have gained particular interest for basic research and preclinical studies in the cardiovascular field. We review here the major applications for lentiviral-vector technology in the cardiovascular field: we will discuss their use in trailing gene expression during the induction of differentiation, in protocols for the isolation of cardiac cells and in the tracking of cardiac cells after transplantation in vivo; we will also describe lentivirally-mediated gene delivery uses, such as the induction of a phenotype of interest in a target cell or the treatment of cardiovascular diseases. In addition, a section of the review will be dedicated to reprogramming approaches, focusing attention on the generation of pluripotent stem cells and on transdifferentiation, two emerging strategies for the production of cardiac myocytes from human cells and for the investigation of human diseases. Finally, in order to give a perspective on their future clinical use we will critically discuss advantages and disadvantages of lentivirus-based strategies for the treatment of cardiovascular diseases.

  15. Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

    PubMed Central

    Zhou, Xunlei; Alvarez-Bolado, Gonzalo

    2013-01-01

    Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body. PMID:23912701

  16. Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass.

    PubMed

    Fu, Chunxiang; Mielenz, Jonathan R; Xiao, Xirong; Ge, Yaxin; Hamilton, Choo Y; Rodriguez, Miguel; Chen, Fang; Foston, Marcus; Ragauskas, Arthur; Bouton, Joseph; Dixon, Richard A; Wang, Zeng-Yu

    2011-03-01

    Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300-400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.

  17. Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems

    PubMed Central

    JIN, Li-Fang; LI, Jin-Song

    2016-01-01

    With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC) approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches. PMID:27469251

  18. Trichinella infestation in mice genetically selected for high and low antibody production.

    PubMed Central

    Perrudet-Badoux, A; Binaghi, R A; Biozzi, G

    1975-01-01

    Mice genetically selected according to their capacity to produce antibodies (high and low responder lines) were infested with Trichinella spiralis. High responder mice produced much higher levels of IgE and gamma1 serum antibodies than low responder mice. IgG and IgM antibodies were detected by immunodiffusion analysis or passive haemagglutination only in animals of the high responder line and in very low concentrations. The number of living larvae present in the muscles was very similar in the animals of both lines 30, 60 or 75 days after infestation. PMID:51004

  19. [Genetic disorders in house mice after the accident at Chernobyl power plant].

    PubMed

    Pomerantseva, M D; Ramaĭa, L K; Chekhovich, A V

    1997-01-01

    Genetic effects were studied in house mice caught from 1986 to 1994 in regions polluted by radionuclides as a result of the Chernobyl disaster. The dose rates of gamma-radiation on the soil surface ranged from 0.02 to 200 mR/h. The frequency of reciprocal translocations in mouse spermatocytes was relatively low, but increased with the dose rate. Embryo mortality was increased only in the progeny of male mice caught in 1987 in the area with maximum contamination. The frequency of mice heterozygous for recessive lethal mutations decreased with the time after the accident.

  20. Genetic modification of iron metabolism in mice affects the gut microbiota.

    PubMed

    Buhnik-Rosenblau, Keren; Moshe-Belizowski, Shirly; Danin-Poleg, Yael; Meyron-Holtz, Esther G

    2012-10-01

    The composition of the gut microbiota is affected by environmental factors as well as host genetics. Iron is one of the important elements essential for bacterial growth, thus we hypothesized that changes in host iron homeostasis, may affect the luminal iron content of the gut and thereby the composition of intestinal bacteria. The iron regulatory protein 2 (Irp2) and one of the genes mutated in hereditary hemochromatosis Hfe , are both proteins involved in the regulation of systemic iron homeostasis. To test our hypothesis, fecal metal content and a selected spectrum of the fecal microbiota were analyzed from Hfe-/-, Irp2-/- and their wild type control mice. Elevated levels of iron as well as other minerals in feces of Irp2-/- mice compared to wild type and Hfe-/- mice were observed. Interestingly significant variation in the general fecal-bacterial population-patterns was observed between Irp2-/- and Hfe-/- mice. Furthermore the relative abundance of five species, mainly lactic acid bacteria, was significantly different among the mouse lines. Lactobacillus (L.) murinus and L. intestinalis were highly abundant in Irp2-/- mice, Enterococcus faecium species cluster and a species most similar to Olsenella were highly abundant in Hfe-/- mice and L. johnsonii was highly abundant in the wild type mice. These results suggest that deletion of iron metabolism genes in the mouse host affects the composition of its intestinal bacteria. Further studying the relationship between gut microbiota and genetic mutations affecting systemic iron metabolism in human should lead to clinical implications.

  1. Genetic variation of iron-induced uroporphyria in mice.

    PubMed Central

    Smith, A G; Francis, J E

    1993-01-01

    Iron overload causes inhibition of hepatic uroporphyrinogen decarboxylase (UROD) and uroporphyria in C57BL/10ScSn but not DBA/2 mice [Smith, Cabral, Carthew, Francis and Manson (1989) Int. J. Cancer 43, 492-496]. We have investigated the induction of uroporphyria in 12 inbred strains of mice 25 weeks after iron treatment (600 mg/kg) to determine if there was any correlation with the Ah locus. Under these conditions, inhibition of UROD occurred to varying degrees in Ahd mice (SWR and AKR) as well as nominally Ahb-1 (C57BL/6J, C57BL/10ScSn and C57BL/10-cc) and Ahb-2 strains (BALB/c and C3H/HeJ). Five other Ahb or Ahd strains (C57BL/Ks, A/J, CBA/J, LP and DBA/2) were unaffected. Thus there appeared to be no correlation with the Ah phenotype and this illustrated that some other variable inherited factors are involved. Comparisons between another susceptible strain, A2G, and the congenic A2G-hr/+strain (carrying the recessive hr gene) showed a modulating influence associated with the hr locus. In contrast with individual mice of inbred strains, which showed consistent responses to iron, those of the outbred MF1 strain showed a spectrum of sensitivities as might be expected for a heterogeneic stock. The rate of porphyria development was accelerated by administration of 5-aminolaevulinic acid (5-ALA) in the drinking water, but this did not overcome strain differences. Among four strains the order of susceptibility was SWR > C57BL/10ScSn > C57B1/6J > DBA/2 (the last strain was completely resistant). With degrees of iron loading greater than 600 mg of Fe/kg (1200-1800 mg of Fe/kg) C57BL/10ScSn mice (after 20 weeks) and SWR mice (after 5 weeks which included 4 weeks of 5-ALA treatment) had less inhibition of UROD and a lower uroporphyric response, showing that there was an optimum level of liver iron concentration. Studies on selected microsomal enzyme activities associated with cytochrome P-450 showed no correlation with the propensities of strains to develop porphyria

  2. Genetic, hormonal, and metabolomic influences on social behavior and sex preference of XXY mice

    PubMed Central

    Erkkila, Krista; Lue, YanHe; Jentsch, J. David; Schwarcz, Monica Dorin; Abuyounes, Deena; Hikim, Amiya Sinha; Wang, Christina; Lee, Paul W.-N.; Swerdloff, Ronald S.

    2010-01-01

    XXY men (Klinefelter syndrome) are testosterone deficient, socially isolated, exhibit impaired gender identity, and may experience more homosexual behaviors. Here, we characterize social behaviors in a validated XXY mouse model to understand mechanisms. Sociability and gender preference were assessed by three-chambered choice tasks before and after castration and after testosterone replacement. Metabolomic activities of brain and blood were quantified through fractional synthesis rates of palmitate and ribose (GC-MS). XXY mice exhibit greater sociability than XY littermates, particularly for male mice. The differences in sociability disappear after matching androgen exposure. Intact XXY, compared with XY, mice prefer male mice odors when the alternatives are ovariectomized female mice odors, but they prefer estrous over male mice odors, suggesting that preference for male mice may be due to social, not sexual, cues. Castration followed by testosterone treatment essentially remove these preferences. Fractional synthesis rates of palmitate are higher in the hypothalamus, amygdala, and hippocampus of XXY compared with XY mice but not with ribose in these brain regions or palmitate in blood. Androgen ablation in XY mice increases fractional synthesis rates of fatty acids in the brain to levels indistinguishable from those in XXY mice. We conclude that intact XXY mice exhibit increased sociability, differences in gender preference for mice and their odors are due to social rather than sexual cues and, these differences are mostly related to androgen deficiency rather than genetics. Specific metabolic changes in brain lipids, which are also regulated by androgens, are observed in brain regions that are involved in these behaviors. PMID:20570823

  3. Genetic analysis of experimental allergic encephalomyelitis in mice

    SciTech Connect

    Baker, D.; Rosenwasser, O.A.; O`Neill, J.K.; Turk, J.L.

    1995-10-15

    Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that exhibits many pathologic similarities with multiple sclerosis. While products of the MHC are known to control the development of EAE, it is clear that non-MHC products also influence susceptibility. The chromosomal locations of these were investigated in selective crosses between MHC class II-compatible, EAE-susceptible Biozzi ABH, and low responder nonobese diabetic (NOD) mice. The disease was dominant and highly influenced by gender in the backcross one (BC{sub 1}) generation. Female mice were significantly more susceptible than male mice. Segregation of disease frequency of female animals in this cross suggested that EAE was controlled by a major locus. Although microsatellite-based exclusion mapping indicated that a number of regions on chromosomes 5, 6, 7, 8, 9, 10, 11, 12, 13, and 18 showed evidence of linkage (p<0.05) compared with expected random distributions of alleles, disease susceptibility was most strongly linked (p<0.05) to chromosome 7. However, by selectively analyzing animals that were either severely affected or almost normal, additional susceptibility loci were mapped on chromosomes 18 and 11 that were linked (p<0.001) to resistance and the development of severe disease, respectively. The data indicate a major locus on chromosome 7, affecting initiation and severity of EAE that is probably modified by several other unlinked loci. These localizations may provide candidate loci for the analysis of human autoimmune-demyelinating disease. 30 refs., 5 tabs.

  4. Effect of genetic background on the dystrophic phenotype in mdx mice

    PubMed Central

    Coley, William D.; Bogdanik, Laurent; Vila, Maria Candida; Yu, Qing; Van Der Meulen, Jack H.; Rayavarapu, Sree; Novak, James S.; Nearing, Marie; Quinn, James L.; Saunders, Allison; Dolan, Connor; Andrews, Whitney; Lammert, Catherine; Austin, Andrew; Partridge, Terence A.; Cox, Gregory A.; Lutz, Cathleen; Nagaraju, Kanneboyina

    2016-01-01

    Genetic background significantly affects phenotype in multiple mouse models of human diseases, including muscular dystrophy. This phenotypic variability is partly attributed to genetic modifiers that regulate the disease process. Studies have demonstrated that introduction of the γ-sarcoglycan-null allele onto the DBA/2J background confers a more severe muscular dystrophy phenotype than the original strain, demonstrating the presence of genetic modifier loci in the DBA/2J background. To characterize the phenotype of dystrophin deficiency on the DBA/2J background, we created and phenotyped DBA/2J-congenic Dmdmdx mice (D2-mdx) and compared them with the original, C57BL/10ScSn-Dmdmdx (B10-mdx) model. These strains were compared with their respective control strains at multiple time points between 6 and 52 weeks of age. Skeletal and cardiac muscle function, inflammation, regeneration, histology and biochemistry were characterized. We found that D2-mdx mice showed significantly reduced skeletal muscle function as early as 7 weeks and reduced cardiac function by 28 weeks, suggesting that the disease phenotype is more severe than in B10-mdx mice. In addition, D2-mdx mice showed fewer central myonuclei and increased calcifications in the skeletal muscle, heart and diaphragm at 7 weeks, suggesting that their pathology is different from the B10-mdx mice. The new D2-mdx model with an earlier onset and more pronounced dystrophy phenotype may be useful for evaluating therapies that target cardiac and skeletal muscle function in dystrophin-deficient mice. Our data align the D2-mdx with Duchenne muscular dystrophy patients with the LTBP4 genetic modifier, making it one of the few instances of cross-species genetic modifiers of monogenic traits. PMID:26566673

  5. Lung defenses against Pseudomonas aeruginosa in C5-deficient mice with different genetic backgrounds.

    PubMed Central

    Cerquetti, M C; Sordelli, D O; Bellanti, J A; Hooke, A M

    1986-01-01

    Lung defenses against Pseudomonas aeruginosa were investigated in C5-deficient strains of mice with different genetic backgrounds. We studied pulmonary clearance and cell responses after aerosol exposure to P. aeruginosa in C5-deficient B10.D2/oSnJ and DBA/2J mice and their closest C5-sufficient counterparts, B10.D2/nSnJ and DBA/1J mice. Different patterns of lung clearance and pulmonary cell responses were found for the two C5-deficient strains. C5-deficient B10.D2/oSnJ mice showed defective lung clearance of P. aeruginosa 4 h after challenge compared with C5-sufficient B10.D2/nSnJ animals. This finding was associated with a decreased number of polymorphonuclear leukocytes recruited into the airways during the same time. Interestingly, C5-deficient DBA/2J mice recruited higher numbers of polymorphonuclear leukocytes than did C5-sufficient DBA/1J mice by 4 h after aerosolization. Nevertheless, lung clearance of P. aeruginosa in DBA/2J mice was not as effective as in C5-sufficient DBA/1J mice, suggesting that other functions of C5 besides chemotaxism could be involved. Lung clearance of P. aeruginosa was also investigated in C5-deficient and -sufficient hybrids sharing the same genetic background (DBA/2J X B10.D2). The results suggested that murine lung clearance of P. aeruginosa is markedly affected by lack of C5 in a specific genetic background (B10.D2). PMID:3086235

  6. Resistance to listeriosis in two lines of mice genetically selected for high and low antibody production.

    PubMed Central

    Berche, P A

    1985-01-01

    Infection by the intracellular parasite Listeria monocytogenes was studied in two inbred lines of mice genetically selected for high and low antibody production against xenogeneic red blood cells. It was revealed that, during the early non-specific phase of infection, bacterial growth in tissues was significantly enhanced in high responder (HR) mice, as opposed to low responder (LR) mice. This is interpreted as the in vivo expression of a genetic impairment of the bactericidal activity of resident macrophages in this line of mice. After Day 2 of infection, the kinetics of bacterial growth in the spleen and the liver was almost identical in the two lines, indicating that mice from both lines generated efficient anti-Listeria immunity. This was confirmed by the fact that no interline difference could be detected in the expression of T-cell mediated immunity, as estimated by the production of protective T cells and delayed sensitivity T cells, and by the level of immunological memory. The genetic impairment in the bactericidal activity of resident macrophages resulted in a significant increase of anti-Listeria antibody production in HR mice and did not prevent T-dependent activation of effector macrophages mobilized in infectious sites. This explains that the overall resistance to listeriosis was similar in LR and HR mice, as shown by the LD50 values respectively estimated as 2.2 X 10(5) and 3.8 X 10(5) bacteria per mouse. This natural resistance was expressed at the same level as that of C57BL/6 mice. PMID:4077102

  7. Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea

    PubMed Central

    Ding, Zhong-Tao; Zhang, Zhi; Luo, Di; Zhou, Jin-Yan; Zhong, Juan; Yang, Jie; Xiao, Liang; Shu, Dan; Tan, Hong

    2015-01-01

    The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain. PMID:25955649

  8. Genetically inbred Balb/c mice differ from outbred Swiss Webster mice on discrete measures of sociability: relevance to a genetic mouse model of autism spectrum disorders.

    PubMed

    Jacome, Luis F; Burket, Jessica A; Herndon, Amy L; Deutsch, Stephen I

    2011-12-01

    The Balb/c mouse is proposed as a model of human disorders with prominent deficits of sociability, such as autism spectrum disorders (ASDs) that may involve pathophysiological disruption of NMDA receptor-mediated neurotransmission. A standard procedure was used to measure sociability in 8-week-old male genetically inbred Balb/c and outbred Swiss Webster mice. Moreover, because impaired sociability may influence the social behavior of stimulus mice, we also measured the proportion of total episodes of social approach made by the stimulus mouse while test and stimulus mice were allowed to interact freely. Three raters with good inter-rater agreement evaluated operationally defined measures of sociability chosen because of their descriptive similarity to deficits of social behavior reported in persons with ASDs. The data support previous reports that the Balb/c mouse is a genetic mouse model of impaired sociability. The data also show that the behavior of the social stimulus mouse is influenced by the impaired sociability of the Balb/c strain. Interestingly, operationally defined measures of sociability did not necessarily correlate with each other within mouse strain and the profile of correlated measures differed between strains. Finally, "stereotypic" behaviors (i.e. rearing, grooming and wall climbing) recorded during the session of free interaction between the test and social stimulus mice were more intensely displayed by Swiss Webster than Balb/c mice, suggesting that the domains of sociability and "restricted repetitive and stereotyped patterns of behavior" are independent of each other in the Balb/c strain.

  9. Subchronic exposure to ethyl tertiary butyl ether resulting in genetic damage in Aldh2 knockout mice.

    PubMed

    Weng, Zuquan; Suda, Megumi; Ohtani, Katsumi; Mei, Nan; Kawamoto, Toshihiro; Nakajima, Tamie; Wang, Rui-Sheng

    2013-09-15

    Ethyl tertiary butyl ether (ETBE) is biofuel additive recently used in Japan and some other countries. Limited evidence shows that ETBE has low toxicity. Acetaldehyde (AA), however, as one primary metabolite of ETBE, is clearly genotoxic and has been considered to be a potential carcinogen. The aim of this study was to evaluate the effects of ALDH2 gene on ETBE-induced genotoxicity and metabolism of its metabolites after inhalation exposure to ETBE. A group of wild-type (WT) and Aldh2 knockout (KO) C57BL/6 mice were exposed to 500ppm ETBE for 1-6h, and the blood concentrations of ETBE metabolites, including AA, tert-butyl alcohol and 2-methyl-1,2-propanediol, were measured. Another group of mice of WT and KO were exposed to 0, 500, 1750, or 5000ppm ETBE for 6h/day with 5 days per weeks for 13 weeks. Genotoxic effects of ETBE in these mice were measured by the alkaline comet assay, 8-hydroxyguanine DNA-glycosylase modified comet assay and micronucleus test. With short-term exposure to ETBE, the blood concentrations of all the three metabolites in KO mice were significantly higher than the corresponding concentrations of those in WT mice of both sexes. After subchronic exposure to ETBE, there was significant increase in DNA damage in a dose-dependent manner in KO male mice, while only 5000ppm exposure significantly increased DNA damage in male WT mice. Overall, there was a significant sex difference in genetic damage in both genetic types of mice. These results showed that ALDH2 is involved in the detoxification of ETBE and lack of enzyme activity may greatly increase the sensitivity to the genotoxic effects of ETBE, and male mice were more sensitive than females.

  10. GENETIC BACKGROUND BUT NOT METALLOTHIONEIN PHENOTYPE DICTATES SENSITIVITY TO CADMIUM-INDUCED TESTICULAR INJURY IN MICE

    EPA Science Inventory

    Genetic Background but not Metallothionein Phenotype Dictates Sensitivity to
    Cadmium-Induced Testicular Injury in Mice

    Jie Liu1,2, Chris Corton3, David J. Dix4, Yaping Liu1, Michael P. Waalkes2
    and Curtis D. Klaassen1

    ABSTRACT

    Parenteral administrati...

  11. GENETIC BACKGROUND BUT NOT METALLOTHIONEIN PHENOTYPE DICTATES SENSITIVITY TO CADMIUM-INDUCED TESTICULAR INJURY IN MICE

    EPA Science Inventory

    Genetic Background but not Metallothionein Phenotype Dictates Sensitivity to
    Cadmium-Induced Testicular Injury in Mice

    Jie Liu1,2, Chris Corton3, David J. Dix4, Yaping Liu1, Michael P. Waalkes2
    and Curtis D. Klaassen1

    ABSTRACT

    Parenteral administrati...

  12. Nicotine addiction and nicotinic receptors: lessons from genetically modified mice.

    PubMed

    Changeux, Jean-Pierre

    2010-06-01

    The past decades have seen a revolution in our understanding of brain diseases and in particular of drug addiction. This has been largely due to the identification of neurotransmitter receptors and the development of animal models, which together have enabled the investigation of brain functions from the molecular to the cognitive level. Tobacco smoking, the principal - yet avoidable - cause of lung cancer is associated with nicotine addiction. Recent studies in mice involving deletion and replacement of nicotinic acetylcholine receptor subunits have begun to identify the molecular mechanisms underlying nicotine addiction and might offer new therapeutic strategies to treat this addiction.

  13. Visualization and genetic manipulation of dendrites and spines in the mouse cerebral cortex and hippocampus using in utero electroporation.

    PubMed

    Pacary, Emilie; Haas, Matilda A; Wildner, Hendrik; Azzarelli, Roberta; Bell, Donald M; Abrous, Djoher Nora; Guillemot, François

    2012-07-26

    In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system (1-5). To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex (6-8). Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions. Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method( 9). However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice (10-11) (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA (12). These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral

  14. Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

    PubMed Central

    Pacary, Emilie; Haas, Matilda A.; Wildner, Hendrik; Azzarelli, Roberta; Bell, Donald M.; Abrous, Djoher Nora; Guillemot, François

    2012-01-01

    In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions. Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures

  15. Murine cytomegalovirus stimulates natural killer cell function but kills genetically resistant mice treated with radioactive strontium

    SciTech Connect

    Masuda, A.; Bennett, M.

    1981-12-01

    Treatment of C3H/St mice with 100 microCi of 89Sr weakened their genetic resistance to murine cytomegalovirus (MCMV) infection. The criteria utilized to detect increased susceptibility were: (i) survival of mice; (ii) numbers of MCMV-infected cells in the spleens and liver; and (iii) serum glutamic pyruvic transaminase levels. The natural killer (NK) cell activity of spleen cells from mice treated with 89Sr is very low. However, the NK activities of spleen cells of both normal and 89Sr-treated mice were greatly augmented 3 days after infection with MCMV. These NK cells lysed a variety of tumor cells and shared several features with conventional NK cells, but were not lysed by anti-Nk-1.2 serum (specific for NK cells) plus complement. Splenic adherent cells did not lyse tumor cells themselves but were necessary for the stimulation of NK cells by MCMV. The paradox of high NK cell function and poor survival in 89Sr-treated mice infected with MCMV was a surprise. We conclude that these augmented NK cells, of themselves, cannot account for the genetic resistance of C3H/St mice to infection with MCMV.

  16. The role of osteopontin in D-galactosamine-induced liver injury in genetically obese mice

    SciTech Connect

    Kwon, Hyo-Jung; Won, Young-Suk; Yoon, Won-Kee; Nam, Ki-Hoan; Kim, Dae-Yong; Kim, Hyoung-Chin

    2010-02-01

    Various epidemiological studies have shown that obesity increases the risk of liver disease, but the precise mechanisms through which this occurs are poorly understood. In the present study, we hypothesized that osteopontin (OPN), an extracellular matrix and proinflammatory cytokine, has an important role in making obese mice more susceptible to inflammatory liver injury. After exposure of genetically obese ob/ob and db/db mice to a single dose of D-galactosamine (GalN), the plasma liver enzyme levels, histology and expression levels of cytokines and OPN were evaluated. The ob/ob and db/db mice, which were more sensitive to GalN-induced inflammatory liver injury compared with wild-type mice, had significantly higher plasma and hepatic OPN expression levels. Increased OPN expression was mainly found in hepatocytes and inflammatory cells and was correlated with markedly up-regulated interleukin (IL)-12 and IL-18 levels. Furthermore, pretreatment with a neutralizing OPN (nOPN) antibody attenuated the GalN-induced inflammatory liver injury in ob/ob and db/db mice, which was accompanied by significantly reduced macrophages recruitment and IL-12 and IL-18 productions. Taken together, these results suggest that up-regulated OPN expression is a contributing factor to increased susceptibility of genetically obese mice to GalN-induced liver injury by promoting inflammation and modulating immune response.

  17. Identification of genetic factors that modify motor performance and body weight using Collaborative Cross mice

    DOE PAGES

    Mao, Jian -Hua; Langley, Sasha A.; Huang, Yurong; ...

    2015-11-09

    Evidence has emerged that suggests a link between motor deficits, obesity and many neurological disorders. However, the contributing genetic risk factors are poorly understood. Here we used the Collaborative Cross (CC), a large panel of newly inbred mice that captures 90% of the known variation among laboratory mice, to identify the genetic loci controlling rotarod performance and its relationship with body weight in a cohort of 365 mice across 16 CC strains. Body weight and rotarod performance varied widely across CC strains and were significantly negatively correlated. Genetic linkage analysis identified 14 loci that were associated with body weight. However,more » 45 loci affected rotarod performance, seven of which were also associated with body weight, suggesting a strong link at the genetic level. As a result, we show that genes identified in this study overlap significantly with those related to neurological disorders and obesity found in human GWA studies. In conclusion, our results provide a genetic framework for studies of the connection between body weight, the central nervous system and behavior.« less

  18. Identification of genetic factors that modify motor performance and body weight using Collaborative Cross mice

    SciTech Connect

    Mao, Jian -Hua; Langley, Sasha A.; Huang, Yurong; Hang, Michael; Bouchard, Kristofer E.; Celniker, Susan E.; Brown, James B.; Jansson, Janet K.; Karpen, Gary H.; Snijders, Antoine M.

    2015-11-09

    Evidence has emerged that suggests a link between motor deficits, obesity and many neurological disorders. However, the contributing genetic risk factors are poorly understood. Here we used the Collaborative Cross (CC), a large panel of newly inbred mice that captures 90% of the known variation among laboratory mice, to identify the genetic loci controlling rotarod performance and its relationship with body weight in a cohort of 365 mice across 16 CC strains. Body weight and rotarod performance varied widely across CC strains and were significantly negatively correlated. Genetic linkage analysis identified 14 loci that were associated with body weight. However, 45 loci affected rotarod performance, seven of which were also associated with body weight, suggesting a strong link at the genetic level. As a result, we show that genes identified in this study overlap significantly with those related to neurological disorders and obesity found in human GWA studies. In conclusion, our results provide a genetic framework for studies of the connection between body weight, the central nervous system and behavior.

  19. Genetics of acute inflammation: inflammatory reactions in inbred lines of mice and in their interline crosses.

    PubMed

    Stiffel, C; Ibanez, O M; Ribeiro, O G; Decreusefond, C; Mouton, D; Siqueira, M; Biozzi, G

    1990-01-01

    Acute inflammation is induced by the subcutaneous injection of swollen polyacrylamide microbeads, its intensity measured by the cell and protein concentration of the local exudates. A large and continuous range of responses is obtained in different inbred strains of mice, which suggests a polygenic control of the inflammatory response. The variable levels of the global dominance observed in F1 hybrids issued from several parental combinations indicated that the pattern of alleles controlling high or low response was different in each parental strain. Balanced intercrossing of the 8 inbred strains studied has provided a genetically heterogeneous F3 population, presenting a high variability of responses. The value of the genetic part of F3 phenotypic variance, the spread of the interstrain differences, as well as the polygenic nature of the regulation of inflammatory responses pointed out the possibility to perform a bidirectional genetic selection by using the F3 mice as the foundation population, and response to microbeads as the selective phenotypic character.

  20. Genetically-directed Sparse Neuronal Labeling in BAC Transgenic Mice through Mononucleotide Repeat Frameshift

    PubMed Central

    Lu, Xiao-Hong; Yang, X. William

    2017-01-01

    Mosaicism with Repeat Frameshift (MORF) allows a single Bacterial Artificial Chromosome (BAC) transgene to direct sparse labeling of genetically-defined neuronal populations in mice. The BAC transgene drives cell-type-specific transcription of an out-of-frame mononucleotide repeat that is placed between a translational start codon and a membrane-bound fluorescent protein lacking its start codon. The stochastic frameshift of the unstable repeat DNA in a subset of BAC-expressing neurons results in the in-frame translation of the reporter protein hence the sparse neuronal labeling. As a proof-of-concept, we generated D1-dopamine receptor (D1) BAC MORF mice that label about 1% striatal D1-expressing medium spiny neurons and allow visualization of their dendrites. These mice enable the study of D1-MSN dendrite development in wildtype mice, and its degeneration in a mouse model of Huntington’s disease. PMID:28272512

  1. Metabolomics of Apc Min/+ mice genetically susceptible to intestinal cancer

    PubMed Central

    2014-01-01

    Background To determine how diets high in saturated fat could increase polyp formation in the mouse model of intestinal neoplasia, Apc Min/+ , we conducted large-scale metabolome analysis and association study of colon and small intestine polyp formation from plasma and liver samples of Apc Min/+ vs. wild-type littermates, kept on low vs. high-fat diet. Label-free mass spectrometry was used to quantify untargeted plasma and acyl-CoA liver compounds, respectively. Differences in contrasts of interest were analyzed statistically by unsupervised and supervised modeling approaches, namely Principal Component Analysis and Linear Model of analysis of variance. Correlation between plasma metabolite concentrations and polyp numbers was analyzed with a zero-inflated Generalized Linear Model. Results Plasma metabolome in parallel to promotion of tumor development comprises a clearly distinct profile in Apc Min/+ mice vs. wild type littermates, which is further altered by high-fat diet. Further, functional metabolomics pathway and network analyses in Apc Min/+ mice on high-fat diet revealed associations between polyp formation and plasma metabolic compounds including those involved in amino-acids metabolism as well as nicotinamide and hippuric acid metabolic pathways. Finally, we also show changes in liver acyl-CoA profiles, which may result from a combination of Apc Min/+ -mediated tumor progression and high fat diet. The biological significance of these findings is discussed in the context of intestinal cancer progression. Conclusions These studies show that high-throughput metabolomics combined with appropriate statistical modeling and large scale functional approaches can be used to monitor and infer changes and interactions in the metabolome and genome of the host under controlled experimental conditions. Further these studies demonstrate the impact of diet on metabolic pathways and its relation to intestinal cancer progression. Based on our results, metabolic signatures

  2. Changes in Gene Expression Foreshadow Diet-Induced Obesity in Genetically Identical Mice

    PubMed Central

    Koza, Robert A; Nikonova, Larissa; Hogan, Jessica; Rim, Jong-Seop; Mendoza, Tamra; Faulk, Christopher; Skaf, Jihad; Kozak, Leslie P

    2006-01-01

    High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet) correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity. PMID:16733553

  3. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice.

    PubMed

    Koza, Robert A; Nikonova, Larissa; Hogan, Jessica; Rim, Jong-Seop; Mendoza, Tamra; Faulk, Christopher; Skaf, Jihad; Kozak, Leslie P

    2006-05-01

    High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet) correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity.

  4. Antidiabetic effect of glycyrrhizin in genetically diabetic KK-Ay mice.

    PubMed

    Takii, H; Kometani, T; Nishimura, T; Nakae, T; Okada, S; Fushiki, T

    2001-05-01

    We, previously demonstrated that one shot administration of glycyrrhizin (Grz) reduced the postprandial blood glucose rise, using Std ddY mice. Subsequently, we evaluated the effects of long-term Grz treatment (2.7, 4.1 g/kg diet) on diabetic symptoms using genetically non-insulin dependent diabetic model mice (KK-Ay). Male KK-Ay mice were divided into 3 groups: the control group, 0.27% Grz diet (2.7 g of Grz/kg diet) group and 0.41% Grz diet (4.1 g of Grz/kg diet) group. The elevation of blood glucose concentration was almost entirely suppressed in mice fed the 0.41% Grz diet 7 weeks after the beginning of test feeding, although it was not suppressed in mice fed the control diet or the 0.27% Grz diet. Water intake in the control and 0.27% Grz diet groups increased gradually, whereas, this was not true in the 0.41% Grz diet group. Grz treatment significantly lowered blood insulin level. Throughout the experiment, Grz did not affect the food intake or body weight among the three groups. The mice fed the 0.41% Grz diet also improved their tolerance to oral glucose loading 9 weeks after the beginning of test feeding. This study shows that Grz has an antidiabetic effect in noninsulin-dependent diabetes model mice.

  5. Ethanol administration exacerbates the abnormalities in hepatic lipid oxidation in genetically obese mice

    PubMed Central

    Everitt, Hannah; Hu, Ming; Ajmo, Joanne M.; Rogers, Christopher Q.; Liang, Xiaomei; Zhang, Ray; Yin, Huquan; Choi, Alison; Bennett, Eric S.

    2013-01-01

    Alcohol consumption synergistically increases the risk and severity of liver damage in obese patients. To gain insight into cellular or molecular mechanisms underlying the development of fatty liver caused by ethanol-obesity synergism, we have carried out animal experiments that examine the effects of ethanol administration in genetically obese mice. Lean wild-type (WT) and obese (ob/ob) mice were subjected to ethanol feeding for 4 wk using a modified Lieber-DeCarli diet. After ethanol feeding, the ob/ob mice displayed much more pronounced changes in terms of liver steatosis and elevated plasma levels of alanine aminotransferase and aspartate aminotransferase, indicators of liver injury, compared with control mice. Mechanistic studies showed that ethanol feeding augmented the impairment of hepatic sirtuin 1 (SIRT1)-AMP-activated kinase (AMPK) signaling in the ob/ob mice. Moreover, the impairment of SIRT1-AMPK signaling was closely associated with altered hepatic functional activity of peroxisome proliferator-activated receptor γ coactivator-α and lipin-1, two vital downstream lipid regulators, which ultimately contributed to aggravated fatty liver observed in ethanol-fed ob/ob mice. Taken together, our novel findings suggest that ethanol administration to obese mice exacerbates fatty liver via impairment of the hepatic lipid metabolism pathways mediated largely by a central signaling system, the SIRT1-AMPK axis. PMID:23139221

  6. Ethanol administration exacerbates the abnormalities in hepatic lipid oxidation in genetically obese mice.

    PubMed

    Everitt, Hannah; Hu, Ming; Ajmo, Joanne M; Rogers, Christopher Q; Liang, Xiaomei; Zhang, Ray; Yin, Huquan; Choi, Alison; Bennett, Eric S; You, Min

    2013-01-01

    Alcohol consumption synergistically increases the risk and severity of liver damage in obese patients. To gain insight into cellular or molecular mechanisms underlying the development of fatty liver caused by ethanol-obesity synergism, we have carried out animal experiments that examine the effects of ethanol administration in genetically obese mice. Lean wild-type (WT) and obese (ob/ob) mice were subjected to ethanol feeding for 4 wk using a modified Lieber-DeCarli diet. After ethanol feeding, the ob/ob mice displayed much more pronounced changes in terms of liver steatosis and elevated plasma levels of alanine aminotransferase and aspartate aminotransferase, indicators of liver injury, compared with control mice. Mechanistic studies showed that ethanol feeding augmented the impairment of hepatic sirtuin 1 (SIRT1)-AMP-activated kinase (AMPK) signaling in the ob/ob mice. Moreover, the impairment of SIRT1-AMPK signaling was closely associated with altered hepatic functional activity of peroxisome proliferator-activated receptor γ coactivator-α and lipin-1, two vital downstream lipid regulators, which ultimately contributed to aggravated fatty liver observed in ethanol-fed ob/ob mice. Taken together, our novel findings suggest that ethanol administration to obese mice exacerbates fatty liver via impairment of the hepatic lipid metabolism pathways mediated largely by a central signaling system, the SIRT1-AMPK axis.

  7. The genetic architecture of NAFLD among inbred strains of mice

    PubMed Central

    Hui, Simon T; Parks, Brian W; Org, Elin; Norheim, Frode; Che, Nam; Pan, Calvin; Castellani, Lawrence W; Charugundla, Sarada; Dirks, Darwin L; Psychogios, Nikolaos; Neuhaus, Isaac; Gerszten, Robert E; Kirchgessner, Todd; Gargalovic, Peter S; Lusis, Aldons J

    2015-01-01

    To identify genetic and environmental factors contributing to the pathogenesis of non-alcoholic fatty liver disease, we examined liver steatosis and related clinical and molecular traits in more than 100 unique inbred mouse strains, which were fed a diet rich in fat and carbohydrates. A >30-fold variation in hepatic TG accumulation was observed among the strains. Genome-wide association studies revealed three loci associated with hepatic TG accumulation. Utilizing transcriptomic data from the liver and adipose tissue, we identified several high-confidence candidate genes for hepatic steatosis, including Gde1, a glycerophosphodiester phosphodiesterase not previously implicated in triglyceride metabolism. We confirmed the role of Gde1 by in vivo hepatic over-expression and shRNA knockdown studies. We hypothesize that Gde1 expression increases TG production by contributing to the production of glycerol-3-phosphate. Our multi-level data, including transcript levels, metabolite levels, and gut microbiota composition, provide a framework for understanding genetic and environmental interactions underlying hepatic steatosis. DOI: http://dx.doi.org/10.7554/eLife.05607.001 PMID:26067236

  8. Mob/oriT, a mobilizable site-specific recombination system for unmarked genetic manipulation in Bacillus thuringiensis and Bacillus cereus.

    PubMed

    Wang, Pengxia; Zhu, Yiguang; Zhang, Yuyang; Zhang, Chunyi; Xu, Jianyi; Deng, Yun; Peng, Donghai; Ruan, Lifang; Sun, Ming

    2016-06-10

    Bacillus thuringiensis and Bacillus cereus are two important species in B. cereus group. The intensive study of these strains at the molecular level and construction of genetically modified bacteria requires the development of efficient genetic tools. To insert genes into or delete genes from bacterial chromosomes, marker-less manipulation methods were employed. We present a novel genetic manipulation method for B. thuringiensis and B. cereus strains that does not leave selection markers. Our approach takes advantage of the relaxase Mob02281 encoded by plasmid pBMB0228 from Bacillus thuringiensis. In addition to its mobilization function, this Mob protein can mediate recombination between oriT sites. The Mob02281 mobilization module was associated with a spectinomycin-resistance gene to form a Mob-Spc cassette, which was flanked by the core 24-bp oriT sequences from pBMB0228. A strain in which the wild-type chromosome was replaced with the modified copy containing the Mob-Spc cassette at the target locus was obtained via homologous recombination. Thus, the spectinomycin-resistance gene can be used to screen for Mob-Spc cassette integration mutants. Recombination between the two oriT sequences mediated by Mob02281, encoded by the Mob-Spc cassette, resulted in the excision of the Mob-Spc cassette, producing the desired chromosomal alteration without introducing unwanted selection markers. We used this system to generate an in-frame deletion of a target gene in B. thuringiensis as well as a gene located in an operon of B. cereus. Moreover, we demonstrated that this system can be used to introduce a single gene or an expression cassette of interest in B. thuringiensis. The Mob/oriT recombination system provides an efficient method for unmarked genetic manipulation and for constructing genetically modified bacteria of B. thuringiensis and B. cereus. Our method extends the available genetic tools for B. thuringiensis and B. cereus strains.

  9. Genetic Dissection of a Key Reproductive Barrier Between Nascent Species of House Mice

    PubMed Central

    White, Michael A.; Steffy, Brian; Wiltshire, Tim; Payseur, Bret A.

    2011-01-01

    Reproductive isolation between species is often caused by deleterious interactions among loci in hybrids. Finding the genes involved in these incompatibilities provides insight into the mechanisms of speciation. With recently diverged subspecies, house mice provide a powerful system for understanding the genetics of reproductive isolation early in the speciation process. Although previous studies have yielded important clues about the genetics of hybrid male sterility in house mice, they have been restricted to F1 sterility or incompatibilities involving the X chromosome. To provide a more complete characterization of this key reproductive barrier, we conducted an F2 intercross between wild-derived inbred strains from two subspecies of house mice, Mus musculus musculus and Mus musculus domesticus. We identified a suite of autosomal and X-linked QTL that underlie measures of hybrid male sterility, including testis weight, sperm density, and sperm morphology. In many cases, the autosomal loci were unique to a specific sterility trait and exhibited an effect only when homozygous, underscoring the importance of examining reproductive barriers beyond the F1 generation. We also found novel two-locus incompatibilities between the M. m. musculus X chromosome and M. m. domesticus autosomal alleles. Our results reveal a complex genetic architecture for hybrid male sterility and suggest a prominent role for reproductive barriers in advanced generations in maintaining subspecies integrity in house mice. PMID:21750261

  10. Wild mice as bountiful resources of novel genetic variants for quantitative traits.

    PubMed

    Ishikawa, Akira

    2013-06-01

    Most traits of biological importance, including traits for human complex diseases (e.g., obesity and diabetes), are continuously distributed. These complex or quantitative traits are controlled by multiple genetic loci called QTLs (quantitative trait loci), environments and their interactions. The laboratory mouse has long been used as a pilot animal model for understanding the genetic architecture of quantitative traits. Next-generation sequencing analyses and genome-wide SNP (single nucleotide polymorphism) analyses of mouse genomes have revealed that classical inbred strains commonly used throughout the world are derived from a few fancy mice with limited and non-randomly distributed genetic diversity that occurs in nature and also indicated that their genomes are predominantly Mus musculus domesticus in origin. Many QTLs for a huge variety of traits have so far been discovered from a very limited gene pool of classical inbred strains. However, wild M. musculus mice consisting of five subspecies widely inhabit areas all over the world, and hence a number of novel QTLs may still lie undiscovered in gene pools of the wild mice. Some of the QTLs are expected to improve our understanding of human complex diseases. Using wild M. musculus subspecies in Asia as examples, this review illustrates that wild mice are untapped natural resources for valuable QTL discovery.

  11. Genetic control of ATGL-mediated lipolysis modulates adipose triglyceride stores in leptin-deficient mice.

    PubMed

    Marcelin, Genevieve; Liu, Shun-Mei; Li, Xiaosong; Schwartz, Gary J; Chua, Streamson

    2012-05-01

    Dissecting the genetics of complex traits such as obesity allows the identification of causal genes for disease. Here, we show that the BALB/c mouse strain carries genetic variants that confer resistance to obesity induced by leptin-deficiency or a high-fat diet (HFD). We set out to identify the physiological and genetic bases underlying this phenotype. When compared with C57BL6/J ob/ob mice (B6), BALB/c ob/ob mice exhibited decreased food intake, increased thermogenic capacity, and improved fat catabolism, each of which can potentially modify obesity. Interestingly, analysis of F1 ob/ob (progeny of B6 ob/+ × BALB/c ob+) mice revealed that obesity resistance in BALB/c ob/ob mice principally relied upon improved fat mobilization. This was mechanistically explained by increased adipose triglyceride lipase (ATGL) content in adipocytes, along with increased lipolysis and fatty acid oxidation. We conducted a genome-wide scan and defined a quantitative trait locus (QTL) on chromosome 2. BALB/c alleles on chromosome 2 not only associated with the obesity resistance phenotype but also supported increased ATGL content in adipose tissue. In summary, our study provides evidence that leptin-independent control of adipocyte lipolysis rates directly modifies the balance of macronutrient handling and is sufficient to regulate fat mass in the absence of alterations in food intake and energy expenditure.-Marcelin, G., S-M. Liu, X. Li, G. J. Schwartz, and S. Chua.

  12. Generating double knockout mice to model genetic intervention for diabetic cardiomyopathy in humans.

    PubMed

    Chavali, Vishalakshi; Nandi, Shyam Sundar; Singh, Shree Ram; Mishra, Paras Kumar

    2014-01-01

    Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/-) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3-4 weeks and show robust hyperglycemia at the age of 10-12 weeks. It is a chronic model of T1D. In Ins2+/- Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/-/Mmp9-/- (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy.

  13. Genetic factors contributing to defective spermatogonial differentiation in juvenile spermatogonial depletion (Utp14bjsd) mice.

    PubMed

    Bolden-Tiller, Olga U; Chiarini-Garcia, Helio; Poirier, Christophe; Alves-Freitas, Daniel; Weng, Connie C; Shetty, Gunapala; Meistrich, Marvin L

    2007-08-01

    Male mice that are homozygous for the juvenile spermatogonial depletion (jsd) mutation in the Utp14b gene undergo several waves of spermatogenesis. However, spermatogonial differentiation ceases and in adults, spermatogonia are the only germ cells that remain. To understand further the blockage in spermatogonial differentiation in Utp14b(jsd) mutant mice, we correlated the rate and severity of spermatogonial depletion and the restoration of spermatogenesis following the suppression of testosterone or elevation of testicular temperature with the genetic background. Testes from Utp14b(jsd) mutant mice on B6, C3H, and mixed C3H-B6-129 (HB129) genetic backgrounds all showed steady decreases in the numbers of normal spermatogonia between 8 wk and 20 wk of age. The percentages of tubules with differentiating germ cells were higher and the spermatogonia were more advanced in C3H- background than in B6- or HB129-background Utp14b(jsd) mice. Genetic crosses showed that the source of the Y chromosome was a major factor in determining the severity of spermatogonial depletion in Utp14b(jsd) mutant mice. When Utp14b(jsd) mutants were subjected to total androgen ablation or unilateral cryptorchidization, spermatogenic development recovered markedly in the C3H and HB129 background but showed less recovery in the B6-background mice. The differences noted between the strains in terms of the severity of spermatogonial depletion were not dependent upon testosterone level or scrotal temperature but correlated with the magnitudes of the effects of elevated temperature on normal and Utp14b(jsd) mutant spermatogenic cells. Thus, the abilities of germ cells in certain strains to survive elevated temperatures may be related to their abilities to maintain some degree of differentiation potential after the Utp14b(jsd) gene is mutated.

  14. 70 years of radiation genetics: Fruit flies, mice and humans

    SciTech Connect

    Abrahamson, S.

    1997-03-01

    Radiation protection`s function is to protect society from the potential hazards that might occur through the human use of radiation, whether it be from energy production, medical uses or other sources of exposure. To do so, various scientific bodies are called upon to develop risk estimates which will provide society with adequate protection to the adverse effects of radiation, as best we can understand those adverse affects. Geneticists have the added burden, in that they must attempt to provide protection not only to the offspring of the present generation but also for all subsequent generations. While most of us have difficulty in thinking of effects that might be manifest only one or two generations into the future, some have projected potential risks for 50 to 100 generations. Here the author reviews work on fruit flies and mice, and studies of human exposures, which has provided much of the foundational information upon which geneticists can derive conclusions with regard to radiation protection questions.

  15. Cure of metastatic growth of EMT6 tumor cells in mice following manipulation of CD200:CD200R signaling.

    PubMed

    Gorczynski, Reginald M; Chen, Zhiqi; Khatri, Ismat; Podnos, Anna; Yu, Kai

    2013-11-01

    In previous studies, we observed that regulation of expression of CD200, both on cells of a transplantable breast cancer, EMT6, and of the host, as well as of the receptor, CD200R in host mice, regulated local tumor growth and metastasis in immunocompetent animals. This in turn led to an improved ability to document immunity to EMT6 in CD200R1KO mice. In the current study, we have explored the ability to cure BALB/c CD200KO or CD200R1KO mice of tumors ≤1 cm(3) in size by surgical resection of localized tumor, followed by immunization with irradiated EMT6 cells along with CpG as adjuvant. While control animals treated in this fashion developed significant pulmonary and liver metastases within 30 days of surgery, significant protection was seen in both CD200KO or CD200R1KO mice, with no macroscopic lung/liver metastases observed in CD200R1KO mice on sacrifice at day 300. Following surgical resection and immunization, draining lymph nodes from control mice contained tumor cells cloned at limiting dilution in vitro even before pulmonary and hepatic metastasis was seen. In contrast, within the limits of detection of the assay used (sensitivity ~1 in 10(7) cells), no tumor cells were detected at limiting dilution in similarly treated CD200R1KO mice, and significant reductions were seen in CD200KO mice. Infusion of anti-CD4, but less so anti-CD8, mAb into surgically treated and immunized CD200R1KO mice attenuated protection from both macroscopic (liver/lung) and microscopic (assayed by limiting dilution of DLN) metastasis. Adoptive transfer of lymphocytes from treated CD200R1KO mice to surgically treated control mice also attenuated metastatic growth of tumor, which was abolished by pretreatment of transferred cells with anti-CD4 mAb. Our data suggest that CD200:CD200R attenuates a potentially tumor-protective CD4 host response to breast cancer.

  16. Genetics of Skeletal Evolution in Unusually Large Mice from Gough Island.

    PubMed

    Parmenter, Michelle D; Gray, Melissa M; Hogan, Caley A; Ford, Irene N; Broman, Karl W; Vinyard, Christopher J; Payseur, Bret A

    2016-12-01

    Organisms on islands often undergo rapid morphological evolution, providing a platform for understanding mechanisms of phenotypic change. Many examples of evolution on islands involve the vertebrate skeleton. Although the genetic basis of skeletal variation has been studied in laboratory strains, especially in the house mouse Mus musculus domesticus, the genetic determinants of skeletal evolution in natural populations remain poorly understood. We used house mice living on the remote Gough Island-the largest wild house mice on record-to understand the genetics of rapid skeletal evolution in nature. Compared to a mainland reference strain from the same subspecies (WSB/EiJ), the skeleton of Gough Island mice is considerably larger, with notable expansions of the pelvis and limbs. The Gough Island mouse skeleton also displays changes in shape, including elongations of the skull and the proximal vs. distal elements in the limbs. Quantitative trait locus (QTL) mapping in a large F2 intercross between Gough Island mice and WSB/EiJ reveals hundreds of QTL that control skeletal dimensions measured at 5, 10, and/or 16 weeks of age. QTL exhibit modest, mostly additive effects, and Gough Island alleles are associated with larger skeletal size at most QTL. The QTL with the largest effects are found on a few chromosomes and affect suites of skeletal traits. Many of these loci also colocalize with QTL for body weight. The high degree of QTL colocalization is consistent with an important contribution of pleiotropy to skeletal evolution. Our results provide a rare portrait of the genetic basis of skeletal evolution in an island population and position the Gough Island mouse as a model system for understanding mechanisms of rapid evolution in nature. Copyright © 2016 by the Genetics Society of America.

  17. Genetic and nutritional deficiencies in folate metabolism influence tumorigenicity in Apcmin/+ mice.

    PubMed

    Lawrance, Andrea K; Deng, Liyuan; Brody, Lawrence C; Finnell, Richard H; Shane, Barry; Rozen, Rima

    2007-05-01

    Epidemiological studies indicate that adequate dietary folate is protective against colon cancer, although mechanisms remain largely elusive. We investigated the effects of genetic disruptions of folate transport and metabolism and of dietary folate deficiency in a mouse model of colon cancer, the Apc(min/+) mouse. Apc(min/+) mice with heterozygous knockout of the gene for reduced folate carrier 1 (Rfc1(+/-)) developed significantly fewer adenomas compared to Rfc1(+/+)Apc(min/+) mice [30.3+/-4.6 vs. 60.4+/-9.4 on a control diet (CD) and 42.6+/-4.4 vs. 55.8+/-7.6 on a folate-deficient diet, respectively]. Rfc1(+/-)Apc(min/+) mice also carried a lower tumor load, an indicator of tumor size as well as of tumor number. In contrast, there were no differences in adenoma formation between Apc(min/+) mice carrying a knockout allele for methionine synthase (Mtr(+/-)), an enzyme that catalyzes folate-dependent homocysteine remethylation, and Mtr(+/+)Apc(min/+) mice. However, in both Mtr groups of mice, dietary folate deficiency significantly increased adenoma number (from 32.3+/-3.8 on a CD to 48.1+/-4.2 on a folate-deficient diet), increased plasma homocysteine, decreased global DNA methylation in preneoplastic intestines and increased apoptosis in tissues. There were no genotype-associated differences in these parameters in the Rfc1 group, suggesting that the protection conferred by Rfc1 deficiency is carried out through a different mechanism. In conclusion, genetic and nutritional disturbances in folate metabolism can have distinct influences on tumorigenesis in Apc(min/+) mice; altered levels of homocysteine, global DNA methylation and apoptosis may contribute mechanistically to dietary influence.

  18. Genetic variation in offspring indirectly influences the quality of maternal behaviour in mice.

    PubMed

    Ashbrook, David George; Gini, Beatrice; Hager, Reinmar

    2015-12-23

    Conflict over parental investment between parent and offspring is predicted to lead to selection on genes expressed in offspring for traits influencing maternal investment, and on parentally expressed genes affecting offspring behaviour. However, the specific genetic variants that indirectly modify maternal or offspring behaviour remain largely unknown. Using a cross-fostered population of mice, we map maternal behaviour in genetically uniform mothers as a function of genetic variation in offspring and identify loci on offspring chromosomes 5 and 7 that modify maternal behaviour. Conversely, we found that genetic variation among mothers influences offspring development, independent of offspring genotype. Offspring solicitation and maternal behaviour show signs of coadaptation as they are negatively correlated between mothers and their biological offspring, which may be linked to costs of increased solicitation on growth found in our study. Overall, our results show levels of parental provisioning and offspring solicitation are unique to specific genotypes.

  19. Genetically modified laboratory mice with sebaceous glands abnormalities.

    PubMed

    Ehrmann, Carmen; Schneider, Marlon R

    2016-12-01

    Sebaceous glands (SG) are exocrine glands that release their product by holocrine secretion, meaning that the whole cell becomes a secretion following disruption of the membrane. SG may be found in association with a hair follicle, forming the pilosebaceous unit, or as modified SG at different body sites such as the eyelids (Meibomian glands) or the preputial glands. Depending on their location, SG fulfill a number of functions, including protection of the skin and fur, thermoregulation, formation of the tear lipid film, and pheromone-based communication. Accordingly, SG abnormalities are associated with several diseases such as acne, cicatricial alopecia, and dry eye disease. An increasing number of genetically modified laboratory mouse lines develop SG abnormalities, and their study may provide important clues regarding the molecular pathways regulating SG development, physiology, and pathology. Here, we summarize in tabulated form the available mouse lines with SG abnormalities and, focusing on selected examples, discuss the insights they provide into SG biology and pathology. We hope this survey will become a helpful information source for researchers with a primary interest in SG but also as for researchers from unrelated fields that are unexpectedly confronted with a SG phenotype in newly generated mouse lines.

  20. Pigmentation, pleiotropy, and genetic pathways in humans and mice

    SciTech Connect

    Barsh, G.S.

    1995-10-01

    Some of the most striking polymorphisms in human populations affect the color of our eyes, hair, or skin. Despite some simple lessons from high school biology (blue eyes are recessive; brown are dominant), the genetic basis of such phenotypic variability has, for the most part, eluded Mendelian description. A logical place to search for the keys to understanding common variation in human pigmentation are genes in which defects cause uncommon conditions such as albinism or piebaldism. The area under this lamppost has recently gotten larger, with two articles, one in this issue of the Journal, that describe the map position for Hermansky-Pudlak syndrome (HPS) and with the recent cloning of a gene that causes X-linked ocular albinism (OA1). In addition, a series of three recent articles in Cell demonstrate (1) that defects in the gene encoding the endothelin B (ET{sub B}) receptor cause hypopigmentation and Hirschsprung disease in a Mennonite population and the mouse mutation piebald(s) and (2) that a defect in the edn3 gene, which encodes one of the ligands for the ET{sub B} receptor, causes the lethal spotting (ls) mouse mutation. 47 refs., 1 fig.

  1. Antidiabetic activity of Lyophyllum decastes in genetically type 2 diabetic mice.

    PubMed

    Miura, Toshihiro; Kubo, Mizue; Itoh, Yasushi; Iwamoto, Naoki; Kato, Motoshi; Park, Sang Rae; Ukawa, Yuuichi; Kita, Yukio; Suzuki, Ikukatsu

    2002-09-01

    The antidiabetic activity of Lyophyllum decastes (Tricholomataceae) was investigated in KK-Ay mice, an animal model of genetically type 2 diabetes with hyperinsulinemia. The water extract of Lyophyllum decastes (LD) (500 mg/kg body weight) reduced the blood glucose of KK-Ay mice 7 h after a single oral administration (p<0.05) when compared with control. LD reduced the blood glucose of KK-Ay mice 3 weeks after repeated administration (p<0.05), and also significantly lowered the serum insulin of KK-Ay mice under similar conditions (p<0.01). However, LD did not affect the blood glucose in normal mice. LD tended to decrease of the blood glucose in an insulin tolerance test. In addition, the muscle content of facilitative glucose transporter isoform 4 (GLUT4) protein content in the plasma membrane fraction from muscle significantly increased in the orally LD-treated KK-Ay mice when compared to that of the controls (p<0.01). These results suggest that the antidiabetic activity of LD is derived, at least in part, from a decrease in insulin resistance, due to the increase of GLUT4 protein content in the plasma membrane of the muscle.

  2. Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity

    NASA Technical Reports Server (NTRS)

    Anterola, Aldwin M.; Lewis, Norman G.

    2002-01-01

    A comprehensive assessment of lignin configuration in transgenic and mutant plants is long overdue. This review thus undertook the systematic analysis of trends manifested through genetic and mutational manipulations of the various steps associated with monolignol biosynthesis; this included consideration of the downstream effects on organized lignin assembly in the various cell types, on vascular function/integrity, and on plant growth and development. As previously noted for dirigent protein (homologs), distinct and sophisticated monolignol forming metabolic networks were operative in various cell types, tissues and organs, and form the cell-specific guaiacyl (G) and guaiacyl-syringyl (G-S) enriched lignin biopolymers, respectively. Regardless of cell type undergoing lignification, carbon allocation to the different monolignol pools is apparently determined by a combination of phenylalanine availability and cinnamate-4-hydroxylase/"p-coumarate-3-hydroxylase" (C4H/C3H) activities, as revealed by transcriptional and metabolic profiling. Downregulation of either phenylalanine ammonia lyase or cinnamate-4-hydroxylase thus predictably results in reduced lignin levels and impaired vascular integrity, as well as affecting related (phenylpropanoid-dependent) metabolism. Depletion of C3H activity also results in reduced lignin deposition, albeit with the latter being derived only from hydroxyphenyl (H) units, due to both the guaiacyl (G) and syringyl (S) pathways being blocked. Apparently the cells affected are unable to compensate for reduced G/S levels by increasing the amounts of H-components. The downstream metabolic networks for G-lignin enriched formation in both angiosperms and gymnosperms utilize specific cinnamoyl CoA O-methyltransferase (CCOMT), 4-coumarate:CoA ligase (4CL), cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) isoforms: however, these steps neither affect carbon allocation nor H/G designations, this being determined by C4H/C3H

  3. Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells.

    PubMed

    Yang, Hui; Shi, Linyu; Wang, Bang-An; Liang, Dan; Zhong, Cuiqing; Liu, Wei; Nie, Yongzhan; Liu, Jie; Zhao, Jing; Gao, Xiang; Li, Dangsheng; Xu, Guo-Liang; Li, Jinsong

    2012-04-27

    Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Serotonin innervation of Lurcher mutant mice: basic data and manipulation with a combination of amantadine, thiamine and L-tryptophan.

    PubMed

    Le Marec, N; Hébert, C; Botez, M I; Botez-Marquard, T; Marchand, L; Reader, T A

    1999-01-15

    The Lurcher (Lc/+) mutant mouse is characterized by a considerable atrophy of the cerebellum due to a massive loss of cerebellar Purkinje and granule cells, as well as of neurons from the inferior olivary nucleus. In this study the effects of a therapeutic combination of amantadine, thiamine and L-tryptophan on the serotonin (5-HT) innervation was assessed in Lurcher mice by autoradiography, using [3H]citalopram to label 5-HT transporters. In wild type mice as well as in both saline-treated and drug-treated Lurcher mutants, [3H]citalopram binding remained unchanged in forebrain and brainstem regions. In the cerebellum, labelling of deep cerebellar nuclei (CBnuc) was about twofold higher than in the cortex (CBctx). In saline-treated Lurcher mutants compared to wild type mice, the densities of [3H]citalopram were 98% higher in CBctx, and 180% higher in CBnuc. In CBctx of drug-treated Lurcher mutants, transporter densities were 89% higher than in the wild type, but did not differ from the saline-treated Lurcher. In the CBnuc of the drug-treated Lurcher mutants, [3H]citalopram binding was 50% higher than in the saline-treated Lurcher group, and 320% higher than in wild type mice. The results show that 5-HT transporters, already upregulated in the CBnuc of Lurcher mutant mice, can be further increased by a pharmacological treatment, possibly altering the availability of 5-HT in some of its target areas.

  5. Genetically engineered insertional mutagenesis in mice to model cancer: Sleeping Beauty.

    PubMed

    Howell, Viive M; Colvin, Emily K

    2014-01-01

    The ability to accurately model human cancer in mice enables in vivo examination of the biological mechanisms related to cancer initiation and progression as well as preclinical testing of new anticancer treatments and potential targets. The emergence of the genetically engineered Sleeping Beauty system of insertional mutagenesis has led to the development of a new generation of genetic mouse models of cancer and identification of novel cancer-causing genes. This chapter reviews the published cancer models of Sleeping Beauty and strategies using available strains to generate several models of cancer.

  6. Antiatherogenic role of high-density lipoproteins: insights from genetically engineered-mice.

    PubMed

    Escola-Gil, Joan Carles; Calpe-Berdiel, Laura; Palomer, Xavier; Ribas, Vicent; Ordonez-Llanos, Jordi; Blanco-Vaca, Francisco

    2006-05-01

    Plasma levels of high-density lipoprotein (HDL) cholesterol are inversely correlated with the incidence of atherosclerotic cardiovascular disease. The cardioprotective effects of HDL have been attributed to its role in reverse cholesterol transport (RCT) and especially the macrophage-dependent RCT, and also to the antioxidant properties of HDL as well as its direct effects on endothelial function. However, few of these effects have been verified in vivo in humans. With the creation and detailed analysis of genetically-engineered mice, a solid body of new information has emerged on the mechanisms controlling these key antiatherogenic functions of HDL and their effects on atherogenesis. This article provides a review of new insights into the molecular mechanisms underlying these three most studied antiatherogenic functions of HDL in vivo with a focus on genetically-engineered mice.

  7. Genetic susceptibility to interstitial pulmonary fibrosis in mice induced by vanadium pentoxide (V2O5)

    PubMed Central

    Walters, Dianne M.; White, Kevin M.; Patel, Ushma; Davis, Martin J.; Veluci-Marlow, Roberta M.; Bhupanapadu Sunkesula, Solomon Raju; Bonner, James C.; Martin, Jessica R.; Gladwell, Wes; Kleeberger, Steven R.

    2014-01-01

    Interstitial lung diseases (ILDs) are characterized by injury, inflammation, and scarring of alveoli, leading to impaired function. The etiology of idiopathic forms of ILD is not understood, making them particularly difficult to study due to the lack of appropriate animal models. Consequently, few effective therapies have emerged. We developed an inbred mouse model of ILD using vanadium pentoxide (V2O5), the most common form of a transition metal found in cigarette smoke, fuel ash, mineral ores, and steel alloys. Pulmonary responses to V2O5, including dose-dependent increases in lung permeability, inflammation, collagen content, and dysfunction, were significantly greater in DBA/2J mice compared to C57BL/6J mice. Inflammatory and fibrotic responses persisted for 4 mo in DBA/2J mice, while limited responses in C57BL/6J mice resolved. We investigated the genetic basis for differential responses through genetic mapping of V2O5-induced lung collagen content in BXD recombinant inbred (RI) strains and identified significant linkage on chromosome 4 with candidate genes that associate with V2O5-induced collagen content across the RI strains. Results suggest that V2O5 may induce pulmonary fibrosis through mechanisms distinct from those in other models of pulmonary fibrosis. These findings should further advance our understanding of mechanisms involved in ILD and thereby aid in identification of new therapeutic targets.—Walters, D. M., White, K. M., Patel, U., Davis, M. J., Veluci-Marlow, R. M., Bhupanapadu Sunkesula, S. R., Bonner, J. C., Martin, J. R., Gladwell, W., Kleeberger, S. R. Genetic susceptibility to interstitial pulmonary fibrosis in mice induced by vanadium pentoxide (V2O5). PMID:24285090

  8. Genetic dissection of horizontal cell inhibitory signaling in mice in complete darkness in vivo.

    PubMed

    Berkowitz, Bruce A; Murphy, Geoffrey G; Craft, Cheryl Mae; Surmeier, D James; Roberts, Robin

    2015-05-01

    To test the hypothesis that horizontal cell (HC) inhibitory signaling controls the degree to which rod cell membranes are depolarized as measured by the extent to which L-type calcium channels (LTCCs) are open in complete darkness in the mouse retina in vivo. Dark-adapted wild-type (wt), CACNA1F (Ca(v)1.4(-/-)), arrestin-1 (Arr1(-/-)), and CACNA1D (Ca(v)1.3(-/-)) C57Bl/6 mice were studied. Manganese-enhanced MRI (MEMRI) evaluated the extent that rod LTCCs are open as an index of loss of HC inhibitory signaling. Subgroups were pretreated with D-cis-diltiazem (DIL) at a dose that specifically antagonizes Ca(v)1.2 channels in vivo. Knockout mice predicted to have impaired HC inhibitory signaling (Ca(v)1.4(-/-) or Arr1(-/-)) exhibited greater than normal rod manganese uptake; inner retinal uptake was also supernormal. Genetically knocking out a closely associated gene not expected to impact HC inhibitory signaling (CACNA1D) did not generate this phenotype. The Arr1(-/-) mice exhibited the largest rod uptake of manganese. Manganese-enhanced MRI of DIL-treated Arr1(-/-) mice suggested a greater number of operant LTCC subtypes (i.e., Ca(v)1.2, 1.3, and 1.4) in rods and inner retina than that in DIL-treated Ca(v)1.4(-/-) mice (i.e., Ca(v)1.3). The Ca(v)1.3(-/-) + DIL-treated mice exhibited evidence for a compensatory contribution from Ca(v)1.2 LTCCs. The data suggest that loss of HC inhibitory signaling is the proximate cause leading to maximally open LTCCs in rods, and possibly inner retinal cells, in mice in total darkness in vivo, regardless of compensatory changes in LTCC subtype manifested in the mutant mice.

  9. Changes in metabolite profiles caused by genetically determined obesity in mice.

    PubMed

    Schäfer, Nadine; Yu, Zhonghao; Wagener, Asja; Millrose, Marion K; Reissmann, Monika; Bortfeldt, Ralf; Dieterich, Christoph; Adamski, Jerzy; Wang-Sattler, Rui; Illig, Thomas; Brockmann, Gudrun A

    2014-01-01

    The Berlin Fat Mouse Inbred (BFMI) line harbors a major recessive gene defect on chromosome 3 (jobes1) leading to juvenile obesity and metabolic syndrome. The present study aimed at the identification of metabolites that might be linked to recessively acting genes in the obesity locus. Firstly, serum metabolites were analyzed between obese BFMI and lean B6 and BFMI × B6 F1 mice to identify metabolites that are different. In a second step, a metabolite-protein network analysis was performed linking metabolites typical for BFMI mice with genes of the jobes1 region. The levels of 22 diacyl-phosphatidylcholines (PC aa), two lyso-PC and three carnitines were found to be significantly lower in obese mice compared with lean mice, while serine, glycine, arginine and hydroxysphingomyelin were higher for the same comparison. The network analysis identified PC aa C42:1 as functionally linked with the genes Ccna2 and Trpc3 via the enzymes choline kinase alpha and phospholipase A2 group 1B (PLA2G1B), respectively. Gene expression analysis revealed elevated Ccna2 expression in adipose tissue of BFMI mice. Furthermore, unique mutations were found in the Ccna2 promoter of BFMI mice which are located in binding sites for transcription factors or micro RNAs and could cause differential Ccna2 mRNA levels between BFMI and B6 mice. Increased expression of Ccna2 was consistent with higher mitotic activity of adipose tissue in BFMI mice. Therefore, we suggest a higher demand for PC necessary for adipose tissue growth and remodeling. This study highlights the relationship between metabolite profiles and the underlying genetics of obesity in the BFMI line.

  10. The genetic basis for susceptibility to Rift Valley fever disease in MBT/Pas mice.

    PubMed

    Tokuda, S; Do Valle, T Z; Batista, L; Simon-Chazottes, D; Guillemot, L; Bouloy, M; Flamand, M; Montagutelli, X; Panthier, J-J

    2015-01-01

    The large variation in individual response to infection with Rift Valley fever virus (RVFV) suggests that host genetic determinants play a role in determining virus-induced disease outcomes. These genetic factors are still unknown. The systemic inoculation of mice with RVFV reproduces major pathological features of severe human disease, notably the hepatitis and encephalitis. A genome scan performed on 546 (BALB/c × MBT) F2 progeny identified three quantitative trait loci (QTLs), denoted Rvfs-1 to Rvfs-3, that were associated with disease susceptibility in MBT/Pas mice. Non-parametric interval-mapping revealed one significant and two suggestive linkages with survival time on chromosomes 2 (Rvfs-1), 5 (Rvfs-3) and 11 (Rvfs-2) with respective logarithm of odds (LOD) scores of 4.58, 2.95 and 2.99. The two-part model, combining survival time and survival/death, identified one significant linkage to Rvfs-2 and one suggestive linkage to Rvfs-1 with respective LOD scores of 5.12 and 4.55. Under a multiple model, with additive effects and sex as a covariate, the three QTLs explained 8.3% of the phenotypic variance. Sex had the strongest influence on susceptibility. The contribution of Rvfs-1, Rvfs-2 and Rvfs-3 to survival time of RVFV-infected mice was further confirmed in congenic mice.

  11. Congenic mice reveal genetic epistasis and overlapping disease loci for autoimmune diabetes and listeriosis.

    PubMed

    Wang, Nancy; Elso, Colleen M; Mackin, Leanne; Mannering, Stuart I; Strugnell, Richard A; Wijburg, Odilia L; Brodnicki, Thomas C

    2014-08-01

    The nonobese diabetic (NOD) mouse strain serves as a genomic standard for assessing how allelic variation for insulin-dependent diabetes (Idd) loci affects the development of autoimmune diabetes. We previously demonstrated that C57BL/6 (B6) mice harbor a more diabetogenic allele than NOD mice for the Idd14 locus when introduced onto the NOD genetic background. New congenic NOD mouse strains, harboring smaller B6-derived intervals on chromosome 13, now localize Idd14 to an ~18-Mb interval and reveal a new locus, Idd31. Notably, the B6 allele for Idd31 confers protection against diabetes, but only in the absence of the diabetogenic B6 allele for Idd14, indicating genetic epistasis between these two loci. Moreover, congenic mice that are more susceptible to diabetes are more resistant to Listeria monocytogenes infection. This result co-localizes Idd14 and Listr2, a resistance locus for listeriosis, to the same genomic interval and indicates that congenic NOD mice may also be useful for localizing resistance loci for infectious disease.

  12. Genetic parameters for canalisation analysis of litter size and litter weight traits at birth in mice

    PubMed Central

    Gutiérrez, Juan Pablo; Nieto, Blanca; Piqueras, Pepa; Ibáñez, Noelia; Salgado, Concepción

    2006-01-01

    The aim of this research was to explore the genetic parameters associated with environmental variability for litter size (LS), litter weight (LW) and mean individual birth weight (IW) in mice before canalisation. The analyses were conducted on an experimental mice population designed to reduce environmental variability for LS. The analysed database included 1976 records for LW and IW and 4129 records for LS. The total number of individuals included in the analysed pedigree was 3997. Heritabilities estimated for the traits under an initial exploratory approach varied from 0.099 to 0.101 for LS, from 0.112 to 0.148 for LW and from 0.028 to 0.033 for IW. The means of the posterior distribution of the heritability under a Bayesian approach were the following: 0.10 (LS), 0.13 (LW) and 0.03 (IW). In general, the heritabilities estimated under the initial exploratory approach for the environmental variability of the analysed traits were low. Genetic correlations estimated between the trait and its variability reached values of -0.929 (LS), -0.815 (LW) and 0.969 (IW). The results presented here for the first time in mice may suggest a genetic basis for variability of the evaluated traits, thus opening the possibility to be implemented in selection schemes. PMID:16954039

  13. Trichinella spiralis infection in mice. Mechanism of the resistance in animals genetically selected for high and low antibody production.

    PubMed Central

    Perrudet-Badoux, A; Binaghi, R A; Boussac-Aron, Y

    1978-01-01

    Mice genetically selected according to their capacity to produce antibody were orally infected with fifty muscle larvae. After 1 month, the number of larvae found in low responder mice was twice the number found in high responder mice. Following a second infection, low responder mice were completely protected while high responder mice showed only partial protection. It is suggested that the better resistance of high responder mice after a primary infection is due to their high and rapid antibody production. However, at the time of a secondary infection both lines of mice possess enough antibody to act on the effector cells (macrophages, eosinophils, etc.) and resistance is then dependent on the metabolic activity of these cells, which is more intense in the low responder mice. PMID:700780

  14. An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice

    PubMed Central

    Kramer, Edgar R.

    2015-01-01

    Background & Aims The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson’s disease (PD) and drug addiction. Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification. Methods & Results Here, we have generated transgenic mice expressing the tetracycline-regulated transactivator (tTA) or the reverse tetracycline-regulated transactivator (rtTA) under control of the tyrosine hydroxylase (TH) promoter, TH-tTA (tet-OFF) and TH-rtTA (tet-ON) mice, to visualize and genetically modify DA neurons. We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre. In combination with mice encoding tet-responsive luciferase, we visualized the DA system in living mice progressively over time. Conclusion These experiments establish TH-tTA and TH-rtTA mice as a powerful tool to generate and monitor mouse models for DA system diseases. PMID:26291828

  15. Relationships between protein and mineral during enamel development in normal and genetically altered mice

    PubMed Central

    Smith, Charles E.; Hu, Yuanyuan; Richardson, Amelia S.; Bartlett, John D.; Hu, Jan C-C.; Simmer, James P.

    2012-01-01

    The purpose of this study was to quantify and compare the amounts of volatiles (mostly protein) and mineral present in developing incisor enamel in normal mice and in those genetically engineered for absence of intact enamelin, ameloblastin, matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4). Data indicated that all mice showed peaks in the gross weight of volatiles and a similar weight of mineral at locations on incisors normally associated with early maturation. Thereafter, the content of volatiles on normal incisors declined rapidly by as much as 62%, but not by 100%, over 2 mm, accompanied by increases of ~threefold in mineral weights. Enamelin heterozygous mice (lower incisors) showed a decrease in volatile content across the maturation stage, yet mineral failed to increase significantly. Mmp20 null mice showed no significant loss of volatiles from maturing enamel, yet the amount of mineral increased. Klk4 null mice showed normal mineral acquisition up to early maturation, but the input of new volatiles in mid to late maturation caused the final mineralization to slow below normal levels. These results suggest that it is not only the amount of protein but also the nature or type of protein or fragments present in the local crystallite environment that affects their volumetric expansion as they mature. PMID:22243238

  16. Genetic functions of the NAIP family of inflammasome receptors for bacterial ligands in mice

    PubMed Central

    Zhao, Yue; Shi, Jianjin; Shi, Xuyan; Wang, Yupeng; Wang, Fengchao

    2016-01-01

    Biochemical studies suggest that the NAIP family of NLR proteins are cytosolic innate receptors that directly recognize bacterial ligands and trigger NLRC4 inflammasome activation. In this study, we generated Naip5−/−, Naip1−/−, and Naip2−/− mice and showed that bone marrow macrophages derived from these knockout mice are specifically deficient in detecting bacterial flagellin, the type III secretion system needle, and the rod protein, respectively. Naip1−/−, Naip2−/−, and Naip5−/− mice also resist lethal inflammasome activation by the corresponding ligand. Furthermore, infections performed in the Naip-deficient macrophages have helped to define the major signal in Legionella pneumophila, Salmonella Typhimurium and Shigella flexneri that is detected by the NAIP/NLRC4 inflammasome. Using an engineered S. Typhimurium infection model, we demonstrate the critical role of NAIPs in clearing bacterial infection and protecting mice from bacterial virulence–induced lethality. These results provide definitive genetic evidence for the important physiological function of NAIPs in antibacterial defense and inflammatory damage–induced lethality in mice. PMID:27114610

  17. Genetic architecture of nest building in mice LG/J × SM/J.

    PubMed

    Sauce, Bruno; de Brito, Reinaldo Alves; Peripato, Andrea Cristina

    2012-01-01

    Maternal care is critical to offspring growth and survival, which is greatly improved by building an effective nest. Some suggest that genetic variation and underlying genetic effects differ between fitness-related traits and other phenotypes. We investigated the genetic architecture of a fitness-related trait, nest building, in F(2) female mice intercrossed from inbred strains SM/J and LG/J using a QTL analysis for six related nest phenotypes (Presence and Structure pre- and postpartum, prepartum Material Used and postpartum Temperature). We found 15 direct-effect QTLs explaining from 4 to 13% of the phenotypic variation in nest building, mostly with non-additive effect. Epistatic analyses revealed 71 significant epistatic interactions which together explain from 28.4 to 75.5% of the variation, indicating an important role for epistasis in the adaptive process of nest building behavior in mice. Our results suggest a genetic architecture with small direct effects and a larger number of epistatic interactions as expected for fitness-related phenotypes.

  18. Magneto-thermal genetic deep brain stimulation of motor behaviors in awake, freely moving mice.

    PubMed

    Munshi, Rahul; Qadri, Shahnaz M; Zhang, Qian; Castellanos Rubio, Idoia; Del Pino, Pablo; Pralle, Arnd

    2017-08-15

    Establishing how neurocircuit activation causes particular behaviors requires modulating the activity of specific neurons. Here, we demonstrate that magnetothermal genetic stimulation provides tetherless deep brain activation sufficient to evoke motor behavior in awake mice. The approach uses alternating magnetic fields to heat superparamagnetic nanoparticles on the neuronal membrane. Neurons heat-sensitized by expressing TRPV1 are activated with magnetic field application. Magnetothermal genetic stimulation in the motor cortex evoked ambulation, deep brain stimulation in the striatum caused rotation around the body-axis, and stimulation near the ridge between ventral and dorsal striatum caused freezing-of-gait. The duration of the behavior correlated tightly with field application. This approach provides genetically and spatially targetable, repeatable and temporarily precise activation of deep-brain circuits without need for surgical implantation of any device.

  19. The genetic basis and fitness consequences of sperm midpiece size in deer mice

    PubMed Central

    Fisher, Heidi S.; Jacobs-Palmer, Emily; Lassance, Jean-Marc; Hoekstra, Hopi E.

    2016-01-01

    An extensive array of reproductive traits varies among species, yet the genetic mechanisms that enable divergence, often over short evolutionary timescales, remain elusive. Here we examine two sister-species of Peromyscus mice with divergent mating systems. We find that the promiscuous species produces sperm with longer midpiece than the monogamous species, and midpiece size correlates positively with competitive ability and swimming performance. Using forward genetics, we identify a gene associated with midpiece length: Prkar1a, which encodes the R1α regulatory subunit of PKA. R1α localizes to midpiece in Peromyscus and is differentially expressed in mature sperm of the two species yet is similarly abundant in the testis. We also show that genetic variation at this locus accurately predicts male reproductive success. Our findings suggest that rapid evolution of reproductive traits can occur through cell type-specific changes to ubiquitously expressed genes and have an important effect on fitness. PMID:27910854

  20. Manipulation of Ovarian Function Significantly Influenced Trabecular and Cortical Bone Volume, Architecture and Density in Mice at Death

    PubMed Central

    Mason, Jeffrey B.; Terry, Boston C.; Merchant, Samer S.; Mason, Holly M.; Nazokkarmaher, Mahdi

    2015-01-01

    Previously, transplantation of ovaries from young, cycling mice into old, postreproductive-age mice increased life span and decreased cardiomyopathy at death. We anticipated that the same factors that increased life span and decreased cardiomyopathy could also influence the progression of orthopedic disease. At 11 months of age, prepubertally ovariectomized and ovary-intact mice (including reproductively cycling and acyclic mice) received new 60-day-old ovaries. At death, epiphyseal bone in the proximal tibia and the distal femur and mid-shaft tibial and femoral diaphyseal bone was analyzed with micro-computed tomography. For qualitative analysis of osteophytosis, we also included mineralized connective tissue within the stifle joint. Prepubertal ovariectomy had the greatest influence on bone volume, ovarian transplantation had the greatest influence on bone architecture and both treatments influenced bone density. Ovarian transplantation increased cortical, but not trabecular bone density and tended to increase osteophytosis and heterotopic mineralization, except in acyclic recipients. These effects may have been dictated by the timing of the treatments, with ovariectomy appearing to influence early development and ovarian transplantation limited to influencing only the postreproductive period. However, major differences observed between cycling, acyclic and ovariectomized recipients of new ovaries may have been, in part due to differences in the levels of hormone receptors present and the responsiveness of specific bone processes to hormone signaling. Changes that resulted from these treatments may represent a compensatory response to normal age-associated, negative, orthopedic changes. Alternatively, differences between treatments may simply be the 'preservation' of unblemished orthopedic conditions, prior to the influence of negative, age-associated effects. These findings may suggest that in women, tailoring hormone replacement therapy to the patient's current

  1. Genetic inducible fate mapping in adult mice using tamoxifen-dependent Cre recombinases.

    PubMed

    Feil, Susanne; Krauss, Jana; Thunemann, Martin; Feil, Robert

    2014-01-01

    The Cre/lox site-specific recombination system allows the control of gene activity in space and time in almost any tissue of the mouse. A major technical advance was the development of tamoxifen-dependent Cre recombinases, such as CreER(T2), that can be activated by administration of tamoxifen to the animal. This powerful tool greatly facilitates the study of gene functions and the generation of more realistic animal models of sporadic human diseases. Another important application of tamoxifen-dependent Cre recombinases is genetic inducible fate mapping (GIFM). In GIFM studies, the inducible Cre/lox system is used to genetically label a defined cell population at a selected time by irreversible activation of the expression of a Cre-responsive reporter transgene. Then, marked cells are detected at later time points to determine how the originally labeled progenitors contribute to specific structures and cell types during pre- and postnatal development. GIFM was initially applied during mouse embryogenesis, but is now increasingly used for cell lineage tracing in adult mice under physiological and pathophysiological conditions. Here we describe the design of GIFM experiments in adult mice as exemplified by CreER(T2)-assisted tracing of vascular smooth muscle cells during the development of atherosclerotic lesions. First, we give an overview of reporter transgenes available for genetic cell marking that are expressed from the Rosa26 locus, such as β-galactosidase and fluorescent proteins. Then we present detailed protocols for the generation of experimental mice for GIFM studies, the induction of cell labeling by tamoxifen treatment, and the detection of marked cells in fixed and live tissues. Each section also provides a discussion of limitations and common pitfalls of GIFM experiments. Most of the protocols can be easily adapted to other developmental stages, cell types, Cre recombinases, and reporter transgenes and, thus, can be used as general guidelines for GIFM

  2. Genetic diminution of circulating prothrombin ameliorates multiorgan pathologies in sickle cell disease mice.

    PubMed

    Arumugam, Paritha I; Mullins, Eric S; Shanmukhappa, Shiva Kumar; Monia, Brett P; Loberg, Anastacia; Shaw, Maureen A; Rizvi, Tilat; Wansapura, Janaka; Degen, Jay L; Malik, Punam

    2015-10-08

    Sickle cell disease (SCD) results in vascular occlusions, chronic hemolytic anemia, and cumulative organ damage. A conspicuous feature of SCD is chronic inflammation and coagulation system activation. Thrombin (factor IIa [FIIa]) is both a central protease in hemostasis and a key modifier of inflammatory processes. To explore the hypothesis that reduced prothrombin (factor II [FII]) levels in SCD will limit vaso-occlusion, vasculopathy, and inflammation, we used 2 strategies to suppress FII in SCD mice. Weekly administration of FII antisense oligonucleotide "gapmer" to Berkeley SCD mice to selectively reduce circulating FII levels to ∼10% of normal for 15 weeks significantly diminished early mortality. More comprehensive, long-term comparative studies were done using mice with genetic diminution of circulating FII. Here, cohorts of FII(lox/-) mice (constitutively carrying ∼10% normal FII) and FII(WT) mice were tracked in parallel for a year following the imposition of SCD via hematopoietic stem cell transplantation. This genetically imposed suppression of FII levels resulted in an impressive reduction in inflammation (reduction in leukocytosis, thrombocytosis, and circulating interleukin-6 levels), reduced endothelial cell dysfunction (reduced endothelial activation and circulating soluble vascular cell adhesion molecule), and a significant improvement in SCD-associated end-organ damage (nephropathy, pulmonary hypertension, pulmonary inflammation, liver function, inflammatory infiltration, and microinfarctions). Notably, all of these benefits were achieved with a relatively modest 1.25-fold increase in prothrombin times, and in the absence of hemorrhagic complications. Taken together, these data establish that prothrombin is a powerful modifier of SCD-induced end-organ damage, and present a novel therapeutic target to ameliorate SCD pathologies.

  3. Genetic biomarkers for ALS disease in transgenic SOD1(G93A) mice.

    PubMed

    Calvo, Ana C; Manzano, Raquel; Atencia-Cibreiro, Gabriela; Oliván, Sara; Muñoz, María J; Zaragoza, Pilar; Cordero-Vázquez, Pilar; Esteban-Pérez, Jesús; García-Redondo, Alberto; Osta, Rosario

    2012-01-01

    The pathophysiological mechanisms of both familial and sporadic Amyotrophic Lateral Sclerosis (ALS) are unknown, although growing evidence suggests that skeletal muscle tissue is a primary target of ALS toxicity. Skeletal muscle biopsies were performed on transgenic SOD1(G93A) mice, a mouse model of ALS, to determine genetic biomarkers of disease longevity. Mice were anesthetized with isoflurane, and three biopsy samples were obtained per animal at the three main stages of the disease. Transcriptional expression levels of seventeen genes, Ankrd1, Calm1, Col19a1, Fbxo32, Gsr, Impa1, Mef2c, Mt2, Myf5, Myod1, Myog, Nnt, Nogo A, Pax7, Rrad, Sln and Snx10, were tested in each muscle biopsy sample. Total RNA was extracted using TRIzol Reagent according to the manufacturer's protocol, and variations in gene expression were assayed by real-time PCR for all of the samples. The Pearson correlation coefficient was used to determine the linear correlation between transcriptional expression levels throughout disease progression and longevity. Consistent with the results obtained from total skeletal muscle of transgenic SOD1(G93A) mice and 74-day-old denervated mice, five genes (Mef2c, Gsr, Col19a1, Calm1 and Snx10) could be considered potential genetic biomarkers of longevity in transgenic SOD1(G93A) mice. These results are important because they may lead to the exploration of previously unexamined tissues in the search for new disease biomarkers and even to the application of these findings in human studies.

  4. Genetic Biomarkers for ALS Disease in Transgenic SOD1G93A Mice

    PubMed Central

    Calvo, Ana C.; Manzano, Raquel; Atencia-Cibreiro, Gabriela; Oliván, Sara; Muñoz, María J.; Zaragoza, Pilar; Cordero-Vázquez, Pilar; Esteban-Pérez, Jesús; García-Redondo, Alberto; Osta, Rosario

    2012-01-01

    The pathophysiological mechanisms of both familial and sporadic Amyotrophic Lateral Sclerosis (ALS) are unknown, although growing evidence suggests that skeletal muscle tissue is a primary target of ALS toxicity. Skeletal muscle biopsies were performed on transgenic SOD1G93A mice, a mouse model of ALS, to determine genetic biomarkers of disease longevity. Mice were anesthetized with isoflurane, and three biopsy samples were obtained per animal at the three main stages of the disease. Transcriptional expression levels of seventeen genes, Ankrd1, Calm1, Col19a1, Fbxo32, Gsr, Impa1, Mef2c, Mt2, Myf5, Myod1, Myog, Nnt, Nogo A, Pax7, Rrad, Sln and Snx10, were tested in each muscle biopsy sample. Total RNA was extracted using TRIzol Reagent according to the manufacturer's protocol, and variations in gene expression were assayed by real-time PCR for all of the samples. The Pearson correlation coefficient was used to determine the linear correlation between transcriptional expression levels throughout disease progression and longevity. Consistent with the results obtained from total skeletal muscle of transgenic SOD1G93A mice and 74-day-old denervated mice, five genes (Mef2c, Gsr, Col19a1, Calm1 and Snx10) could be considered potential genetic biomarkers of longevity in transgenic SOD1G93A mice. These results are important because they may lead to the exploration of previously unexamined tissues in the search for new disease biomarkers and even to the application of these findings in human studies. PMID:22412900

  5. Genetic diminution of circulating prothrombin ameliorates multiorgan pathologies in sickle cell disease mice

    PubMed Central

    Arumugam, Paritha I.; Mullins, Eric S.; Shanmukhappa, Shiva Kumar; Monia, Brett P.; Loberg, Anastacia; Shaw, Maureen A.; Rizvi, Tilat; Wansapura, Janaka; Degen, Jay L.

    2015-01-01

    Sickle cell disease (SCD) results in vascular occlusions, chronic hemolytic anemia, and cumulative organ damage. A conspicuous feature of SCD is chronic inflammation and coagulation system activation. Thrombin (factor IIa [FIIa]) is both a central protease in hemostasis and a key modifier of inflammatory processes. To explore the hypothesis that reduced prothrombin (factor II [FII]) levels in SCD will limit vaso-occlusion, vasculopathy, and inflammation, we used 2 strategies to suppress FII in SCD mice. Weekly administration of FII antisense oligonucleotide “gapmer” to Berkeley SCD mice to selectively reduce circulating FII levels to ∼10% of normal for 15 weeks significantly diminished early mortality. More comprehensive, long-term comparative studies were done using mice with genetic diminution of circulating FII. Here, cohorts of FIIlox/− mice (constitutively carrying ∼10% normal FII) and FIIWT mice were tracked in parallel for a year following the imposition of SCD via hematopoietic stem cell transplantation. This genetically imposed suppression of FII levels resulted in an impressive reduction in inflammation (reduction in leukocytosis, thrombocytosis, and circulating interleukin-6 levels), reduced endothelial cell dysfunction (reduced endothelial activation and circulating soluble vascular cell adhesion molecule), and a significant improvement in SCD-associated end-organ damage (nephropathy, pulmonary hypertension, pulmonary inflammation, liver function, inflammatory infiltration, and microinfarctions). Notably, all of these benefits were achieved with a relatively modest 1.25-fold increase in prothrombin times, and in the absence of hemorrhagic complications. Taken together, these data establish that prothrombin is a powerful modifier of SCD-induced end-organ damage, and present a novel therapeutic target to ameliorate SCD pathologies. PMID:26286849

  6. Genetic analysis of intestinal polyp development in Collaborative Cross mice carrying the Apc (Min/+) mutation.

    PubMed

    Dorman, Alexandra; Baer, Daria; Tomlinson, Ian; Mott, Richard; Iraqi, Fuad A

    2016-02-19

    Colorectal cancer is an abnormal tissue development in the colon or rectum. Most of CRCs develop due to somatic mutations, while only a small proportion is caused by inherited mutations. Familial adenomatous polyposis is an inherited genetic disease, which is characterized by colorectal polyps. It is caused by inactivating mutations in the Adenomatous polyposis coli gene. Mice carrying and non-sense mutation in Adenomatous polyposis coli gene at site R850, which designated Apc (R850X/+) (Min), develop intestinal adenomas, while the bulk of the disease is in the small intestine. A number of genetic modifier loci of Min have been mapped, but so far most of the underlying genes have not been identified. In our previous studies, we have shown that Collaborative Cross mice are a powerful tool for mapping loci responsible for phenotypic variation. As a first step towards identification of novel modifiers of Min, we assessed the phenotypic variation between 27 F1 crosses between different Collaborative cross mice and C57BL/6-Min lines. Here, C57BL/6-Min male mice were mated with females from 27 Collaborative cross lines. F1 offspring were terminated at 23 weeks old and multiple phenotypes were collected: polyp counts, intestine length, intestine weight, packed cell volume and spleen weight. Additionally, in eight selected F1 Collaborative cross-C57BL/6-Min lines, body weight was monitored and compared to control mice carry wildtype Adenomatous polyposis coli gene. We found significant (p < 0.05) phenotypic variation between the 27 F1 Collaborative cross-C57BL/6-Min lines for all the tested phenotypes, and sex differences with traits; Colon, body weight and intestine length phenotypes, only. Heritability calculation showed that these phenotypes are mainly controlled by genetic factors. Variation in polyp development is controlled, an appreciable extent, by genetic factors segregating in the Collaborative cross population and suggests that it is suited for identifying

  7. Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity

    PubMed Central

    Gatti, Daniel M.; Morgan, Daniel L.; Kissling, Grace E.; Shockley, Keith R.; Knudsen, Gabriel A.; Shepard, Kim G.; Price, Herman C.; King, Deborah; Witt, Kristine L.; Pedersen, Lars C.; Munger, Steven C.; Svenson, Karen L.; Churchill, Gary A.

    2014-01-01

    Background Inhalation of benzene at levels below the current exposure limit values leads to hematotoxicity in occupationally exposed workers. Objective We sought to evaluate Diversity Outbred (DO) mice as a tool for exposure threshold assessment and to identify genetic factors that influence benzene-induced genotoxicity. Methods We exposed male DO mice to benzene (0, 1, 10, or 100 ppm; 75 mice/exposure group) via inhalation for 28 days (6 hr/day for 5 days/week). The study was repeated using two independent cohorts of 300 animals each. We measured micronuclei frequency in reticulocytes from peripheral blood and bone marrow and applied benchmark concentration modeling to estimate exposure thresholds. We genotyped the mice and performed linkage analysis. Results We observed a dose-dependent increase in benzene-induced chromosomal damage and estimated a benchmark concentration limit of 0.205 ppm benzene using DO mice. This estimate is an order of magnitude below the value estimated using B6C3F1 mice. We identified a locus on Chr 10 (31.87 Mb) that contained a pair of overexpressed sulfotransferases that were inversely correlated with genotoxicity. Conclusions The genetically diverse DO mice provided a reproducible response to benzene exposure. The DO mice display interindividual variation in toxicity response and, as such, may more accurately reflect the range of response that is observed in human populations. Studies using DO mice can localize genetic associations with high precision. The identification of sulfotransferases as candidate genes suggests that DO mice may provide additional insight into benzene-induced genotoxicity. Citation French JE, Gatti DM, Morgan DL, Kissling GE, Shockley KR, Knudsen GA, Shepard KG, Price HC, King D, Witt KL, Pedersen LC, Munger SC, Svenson KL, Churchill GA. 2015. Diversity Outbred mice identify population-based exposure thresholds and genetic factors that influence benzene-induced genotoxicity. Environ Health Perspect 123:237

  8. Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice.

    PubMed

    Sellers, R S; Clifford, C B; Treuting, P M; Brayton, C

    2012-01-01

    Inbred laboratory mouse strains are highly divergent in their immune response patterns as a result of genetic mutations and polymorphisms. The generation of genetically engineered mice (GEM) has, in the past, used embryonic stem (ES) cells for gene targeting from various 129 substrains followed by backcrossing into more fecund mouse strains. Although common inbred mice are considered "immune competent," many have variations in their immune system-some of which have been described-that may affect the phenotype. Recognition of these immune variations among commonly used inbred mouse strains is essential for the accurate interpretation of expected phenotypes or those that may arise unexpectedly. In GEM developed to study specific components of the immune system, accurate evaluation of immune responses must take into consideration not only the gene of interest but also how the background strain and microbial milieu contribute to the manifestation of findings in these mice. This article discusses points to consider regarding immunological differences between the common inbred laboratory mouse strains, particularly in their use as background strains in GEM.

  9. Genetic selection for resistance or susceptibility to oral tolerance to ovalbumin affects general mechanisms of tolerance induction in mice.

    PubMed

    Kamphorst, Alice O; da Silva, Maria F S; da Silva, Antônio C; Carvalho, Claudia R; Faria, Ana Maria C

    2004-12-01

    To study the genes involved in oral tolerance susceptibility, two strains of mice were genetically selected for susceptibility (TS) and resistance (TR) to oral tolerance to ovalbumin by bidirectional breeding. Herein we show that the genetic selection process is restricted neither to ovalbumin nor to oral tolerance. It affected oral tolerance to other proteins, such as casein, and tolerance induced the intravenous route.

  10. Genetic interactions between planar cell polarity genes cause diverse neural tube defects in mice

    PubMed Central

    Murdoch, Jennifer N.; Damrau, Christine; Paudyal, Anju; Bogani, Debora; Wells, Sara; Greene, Nicholas D. E.; Stanier, Philip; Copp, Andrew J.

    2014-01-01

    Neural tube defects (NTDs) are among the commonest and most severe forms of developmental defect, characterized by disruption of the early embryonic events of central nervous system formation. NTDs have long been known to exhibit a strong genetic dependence, yet the identity of the genetic determinants remains largely undiscovered. Initiation of neural tube closure is disrupted in mice homozygous for mutations in planar cell polarity (PCP) pathway genes, providing a strong link between NTDs and PCP signaling. Recently, missense gene variants have been identified in PCP genes in humans with NTDs, although the range of phenotypes is greater than in the mouse mutants. In addition, the sequence variants detected in affected humans are heterozygous, and can often be detected in unaffected individuals. It has been suggested that interactions between multiple heterozygous gene mutations cause the NTDs in humans. To determine the phenotypes produced in double heterozygotes, we bred mice with all three pairwise combinations of Vangl2Lp, ScribCrc and Celsr1Crsh mutations, the most intensively studied PCP mutants. The majority of double-mutant embryos had open NTDs, with the range of phenotypes including anencephaly and spina bifida, therefore reflecting the defects observed in humans. Strikingly, even on a uniform genetic background, variability in the penetrance and severity of the mutant phenotypes was observed between the different double-heterozygote combinations. Phenotypically, Celsr1Crsh;Vangl2Lp;ScribCrc triply heterozygous mutants were no more severe than doubly heterozygous or singly homozygous mutants. We propose that some of the variation between double-mutant phenotypes could be attributed to the nature of the protein disruption in each allele: whereas ScribCrc is a null mutant and produces no Scrib protein, Celsr1Crsh and Vangl2Lp homozygotes both express mutant proteins, consistent with dominant effects. The variable outcomes of these genetic interactions are

  11. Genetic interactions between planar cell polarity genes cause diverse neural tube defects in mice.

    PubMed

    Murdoch, Jennifer N; Damrau, Christine; Paudyal, Anju; Bogani, Debora; Wells, Sara; Greene, Nicholas D E; Stanier, Philip; Copp, Andrew J

    2014-10-01

    Neural tube defects (NTDs) are among the commonest and most severe forms of developmental defect, characterized by disruption of the early embryonic events of central nervous system formation. NTDs have long been known to exhibit a strong genetic dependence, yet the identity of the genetic determinants remains largely undiscovered. Initiation of neural tube closure is disrupted in mice homozygous for mutations in planar cell polarity (PCP) pathway genes, providing a strong link between NTDs and PCP signaling. Recently, missense gene variants have been identified in PCP genes in humans with NTDs, although the range of phenotypes is greater than in the mouse mutants. In addition, the sequence variants detected in affected humans are heterozygous, and can often be detected in unaffected individuals. It has been suggested that interactions between multiple heterozygous gene mutations cause the NTDs in humans. To determine the phenotypes produced in double heterozygotes, we bred mice with all three pairwise combinations of Vangl2(Lp), Scrib(Crc) and Celsr1(Crsh) mutations, the most intensively studied PCP mutants. The majority of double-mutant embryos had open NTDs, with the range of phenotypes including anencephaly and spina bifida, therefore reflecting the defects observed in humans. Strikingly, even on a uniform genetic background, variability in the penetrance and severity of the mutant phenotypes was observed between the different double-heterozygote combinations. Phenotypically, Celsr1(Crsh);Vangl2(Lp);Scrib(Crc) triply heterozygous mutants were no more severe than doubly heterozygous or singly homozygous mutants. We propose that some of the variation between double-mutant phenotypes could be attributed to the nature of the protein disruption in each allele: whereas Scrib(Crc) is a null mutant and produces no Scrib protein, Celsr1(Crsh) and Vangl2(Lp) homozygotes both express mutant proteins, consistent with dominant effects. The variable outcomes of these genetic

  12. Landscape models for nuclear genetic diversity and genetic structure in white-footed mice (Peromyscus leucopus)

    PubMed Central

    Taylor, Z S; Hoffman, S M G

    2014-01-01

    Dramatic changes in the North American landscape over the last 12 000 years have shaped the genomes of the small mammals, such as the white-footed mouse (Peromyscus leucopus), which currently inhabit the region. However, very recent interactions of populations with each other and the environment are expected to leave the most pronounced signature on rapidly evolving nuclear microsatellite loci. We analyzed landscape characteristics and microsatellite markers of P. leucopus populations along a transect from southern Ohio to northern Michigan, in order to evaluate hypotheses about the spatial distribution of genetic heterogeneity. Genetic diversity increased to the north and was best approximated by a single-variable model based on habitat availability within a 0.5-km radius of trapping sites. Interpopulation differentiation measured by clustering analysis was highly variable and not significantly related to latitude or habitat availability. Interpopulation differentiation measured as FST values and chord distance was correlated with the proportion of habitat intervening, but was best explained by agricultural distance and by latitude. The observed gradients in diversity and interpopulation differentiation were consistent with recent habitat availability being the major constraint on effective population size in this system, and contradicted the predictions of both the postglacial expansion and core-periphery hypotheses. PMID:24448564

  13. Landscape models for nuclear genetic diversity and genetic structure in white-footed mice (Peromyscus leucopus).

    PubMed

    Taylor, Z S; Hoffman, S M G

    2014-06-01

    Dramatic changes in the North American landscape over the last 12 000 years have shaped the genomes of the small mammals, such as the white-footed mouse (Peromyscus leucopus), which currently inhabit the region. However, very recent interactions of populations with each other and the environment are expected to leave the most pronounced signature on rapidly evolving nuclear microsatellite loci. We analyzed landscape characteristics and microsatellite markers of P. leucopus populations along a transect from southern Ohio to northern Michigan, in order to evaluate hypotheses about the spatial distribution of genetic heterogeneity. Genetic diversity increased to the north and was best approximated by a single-variable model based on habitat availability within a 0.5-km radius of trapping sites. Interpopulation differentiation measured by clustering analysis was highly variable and not significantly related to latitude or habitat availability. Interpopulation differentiation measured as FST values and chord distance was correlated with the proportion of habitat intervening, but was best explained by agricultural distance and by latitude. The observed gradients in diversity and interpopulation differentiation were consistent with recent habitat availability being the major constraint on effective population size in this system, and contradicted the predictions of both the postglacial expansion and core-periphery hypotheses.

  14. Detrimental effects of an autosomal selfish genetic element on sperm competitiveness in house mice

    PubMed Central

    Sutter, Andreas; Lindholm, Anna K.

    2015-01-01

    Female multiple mating (polyandry) is widespread across many animal taxa and indirect genetic benefits are a major evolutionary force favouring polyandry. An incentive for polyandry arises when multiple mating leads to sperm competition that disadvantages sperm from genetically inferior mates. A reduction in genetic quality is associated with costly selfish genetic elements (SGEs), and studies in invertebrates have shown that males bearing sex ratio distorting SGEs are worse sperm competitors than wild-type males. We used a vertebrate model species to test whether females can avoid an autosomal SGE, the t haplotype, through polyandry. The t haplotype in house mice exhibits strong drive in t heterozygous males by affecting spermatogenesis and is associated with homozygous in utero lethality. We used controlled matings to test the effect of the t haplotype on sperm competitiveness. Regardless of mating order, t heterozygous males sired only 11% of zygotes when competing against wild-type males, suggesting a very strong effect of the t haplotype on sperm quality. We provide, to our knowledge, the first substantial evidence that polyandry ameliorates the harmful effects of an autosomal SGE arising through genetic incompatibility. We discuss potential mechanisms in our study species and the broader implications for the benefits of polyandry. PMID:26136452

  15. A multivariate quantitative-genetic analysis of behavioral development in mice.

    PubMed

    Crusio, W E; Schmitt, A

    1998-05-01

    The present experiment attempted a behavior-genetic dissection of early behavioral development in laboratory mice. To this end, we used a full, replicated diallel cross to uncover the genetical architecture as well as the multivariate genetic structure underlying early behavioral ontogeny. A number of standard sensorimotor tests were administered on postnatal Days 3, 5, 8, 10, 13, 17, and 22 to a total of 622 pups from 120 litters (4-6 pups per litter) from a four times replicated complete diallel cross between five inbred mouse strains. The first day on which an animal showed adult performance was taken as its score on that test. MANOVA did not show any effects of the pup's sex on the speed of development. Hayman's analysis of variance for diallel tables indicated no or only weak additive-genetic effects. Dominance was absent in almost all cases, except for the auricular startle response, where weak directional dominance for fast development was found. These results are in accordance with an evolutionary past of directional selection for well-canalized development. Factor analyses of the phenotypic and additive-genetic correlation matrices indicate that at least two factors are necessary to describe the behavioral variation.

  16. Detrimental effects of an autosomal selfish genetic element on sperm competitiveness in house mice.

    PubMed

    Sutter, Andreas; Lindholm, Anna K

    2015-07-22

    Female multiple mating (polyandry) is widespread across many animal taxa and indirect genetic benefits are a major evolutionary force favouring polyandry. An incentive for polyandry arises when multiple mating leads to sperm competition that disadvantages sperm from genetically inferior mates. A reduction in genetic quality is associated with costly selfish genetic elements (SGEs), and studies in invertebrates have shown that males bearing sex ratio distorting SGEs are worse sperm competitors than wild-type males.We used a vertebrate model species to test whether females can avoid an autosomal SGE, the t haplotype, through polyandry. The t haplotype inhouse mice exhibits strong drive in t heterozygous males by affecting spermatogenesis and is associated with homozygous in utero lethality. We used controlled matings to test the effect of the t haplotype on sperm competitiveness. Regardless of mating order, t heterozygous males sired only 11% of zygotes when competing against wild-type males, suggesting a very strong effect of the t haplotype on sperm quality. We provide, to our knowledge,the first substantial evidence that polyandry ameliorates the harmful effects of an autosomal SGE arising through genetic incompatibility. We discuss potential mechanisms in our study species and the broader implications for the benefits of polyandry.

  17. Genetic recombination is directed away from functional genomic elements in mice.

    PubMed

    Brick, Kevin; Smagulova, Fatima; Khil, Pavel; Camerini-Otero, R Daniel; Petukhova, Galina V

    2012-05-13

    Genetic recombination occurs during meiosis, the key developmental programme of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyltransferase, PR domain containing 9 (PRDM9), the product of the only known speciation-associated gene in mammals. PRDM9 is thought to determine the preferred recombination sites--recombination hotspots--through sequence-specific binding of its highly polymorphic multi-Zn-finger domain. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination. Here we map and analyse the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F(1) hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the exception of the pseudo-autosomal region (PAR)--the only area of the genome that undergoes recombination in 100% of cells. Surprisingly, hotspots are still observed in Prdm9 knockout mice, and as in wild type, these hotspots are found at H3 lysine 4 (H3K4) trimethylation marks. However, in the absence of PRDM9, most recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation. Such sites are rarely targeted in wild-type mice, indicating an unexpected role of the PRDM9 protein in sequestering the recombination machinery away from gene-promoter regions and other functional genomic elements.

  18. Genetically Modified α-Amylase Inhibitor Peas Are Not Specifically Allergenic in Mice

    PubMed Central

    Dekan, Gerhard; Moore, Andrew E.; Higgins, T. J. V.; Epstein, Michelle M.

    2013-01-01

    Weevils can devastate food legumes in developing countries, but genetically modified peas (Pisum sativum), chickpeas and cowpeas expressing the gene for alpha-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are completely protected from weevil destruction. αAI is seed-specific, accumulated at high levels and undergoes post-translational modification as it traverses the seed endomembrane system. This modification was thought to be responsible for the reported allergenicity in mice of the transgenic pea but not the bean. Here, we observed that transgenic αAI peas, chickpeas and cowpeas as well as non-transgenic beans were all allergenic in BALB/c mice. Even consuming non-transgenic peas lacking αAI led to an anti-αAI response due to a cross-reactive response to pea lectin. Our data demonstrate that αAI transgenic peas are not more allergenic than beans or non-transgenic peas in mice. This study illustrates the importance of repeat experiments in independent laboratories and the potential for unexpected cross-reactive allergic responses upon consumption of plant products in mice. PMID:23326368

  19. Genetically modified α-amylase inhibitor peas are not specifically allergenic in mice.

    PubMed

    Lee, Rui-Yun; Reiner, Daniela; Dekan, Gerhard; Moore, Andrew E; Higgins, T J V; Epstein, Michelle M

    2013-01-01

    Weevils can devastate food legumes in developing countries, but genetically modified peas (Pisum sativum), chickpeas and cowpeas expressing the gene for alpha-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are completely protected from weevil destruction. αAI is seed-specific, accumulated at high levels and undergoes post-translational modification as it traverses the seed endomembrane system. This modification was thought to be responsible for the reported allergenicity in mice of the transgenic pea but not the bean. Here, we observed that transgenic αAI peas, chickpeas and cowpeas as well as non-transgenic beans were all allergenic in BALB/c mice. Even consuming non-transgenic peas lacking αAI led to an anti-αAI response due to a cross-reactive response to pea lectin. Our data demonstrate that αAI transgenic peas are not more allergenic than beans or non-transgenic peas in mice. This study illustrates the importance of repeat experiments in independent laboratories and the potential for unexpected cross-reactive allergic responses upon consumption of plant products in mice.

  20. Genetic susceptibility to systemic lupus erythematosus protects against cerebral malaria in mice

    PubMed Central

    Waisberg, Michael; Tarasenko, Tatyana; Vickers, Brandi K.; Scott, Bethany L.; Willcocks, Lisa C.; Molina-Cruz, Alvaro; Pierce, Matthew A.; Huang, Chiung-yu; Torres-Velez, Fernando J.; Smith, Kenneth G. C.; Barillas-Mury, Carolina; Miller, Louis H.; Pierce, Susan K.; Bolland, Silvia

    2011-01-01

    Plasmodium falciparum has exerted tremendous selective pressure on genes that improve survival in severe malarial infections. Systemic lupus erythematosus (SLE) is an autoimmune disease that is six to eight times more prevalent in women of African descent than in women of European descent. Here we provide evidence that a genetic susceptibility to SLE protects against cerebral malaria. Mice that are prone to SLE because of a deficiency in FcγRIIB or overexpression of Toll-like receptor 7 are protected from death caused by cerebral malaria. Protection appears to be by immune mechanisms that allow SLE-prone mice better to control their overall inflammatory responses to parasite infections. These findings suggest that the high prevalence of SLE in women of African descent living outside of Africa may result from the inheritance of genes that are beneficial in the immune control of cerebral malaria but that, in the absence of malaria, contribute to autoimmune disease. PMID:21187399

  1. Rapid phenotyping of knockout mice to identify genetic determinants of bone strength

    PubMed Central

    Freudenthal, Bernard; Logan, John; Croucher, Peter I

    2016-01-01

    The genetic determinants of osteoporosis remain poorly understood, and there is a large unmet need for new treatments in our ageing society. Thus, new approaches for gene discovery in skeletal disease are required to complement the current genome-wide association studies in human populations. The International Knockout Mouse Consortium (IKMC) and the International Mouse Phenotyping Consortium (IMPC) provide such an opportunity. The IKMC generates knockout mice representing each of the known protein-coding genes in C57BL/6 mice and, as part of the IMPC initiative, the Origins of Bone and Cartilage Disease project identifies mutants with significant outlier skeletal phenotypes. This initiative will add value to data from large human cohorts and provide a new understanding of bone and cartilage pathophysiology, ultimately leading to the identification of novel drug targets for the treatment of skeletal disease. PMID:27535945

  2. Rapid phenotyping of knockout mice to identify genetic determinants of bone strength.

    PubMed

    Freudenthal, Bernard; Logan, John; Croucher, Peter I; Williams, Graham R; Bassett, J H Duncan

    2016-10-01

    The genetic determinants of osteoporosis remain poorly understood, and there is a large unmet need for new treatments in our ageing society. Thus, new approaches for gene discovery in skeletal disease are required to complement the current genome-wide association studies in human populations. The International Knockout Mouse Consortium (IKMC) and the International Mouse Phenotyping Consortium (IMPC) provide such an opportunity. The IKMC generates knockout mice representing each of the known protein-coding genes in C57BL/6 mice and, as part of the IMPC initiative, the Origins of Bone and Cartilage Disease project identifies mutants with significant outlier skeletal phenotypes. This initiative will add value to data from large human cohorts and provide a new understanding of bone and cartilage pathophysiology, ultimately leading to the identification of novel drug targets for the treatment of skeletal disease. © 2016 Society for Endocrinology.

  3. Increased calcium bioavailability in mice fed genetically engineered plants lacking calcium oxalate.

    PubMed

    Morris, Jay; Nakata, Paul A; McConn, Michele; Brock, Amanda; Hirschi, Kendal D

    2007-07-01

    Bioavailable calcium affects bone formation and calcification. Here we investigate how a single gene mutation altering calcium partitioning in the model forage crop Medicago truncatula affects calcium bioavailability. Previously, the cod5 M. truncatula mutant was identified which contains identical calcium concentrations to wild-type, but contains no oxalate crystals. In this study, equal number of male and female mice were randomly grouped and then fed one of four 45Ca-containing diets: M. truncatula extrinsically or intrinsically labeled, and cod5 extrinsically or intrinsically labeled. Absorption of the tracer was determined in the legs one day after consumption. The absorption was similar in the M. truncatula and cod5 extrinsically labeled diets; however, in the intrinsically labeled diets, calcium absorption was 22.87% (P < 0.001) higher in mice fed cod5. Our study presents the first genetic evidence demonstrating the nutritional impact of removing oxalate crystals from foods.

  4. Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale

    PubMed Central

    Kui, Ling; Chen, Haitao; Zhang, Weixiong; He, Simei; Xiong, Zijun; Zhang, Yesheng; Yan, Liang; Zhong, Chaofang; He, Fengmei; Chen, Junwen; Zeng, Peng; Zhang, Guanghui; Yang, Shengchao; Dong, Yang; Wang, Wen; Cai, Jing

    2017-01-01

    Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D

  5. Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale.

    PubMed

    Kui, Ling; Chen, Haitao; Zhang, Weixiong; He, Simei; Xiong, Zijun; Zhang, Yesheng; Yan, Liang; Zhong, Chaofang; He, Fengmei; Chen, Junwen; Zeng, Peng; Zhang, Guanghui; Yang, Shengchao; Dong, Yang; Wang, Wen; Cai, Jing

    2016-01-01

    Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D

  6. Hürthle Cells Predict Hypothyroidism in Interferon-γ Transgenic Mice of Different Genetic Backgrounds

    PubMed Central

    Iwama, Shintaro; De Remigis, Alessandra; Bishop, Justin A.; Kimura, Hiroaki J.

    2012-01-01

    Hürthle cells have long been described in Hashimoto thyroiditis but remain of undetermined significance. We have previously shown that Hürthle cells and hypothyroidism develop in C57BL/6J mice expressing interferon-γ (IFNγ) in the thyroid. To assess the influence of genetic backgrounds on Hürthle cell development, we crossed C57BL/6J IFNγ transgenic mice to 14 strains and analyzed thyroid histopathology and function in a cohort of 389 mice (225 transgenic and 164 wild type) using a multiple linear regression model that also included strain, sex, genotype, and major histocompatibility complex haplotype. We then queried the Johns Hopkins surgical pathology electronic archive for “Hashimoto” and/or “thyroiditis” keywords, reviewed the reports, and reexamined the Hashimoto slides. Hürthle cells were markedly affected by the genetic background: they were prominent and associated with hypothyroidism in the C57BL/6J, C57BL/6ByJ, C57BL/10J, C57BLKS/J, C57L/J, C58/J, and BPN/3J IFNγ transgenic strains, whereas they are mild or absent in the BPH/2J, BPL/1J, LP/J, CBA/J, Balb/cJ, DBA/1J, and NOD/ShiLtJ strains. Hürthle cells were the strongest predictor of hypothyroidism after adjusting for all the other covariates in the regression model. Interestingly, transgenic mice of the BPL/1J, DBA/1J, and NOD/ShiLtJ strains developed a marked accumulation of intrathyroidal brown adipocytes that was significantly associated with improved thyroid function. Hürthle cells were mentioned in 23% of the Hashimoto reports but increased to 79% upon our slide review. This study reports a novel association of Hürhtle cells and brown adipocytes on thyroid function that should prompt a reconsideration of their significance and role in pathogenesis of autoimmune thyroiditis. PMID:22719056

  7. Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity

    NASA Technical Reports Server (NTRS)

    Anterola, Aldwin M.; Lewis, Norman G.

    2002-01-01

    A comprehensive assessment of lignin configuration in transgenic and mutant plants is long overdue. This review thus undertook the systematic analysis of trends manifested through genetic and mutational manipulations of the various steps associated with monolignol biosynthesis; this included consideration of the downstream effects on organized lignin assembly in the various cell types, on vascular function/integrity, and on plant growth and development. As previously noted for dirigent protein (homologs), distinct and sophisticated monolignol forming metabolic networks were operative in various cell types, tissues and organs, and form the cell-specific guaiacyl (G) and guaiacyl-syringyl (G-S) enriched lignin biopolymers, respectively. Regardless of cell type undergoing lignification, carbon allocation to the different monolignol pools is apparently determined by a combination of phenylalanine availability and cinnamate-4-hydroxylase/"p-coumarate-3-hydroxylase" (C4H/C3H) activities, as revealed by transcriptional and metabolic profiling. Downregulation of either phenylalanine ammonia lyase or cinnamate-4-hydroxylase thus predictably results in reduced lignin levels and impaired vascular integrity, as well as affecting related (phenylpropanoid-dependent) metabolism. Depletion of C3H activity also results in reduced lignin deposition, albeit with the latter being derived only from hydroxyphenyl (H) units, due to both the guaiacyl (G) and syringyl (S) pathways being blocked. Apparently the cells affected are unable to compensate for reduced G/S levels by increasing the amounts of H-components. The downstream metabolic networks for G-lignin enriched formation in both angiosperms and gymnosperms utilize specific cinnamoyl CoA O-methyltransferase (CCOMT), 4-coumarate:CoA ligase (4CL), cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) isoforms: however, these steps neither affect carbon allocation nor H/G designations, this being determined by C4H/C3H

  8. Investigation of the relationship between the onset of arthritis and uveitis in genetically predisposed SKG mice.

    PubMed

    Lee, Ellen J; Vance, Emily E; Brown, Brieanna R; Snow, Paige S; Clowers, Jenna S; Sakaguchi, Shimon; Rosenzweig, Holly L

    2015-08-19

    Systemic rheumatic conditions are often accompanied by intraocular inflammatory disease (termed uveitis). Despite the frequent manifestation of uveitis with arthritis, very little is understood of the underlying mechanisms that mediate the eye's susceptibility to disease. The genetically susceptible SKG mouse strain develops arthritis that arises from an inherent mutation that disrupts T-cell antigen receptor signal transduction and thymic selection. The ensuing T-cell-mediated disease is further modulated through exposure to microbial triggers. The purpose of this study was to elucidate how a genetically determined shift in the T-cell repertoire toward self-reactive T cells that drive arthritis influences uveitis in SKG mice. SKG mice (BALB/c mice that harbor the W163C point mutation in zeta-chain-associated protein kinase 70 [i.e., ZAP-70]) were housed under arthritis-resistant, specific pathogen-free conditions. Arthritis was induced by intraperitoneal injection with fungal glucans (zymosan or curdlan). Arthritis onset and severity were evaluated by clinical scoring, histopathology and infrared imaging within the joints. Periocular traits involving blepharoconjunctivitis were evaluated by clinical scoring and histology. Eyes were evaluated for signs of anterior uveitis using intravital videomicroscopy to document cell-trafficking responses within the iris vasculature and stroma and by histology to detect inflammatory infiltrate and tissue damage within the anterior and posterior eye segments. Exposure to zymosan resulted in the predicted arthritic, sexually dimorphic phenotype in SKG mice. The eyes of SKG mice exhibited episodic intravascular cellular responses to zymosan or curdlan as indicated by significant increases in leukocyte-endothelium interactions akin to ocular vasculitis. However, despite the significant increase in early cell-trafficking responses, cellular infiltration into the iris stroma was not observed and histopathological signs indicative of

  9. Genetically epilepsy-prone rats (GEPRs) and DBA/2 mice: Two animal models of audiogenic reflex epilepsy for the evaluation of new generation AEDs.

    PubMed

    De Sarro, Giovambattista; Russo, Emilio; Citraro, Rita; Meldrum, Brian S

    2015-08-06

    This review summarizes the current knowledge about DBA/2 mice and genetically epilepsy-prone rats (GEPRs) and discusses the contribution of such animal models on the investigation of possible new therapeutic targets and new anticonvulsant compounds for the treatment of epilepsy. Also, possible chemical or physical agents acting as proconvulsant agents are described. Abnormal activities of enzymes involved in catecholamine and serotonin synthesis and metabolism were reported in these models, and as a result of all these abnormalities, seizure susceptibility in both animals is greatly affected by pharmacological manipulations of the brain levels of monoamines and, prevalently, serotonin. In addition, both genetic epileptic models permit the evaluation of pharmacodynamic and pharmacokinetic interactions among several drugs measuring plasma and/or brain level of each compound. Audiogenic models of epilepsy have been used not only for reflex epilepsy studies, but also as animal models of epileptogenesis. The seizure predisposition (epileptiform response to sound stimulation) and substantial characterization of behavioral, cellular, and molecular alterations in both acute and chronic (kindling) protocols potentiate the usefulness of these models in elucidating ictogenesis, epileptogenesis, and their mechanisms. This article is part of a Special Issue entitled "Genetic Models-Epilepsy".

  10. [Transfer of genetic constructions through the transplacental barrier into mice embryos].

    PubMed

    Efremov, A M; Buglaeva, A O; Orlov, S V; Burov, S V; Ignatovich, I A; Dizhe, E B; Shavva, V S; Perevozchikov, A P

    2010-01-01

    Genetic modification of mammalian embryos is an important way to model various changes in human development; also, it is an instrument for studying the functions of certain genes in mammals. Using our own experience in developing modes of delivery of genetic constructions to mammals in a nonviral way, we present here data on the delivery of a eukaryotic expression vector to mice embryos through the transplacental barrier with the use of hydrodynamic intravenous injections of DNA-hybrid peptide complexes to pregnant females. The peptide has a cationic part for interaction with DNA and includes a ligand structure towards receptors of the releasing factor of luteinizing hormone (RFLH, luliberin). Advantages of the suggested method are simplicity, economy, nonimmunogenicity for females, and the ability to multiply repeat the procedure. On the basis of the method, systemic gene delivery into tissues of mammalian embryos may be developed.

  11. Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles.

    PubMed

    Stanley, Sarah A; Sauer, Jeremy; Kane, Ravi S; Dordick, Jonathan S; Friedman, Jeffrey M

    2015-01-01

    Means for temporally regulating gene expression and cellular activity are invaluable for elucidating underlying physiological processes and would have therapeutic implications. Here we report the development of a genetically encoded system for remote regulation of gene expression by low-frequency radio waves (RFs) or a magnetic field. Iron oxide nanoparticles are synthesized intracellularly as a GFP-tagged ferritin heavy and light chain fusion. The ferritin nanoparticles associate with a camelid anti-GFP-transient receptor potential vanilloid 1 fusion protein, αGFP-TRPV1, and can transduce noninvasive RF or magnetic fields into channel activation, also showing that TRPV1 can transduce a mechanical stimulus. This, in turn, initiates calcium-dependent transgene expression. In mice with stem cell or viral expression of these genetically encoded components, remote stimulation of insulin transgene expression with RF or a magnet lowers blood glucose. This robust, repeatable method for remote regulation in vivo may ultimately have applications in basic science, technology and therapeutics.

  12. Knockout mice: simple solutions to the problems of genetic background and flanking genes.

    PubMed

    Wolfer, David P; Crusio, Wim E; Lipp, Hans Peter

    2002-07-01

    Inducing null mutations by means of homologous recombination provides a powerful technique to investigate gene function and has found wide application in many different fields. However, it was realized some time ago that the specific way in which such knockout mutants are generated can be confounding, making it impossible to separate the effects of the induced null mutation from those of alleles originating from the embryonic stem cell donor. In addition, effects from null mutations can be altered on different genetic backgrounds. Here we present some simple breeding strategies to test for flanking gene effects that are compatible with the recommendations of the Banbury Conference on Genetic Background in Mice and with common practices of creating and maintaining mouse knockout lines.

  13. Retrospective on reverse genetics in mice around the world and in Japan.

    PubMed

    Aizawa, Shinichi

    2008-06-01

    The 2007 Nobel Prize for Physiology or Medicine was awarded to Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their contribution in generating mutant mice by gene targeting in embryonic stem (ES) cells. Although there are many experimental animals, it is yet only in mouse that one can genetically examine functions of genes at will. It was merely a dream in the early 1980s that genetic studies with mutants would one day become a reality in mammals. The story began with tetratocarcinoma/embryonal carcinoma cells. Now, through the successes of cloning in mammals, somatic cells such as our skin cells will shortly be transformed into ES-like (induced pluripotent stem) cells by the proper activation of endogenous genes such as Oct4 and Sox2 with chemicals. How have times changed?

  14. Concomitant manipulation of murine NMDA- and AMPA-receptors to produce pro-cognitive drug effects in mice.

    PubMed

    Vignisse, Julie; Steinbusch, Harry W M; Grigoriev, Vladimir; Bolkunov, Alexei; Proshin, Alexey; Bettendorff, Lucien; Bachurin, Sergey; Strekalova, Tatyana

    2014-02-01

    Bifunctional drug therapy targeting distinct receptor signalling systems can generate increased efficacy at lower concentrations compared to monofunctional therapy. Non-competitive blockade of the NMDA receptors or the potentiation of AMPA receptors is well documented to result in memory enhancement. Here, we compared the efficacy of the low-affinity NMDA receptor blocker memantine or the positive modulator of AMPA receptor QXX (in C57BL/6J at 1 or 5mg/kg, ip) with new derivatives of isothiourea (0.5-1 mg/kg, ip) that have bifunctional efficacy. Low-affinity NMDA blockade by these derivatives was achieved by introducing greater flexibility into the molecule, and AMPA receptor stimulation was produced by a sulfamide-containing derivative of isothiourea. Contextual learning was examined in a step-down avoidance task and extinction of contextual memory was studied in a fear-conditioning paradigm. Memantine enhanced contextual learning while QXX facilitated memory extinction; both drugs were effective at 5 mg/kg. The new derivative IPAC-5 elevated memory scores in both tasks at the dose 0.5 mg/kg and exhibited the lowest IC₅₀ values of NMDA receptor blockade and highest potency of AMPA receptor stimulation. Thus, among the new drugs tested, IPAC-5 replicated the properties of memantine and QXX in one administration with increased potency. Our data suggest that a concomitant manipulation of NMDA- and AMPA-receptors results in pro-cognitive effects and supports the concept bifunctional drug therapy as a promising strategy to replace monofunctional therapies with greater efficacy and improved compliance.

  15. Genetic loss of D-amino acid oxidase activity reverses schizophrenia-like phenotypes in mice

    PubMed Central

    Labrie, V.; Wang, W.; Barger, S. W.; Baker, G. B.; Roder, J. C.

    2009-01-01

    Reduced function of the N-methyl-D-aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine binding site in its NR1 subunit that may be a useful target for the treatment of schizophrenia. In this study, we assessed the therapeutic potential of long-term increases in the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation of its catabolic enzyme D-amino acid oxidase (DAO) in mice. The effects of eliminating DAO function were investigated in mice that display schizophrenia-related behavioral deficits due to a mutation (Grin1D481N) in the NR1 subunit that results in a reduction in NMDAR glycine affinity. Grin1D481N mice show deficits in sociability, prolonged latent inhibition, enhanced startle reactivity, and impaired spatial memory. The hypofunctional Dao1G181R mutation elevated brain levels of D-serine, but alone it did not affect performance in the behavioral measures. Compared to animals with only the Grin1D481N mutation, mice with both the Dao1G181R and Grin1D481N mutations displayed an improvement in social approach and spatial memory retention, as well as a reversal of abnormally persistent latent inhibition and a partial normalization of startle responses. Thus, an increased level of D-serine resulting from decreased catalysis corrected the performance of mice with deficient NMDAR glycine site activation in behavioral tasks relevant to the negative and cognitive symptoms of schizophrenia. Diminished DAO activity and elevations in D-serine may serve as an effective therapeutic intervention for the treatment of psychiatric symptoms. PMID:19751394

  16. Genetic variation within the Chrna7 gene modulates nicotine reward-like phenotypes in mice

    PubMed Central

    Harenza, Jo Lynne; Muldoon, Pretal P.; De Biasi, Mariella; Damaj, M. Imad; Miles, Michael F.

    2014-01-01

    Mortality from tobacco smoking remains the leading cause of preventable death in the world, yet current cessation therapies are only modestly successful, suggesting new molecular targets are needed. Genetic analysis of gene expression and behavior identified Chrna7 as potentially modulating nicotine place conditioning in the BXD panel of inbred mice. We used gene targeting and pharmacological tools to confirm the role of Chrna7 in nicotine CPP. To identify molecular events downstream of Chrna7 that may modulate nicotine preference, we performed microarray analysis of α7 KO and WT nucleus accumbens tissue, followed by confirmation with quantitative PCR and immunoblotting. In the BXD panel, we found a putative cis eQTL for Chrna7 in nucleus accumbens that correlated inversely to nicotine CPP. We observed that gain-of-function α7 mice did not display nicotine preference at any dose tested, while conversely, α7 KO mice showed nicotine place preference at a dose below that routinely required to produce preference. In B6 mice, the α7 nAChR-selective agonist, PHA-543613, dose-dependently blocked nicotine CPP, which was restored using the α7 nAChR-selective antagonist, MLA. Our genomic studies implicated an mRNA co-expression network regulated by Chrna7 in nucleus accumbens. Mice lacking Chrna7 demonstrate increased insulin signaling in the nucleus accumbens, which may modulate nicotine place preference. Our studies provide novel targets for future work on development of more effective therapeutic approaches to counteract the rewarding properties of nicotine for smoking cessation. PMID:24289814

  17. Nordihydroguaiaretic acid and aspirin increase lifespan of genetically heterogeneous male mice

    PubMed Central

    Strong, Randy; Miller, Richard A.; Astle, Clinton M.; Floyd, Robert A.; Flurkey, Kevin; Hensley, Kenneth L.; Javors, Martin A.; Leeuwenburgh, Christiaan; Nelson, James F.; Ongini, Ennio; Nadon, Nancy L.; Warner, Huber R.; Harrison, David E.

    2009-01-01

    The National Institute on Aging Interventions Testing Program (ITP) was established to evaluate agents that are purported to increase lifespan and delay the appearance of age-related disease in genetically heterogeneous mice. Up to five compounds are added to the study each year and each compound is tested at three test sites (The Jackson Laboratory, TJL; University of Michigan, UM; and University of Texas Health Science Center, San Antonio, UT). Mice in the first cohort were exposed to one of four agents: aspirin, nitroflurbiprofen (NFP), 4-OH-α-phenyl-N-tert-butyl nitrone (4-OH-PBN), or nordihydroguiaretic acid (NDGA). Sample size was sufficient to detect a 10% difference in lifespan in either sex, with 80% power, using data from two of the three sites. Pooling data from all three sites, a log-rank test showed that both NDGA (p = 0.0006) and aspirin (p = 0.01) led to increased lifespan of male mice. Comparison of the proportion of live mice at the age of 90% mortality was used as a surrogate for measurement of maximum lifespan; neither NDGA (p = 0.12) nor aspirin (p = 0.16) had a significant effect in this test. Measures of blood levels of NDGA or aspirin and its salicylic acid metabolite suggest that the observed lack of effects of NDGA or aspirin on lifespan in females could be related to gender differences in drug disposition or metabolism. Further studies are warranted to find whether NDGA or aspirin, over a range of doses, might prove to postpone death and various age-related outcomes reproducibly in mice. PMID:18631321

  18. Global genetic analysis in mice unveils central role for cilia in congenital heart disease

    PubMed Central

    Li, You; Klena, Nikolai T.; Gabriel, George C; Liu, Xiaoqin; Kim, Andrew J.; Lemke, Kristi; Chen, Yu; Chatterjee, Bishwanath; Devine, William; Damerla, Rama Rao; Chang, Chien-fu; Yagi, Hisato; San Agustin, Jovenal T.; Thahir, Mohamed; Anderton, Shane; Lawhead, Caroline; Vescovi, Anita; Pratt, Herbert; Morgan, Judy; Haynes, Leslie; Smith, Cynthia L.; Eppig, Janan T.; Reinholdt, Laura; Francis, Richard; Leatherbury, Linda; Ganapathiraju, Madhavi K.; Tobita, Kimimasa; Pazour, Gregory J.; Lo, Cecilia W.

    2015-01-01

    Congenital heart disease (CHD) is the most prevalent birth defect, affecting nearly 1% of live births1, but the incidence of CHD is up to ten fold higher in human fetuses2,3. A genetic contribution is strongly suggested by the association of CHD with chromosome abnormalities and high recurrence risk4. Here we report findings from a recessive forward genetic screen in fetal mice, showing the cilium and cilia transduced cell signaling play important roles in the pathogenesis of CHD. The cilium is an evolutionarily conserved organelle projecting from the cell surface with essential roles in diverse cellular processes. Using echocardiography, we ultrasound scanned 87,355 chemically mutagenized C57BL/6J fetal mice and recovered 218 CHD mouse models. Whole exome sequencing identified 91 recessive CHD mutations in 61 genes. This included 34 cilia-related genes, 16 genes involved in cilia transduced cell signaling, and 10 genes regulating vesicular trafficking, a pathway important for ciliogenesis and cell signaling. Surprisingly, many CHD genes encoded interacting proteins, suggesting an interactome protein network may provide a larger genomic context for CHD pathogenesis. These findings provide novel insights into the potential Mendelian genetic contribution to CHD in the fetal population, a segment of the human population not well studied. We note pathways identified show overlap with CHD candidate genes recovered in CHD patients5, suggesting they may have relevance to the more complex genetics of CHD overall. These CHD mouse models and >8,000 incidental mutations are sperm archived, creating a rich public resource for human disease modeling. PMID:25807483

  19. Weak Genetic Relationship Between Trabecular Bone Morphology and Obesity in Mice

    PubMed Central

    Carson, E. Ann; Kenney-Hunt, Jane P; Pavlicev, Mihaela; Bouckaert, Kristine A; Chinn, Alex J; Silva, Matthew J; Cheverud, James M

    2012-01-01

    Obesity, in addition to being associated with metabolic diseases, such as diabetes, has also been found to lower the risk of osteoporotic fractures. The relationship between obesity and bone trabecular structure is complex, involving responses to mechanical loading and the effects of adipocyte-derived hormones, both directly interacting with bone tissue and indirectly through central nervous system signaling. Here we examine the effects of sex, a high fat diet, and genetics on the trabecular density and structure of the lumbar and caudal vertebra and the proximal tibia along with body weight, fat pad weight, and serum leptin levels in a murine obesity model, the LGXSM Recombinant Inbred (RI) mouse strains. The sample included 481 mice from 16 RI strains. We found that vertebral trabecular density was higher in males while the females had higher tibial trabecular density. The high fat diet led to only slightly higher trabecular density in both sexes despite its extreme effects on obesity and serum leptin levels. Trait heritabilities are moderate to strong and genetic correlations among trabecular features are high. Most genetic variation contrasts strains with large numbers of thick, closely-spaced, highly interconnected, plate-like trabeculae with a high bone volume to total volume ratio against strains displaying small numbers of thin, widely-spaced, sparsely connected, rod-like trabeculae with a low bone volume to total volume ratio. Genetic correlations between trabecular and obesity-related traits were low and not statistically significant. We mapped trabecular properties to 20 genomic locations. Only one-quarter of these locations also had effects on obesity. In this population obesity has a relatively minor effect on trabecular bone morphology. Key Words: bone; trabecular morphology; obesity; quantitative trait loci; mice PMID:22503703

  20. High-resolution genetic mapping of mammalian motor activity levels in mice.

    PubMed

    Kas, M J H; de Mooij-van Malsen, J G; de Krom, M; van Gassen, K L I; van Lith, H A; Olivier, B; Oppelaar, H; Hendriks, J; de Wit, M; Groot Koerkamp, M J A; Holstege, F C P; van Oost, B A; de Graan, P N E

    2009-02-01

    The generation of motor activity levels is under tight neural control to execute essential behaviors, such as movement toward food or for social interaction. To identify novel neurobiological mechanisms underlying motor activity levels, we studied a panel of chromosome substitution (CS) strains derived from mice with high (C57BL/6J strain) or low motor activity levels (A/J strain) using automated home cage behavioral registration. In this study, we genetically mapped the expression of baseline motor activity levels (horizontal distance moved) to mouse chromosome 1. Further genetic mapping of this trait revealed an 8.3-Mb quantitative trait locus (QTL) interval. This locus is distinct from the QTL interval for open-field anxiety-related motor behavior on this chromosome. By data mining, an existing phenotypic and genotypic data set of 2445 genetically heterogeneous mice (http://gscan.well.ox.ac.uk/), we confirmed linkage to the peak marker at 79 970 253 bp and refined the QTL to a 312-kb interval containing a single gene (A830043J08Rik). Sequence analysis showed a nucleotide deletion in the 3' untranslated region of the Riken gene. Genome-wide microarray gene expression profiling in brains of discordant F(2) individuals from CS strain 1 showed a significant upregulation of Epha4 in low-active F(2) individuals. Inclusion of a genetic marker for Epha4 confirmed that this gene is located outside of the QTL interval. Both Epha4 and A830043J08Rik are expressed in brain motor circuits, and similar to Epha4 mutants, we found linkage between reduced motor neurons number and A/J chromosome 1. Our findings provide a novel QTL and a potential downstream target underlying motor circuitry development and the expression of physical activity levels.

  1. The Genetic Architecture of Hearing Impairment in Mice: Evidence for Frequency-Specific Genetic Determinants.

    PubMed

    Crow, Amanda L; Ohmen, Jeffrey; Wang, Juemei; Lavinsky, Joel; Hartiala, Jaana; Li, Qingzhong; Li, Xin; Salehide, Pezhman; Eskin, Eleazar; Pan, Calvin; Lusis, Aldons J; Allayee, Hooman; Friedman, Rick A

    2015-09-04

    Genome-wide association studies (GWAS) have been successfully applied in humans for the study of many complex phenotypes. However, identification of the genetic determinants of hearing in adults has been hampered, in part, by the relative inability to control for environmental factors that might affect hearing throughout the lifetime, as well as a large degree of phenotypic heterogeneity. These and other factors have limited the number of large-scale studies performed in humans that have identified candidate genes that contribute to the etiology of this complex trait. To address these limitations, we performed a GWAS analysis using a set of inbred mouse strains from the Hybrid Mouse Diversity Panel. Among 99 strains characterized, we observed approximately two-fold to five-fold variation in hearing at six different frequencies, which are differentiated biologically from each other by the location in the cochlea where each frequency is registered. Among all frequencies tested, we identified a total of nine significant loci, several of which contained promising candidate genes for follow-up study. Taken together, our results indicate the existence of both genes that affect global cochlear function, as well as anatomical- and frequency-specific genes, and further demonstrate the complex nature of mammalian hearing variation. Copyright © 2015 Crow et al.

  2. The Genetic Architecture of Hearing Impairment in Mice: Evidence for Frequency-Specific Genetic Determinants

    PubMed Central

    Crow, Amanda L.; Ohmen, Jeffrey; Wang, Juemei; Lavinsky, Joel; Hartiala, Jaana; Li, Qingzhong; Li, Xin; Salehide, Pezhman; Eskin, Eleazar; Pan, Calvin; Lusis, Aldons J.; Allayee, Hooman; Friedman, Rick A.

    2015-01-01

    Genome-wide association studies (GWAS) have been successfully applied in humans for the study of many complex phenotypes. However, identification of the genetic determinants of hearing in adults has been hampered, in part, by the relative inability to control for environmental factors that might affect hearing throughout the lifetime, as well as a large degree of phenotypic heterogeneity. These and other factors have limited the number of large-scale studies performed in humans that have identified candidate genes that contribute to the etiology of this complex trait. To address these limitations, we performed a GWAS analysis using a set of inbred mouse strains from the Hybrid Mouse Diversity Panel. Among 99 strains characterized, we observed approximately two-fold to five-fold variation in hearing at six different frequencies, which are differentiated biologically from each other by the location in the cochlea where each frequency is registered. Among all frequencies tested, we identified a total of nine significant loci, several of which contained promising candidate genes for follow-up study. Taken together, our results indicate the existence of both genes that affect global cochlear function, as well as anatomical- and frequency-specific genes, and further demonstrate the complex nature of mammalian hearing variation. PMID:26342000

  3. Cell manipulation in microfluidics.

    PubMed

    Yun, Hoyoung; Kim, Kisoo; Lee, Won Gu

    2013-06-01

    Recent advances in the lab-on-a-chip field in association with nano/microfluidics have been made for new applications and functionalities to the fields of molecular biology, genetic analysis and proteomics, enabling the expansion of the cell biology field. Specifically, microfluidics has provided promising tools for enhancing cell biological research, since it has the ability to precisely control the cellular environment, to easily mimic heterogeneous cellular environment by multiplexing, and to analyze sub-cellular information by high-contents screening assays at the single-cell level. Various cell manipulation techniques in microfluidics have been developed in accordance with specific objectives and applications. In this review, we examine the latest achievements of cell manipulation techniques in microfluidics by categorizing externally applied forces for manipulation: (i) optical, (ii) magnetic, (iii) electrical, (iv) mechanical and (v) other manipulations. We furthermore focus on history where the manipulation techniques originate and also discuss future perspectives with key examples where available.

  4. Host Genetics in Granuloma Formation: Human-Like Lung Pathology in Mice with Reciprocal Genetic Susceptibility to M. tuberculosis and M. avium

    PubMed Central

    Kondratieva, Elena; Logunova, Nadya; Majorov, Konstantin; Averbakh, Mikhail; Apt, Alexander

    2010-01-01

    Development of lung granulomata is a hallmark of infections caused by virulent mycobacteria, reflecting both protective host response that restricts infection spreading and inflammatory pathology. The role of host genetics in granuloma formation is not well defined. Earlier we have shown that mice of the I/St strain are extremely susceptible to Mycobacterium tuberculosis but resistant to M. avium infection, whereas B6 mice show a reversed pattern of susceptibility. Here, by directly comparing: (i) characteristics of susceptibility to two infections in vivo; (ii) architecture of lung granulomata assessed by immune staining; and (iii) expression of genes encoding regulatory factors of neutrophil influx in the lung tissue, we demonstrate that genetic susceptibility of the host largely determines the pattern of lung pathology. Necrotizing granuloma surrounded by hypoxic zones, as well as a massive neutrophil influx, develop in the lungs of M. avium-infected B6 mice and in the lungs of M. tuberculosis-infected I/St mice, but not in the lungs of corresponding genetically resistant counterparts. The mirror-type lung tissue responses to two virulent mycobacteria indicate that the level of genetic susceptibility of the host to a given mycobacterial species largely determines characteristics of pathology, and directly demonstrate the importance of host genetics in pathogenesis. PMID:20463893

  5. Modifier genes and non-genetic factors reshape anatomical deficits in Zfp423-deficient mice.

    PubMed

    Alcaraz, Wendy A; Chen, Edward; Valdes, Phoebe; Kim, Eunnie; Lo, Yuan Hung; Vo, Jennifer; Hamilton, Bruce A

    2011-10-01

    Development of neural circuitry depends on the integration of signaling pathways to coordinate specification, proliferation and differentiation of cell types in the right number, in the right place, at the right time. Zinc finger protein 423 (Zfp423), a 30-zinc finger transcription factor, forms alternate complexes with components of several developmental signaling pathways, suggesting it as a point of signal integration during brain development. We previously showed that mice lacking Zfp423 have reduced proliferation of cerebellar precursor cells, resulting in complete loss of vermis and variable hypoplasia of cerebellar hemispheres. Here, we show that Zfp423(-/-) hemisphere malformations are shaped by both genetic and non-genetic factors, producing distinct phenotype distributions in different inbred genetic backgrounds. In genetic mapping studies, we identify four additive modifier loci (Amzn1-4) and seven synthetically interacting loci (Smzn1.1-3.1) that together explain approximately one-third of the phenotypic variance. Strain-specific sequence polymorphism and expression data provide a reduced list of functional variant candidate genes at each modifier locus. Environmental covariates add only modest explanatory power, suggesting an additional stochastic component. These results provide a comprehensive analysis of sources of phenotype variation in a model of hindbrain malformation.

  6. Measuring aging rates of mice subjected to caloric restriction and genetic disruption of growth hormone signaling

    PubMed Central

    Koopman, Jacob J.E.; van Heemst, Diana; van Bodegom, David; Bonkowski, Michael S.; Sun, Liou Y.; Bartke, Andrzej

    2016-01-01

    Caloric restriction and genetic disruption of growth hormone signaling have been shown to counteract aging in mice. The effects of these interventions on aging are examined through age-dependent survival or through the increase in age-dependent mortality rates on a logarithmic scale fitted to the Gompertz model. However, these methods have limitations that impede a fully comprehensive disclosure of these effects. Here we examine the effects of these interventions on murine aging through the increase in age-dependent mortality rates on a linear scale without fitting them to a model like the Gompertz model. Whereas these interventions negligibly and non-consistently affected the aging rates when examined through the age-dependent mortality rates on a logarithmic scale, they caused the aging rates to increase at higher ages and to higher levels when examined through the age-dependent mortality rates on a linear scale. These results add to the debate whether these interventions postpone or slow aging and to the understanding of the mechanisms by which they affect aging. Since different methods yield different results, it is worthwhile to compare their results in future research to obtain further insights into the effects of dietary, genetic, and other interventions on the aging of mice and other species. PMID:26959761

  7. Continues administration of Nano-PSO significantly increased survival of genetic CJD mice.

    PubMed

    Binyamin, Orli; Keller, Guy; Frid, Kati; Larush, Liraz; Magdassi, Shlomo; Gabizon, Ruth

    2017-08-25

    We have shown previously that Nano-PSO, a nanodroplet formulation of pomegranate seed oil, delayed progression of neurodegeneration signs when administered for a designated period of time to TgMHu2ME199K mice, modeling for genetic prion disease. In the present work, we treated these mice with a self-emulsion formulation of Nano-PSO or a parallel Soybean oil formulation from their day of birth until a terminal disease stage. We found that long term Nano-PSO administration resulted in increased survival of TgMHu2ME199K lines by several months. Interestingly, initiation of treatment at day 1 had no clinical advantage over initiation at day 70, however cessation of treatment at 9months of age resulted in the rapid loss of the beneficial clinical effect. Pathological studies revealed that treatment with Nano-PSO resulted in the reduction of GAG accumulation and lipid oxidation, indicating a strong neuroprotective effect. Contrarily, the clinical effect of Nano-PSO did not correlate with reduction in the levels of disease related PrP, the main prion marker. We conclude that long term administration of Nano-PSO is safe and may be effective in the prevention/delay of onset of neurodegenerative conditions such as genetic CJD. Copyright © 2017. Published by Elsevier Inc.

  8. Host genetic influences on the anthelmintic efficacy of papaya-derived cysteine proteinases in mice.

    PubMed

    Luoga, Wenceslaus; Mansur, Fadlul; Stepek, Gillian; Lowe, Ann; Duce, Ian R; Buttle, David J; Behnke, Jerzy M

    2015-06-01

    Eight strains of mice, of contrasting genotypes, infected with Heligmosomoides bakeri were studied to determine whether the anthelmintic efficacy of papaya latex varied between inbred mouse strains and therefore whether there is an underlying genetic influence on the effectiveness of removing the intestinal nematode. Infected mice were treated with 330 nmol of crude papaya latex or with 240 nmol of papaya latex supernatant (PLS). Wide variation of response between different mouse strains was detected. Treatment was most effective in C3H (90·5-99·3% reduction in worm counts) and least effective in CD1 and BALB/c strains (36·0 and 40·5%, respectively). Cimetidine treatment did not improve anthelmintic efficacy of PLS in a poor drug responder mouse strain. Trypsin activity, pH and PLS activity did not differ significantly along the length of the gastro-intestinal (GI) tract between poor (BALB/c) and high (C3H) drug responder mouse strains. Our data indicate that there is a genetic component explaining between-mouse variation in the efficacy of a standard dose of PLS in removing worms, and therefore warrant some caution in developing this therapy for wider scale use in the livestock industry, and even in human medicine.

  9. A genetically adjuvanted influenza B virus vector increases immunogenicity and protective efficacy in mice.

    PubMed

    Kittel, Christian; Wressnigg, Nina; Shurygina, Anna Polina; Wolschek, Markus; Stukova, Marina; Romanovskaya-Romanko, Ekatherina; Romanova, Julia; Kiselev, Oleg; Muster, Thomas; Egorov, Andrej

    2015-10-01

    The existence of multiple antigenically distinct types and subtypes of influenza viruses allows the construction of a multivalent vector system for the mucosal delivery of foreign sequences. Influenza A viruses have been exploited successfully for the expression of extraneous antigens as well as immunostimulatory molecules. In this study, we describe the development of an influenza B virus vector whose functional part of the interferon antagonist NS1 was replaced by human interleukin 2 (IL2) as a genetic adjuvant. We demonstrate that IL2 expressed by this viral vector displays immune adjuvant activity in immunized mice. Animals vaccinated with the IL2 viral vector showed an increased hemagglutination inhibition antibody response and higher protective efficacy after challenge with a wild-type influenza B virus when compared to mice vaccinated with a control virus. Our results demonstrate that it is feasible to construct influenza B vaccine strains expressing immune-potentiating foreign sequences from the NS genomic segment. Based on these data, it is now hypothetically possible to create a trivalent (or quadrivalent) live attenuated influenza vaccine in which each component expresses a selected genetic adjuvant with tailored expression levels.

  10. Measuring aging rates of mice subjected to caloric restriction and genetic disruption of growth hormone signaling.

    PubMed

    Koopman, Jacob J E; van Heemst, Diana; van Bodegom, David; Bonkowski, Michael S; Sun, Liou Y; Bartke, Andrzej

    2016-03-01

    Caloric restriction and genetic disruption of growth hormone signaling have been shown to counteract aging in mice. The effects of these interventions on aging are examined through age-dependent survival or through the increase in age-dependent mortality rates on a logarithmic scale fitted to the Gompertz model. However, these methods have limitations that impede a fully comprehensive disclosure of these effects. Here we examine the effects of these interventions on murine aging through the increase in age-dependent mortality rates on a linear scale without fitting them to a model like the Gompertz model. Whereas these interventions negligibly and non-consistently affected the aging rates when examined through the age-dependent mortality rates on a logarithmic scale, they caused the aging rates to increase at higher ages and to higher levels when examined through the age-dependent mortality rates on a linear scale. These results add to the debate whether these interventions postpone or slow aging and to the understanding of the mechanisms by which they affect aging. Since different methods yield different results, it is worthwhile to compare their results in future research to obtain further insights into the effects of dietary, genetic, and other interventions on the aging of mice and other species.

  11. Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity.

    PubMed

    French, John E; Gatti, Daniel M; Morgan, Daniel L; Kissling, Grace E; Shockley, Keith R; Knudsen, Gabriel A; Shepard, Kim G; Price, Herman C; King, Deborah; Witt, Kristine L; Pedersen, Lars C; Munger, Steven C; Svenson, Karen L; Churchill, Gary A

    2015-03-01

    Inhalation of benzene at levels below the current exposure limit values leads to hematotoxicity in occupationally exposed workers. We sought to evaluate Diversity Outbred (DO) mice as a tool for exposure threshold assessment and to identify genetic factors that influence benzene-induced genotoxicity. We exposed male DO mice to benzene (0, 1, 10, or 100 ppm; 75 mice/exposure group) via inhalation for 28 days (6 hr/day for 5 days/week). The study was repeated using two independent cohorts of 300 animals each. We measured micronuclei frequency in reticulocytes from peripheral blood and bone marrow and applied benchmark concentration modeling to estimate exposure thresholds. We genotyped the mice and performed linkage analysis. We observed a dose-dependent increase in benzene-induced chromosomal damage and estimated a benchmark concentration limit of 0.205 ppm benzene using DO mice. This estimate is an order of magnitude below the value estimated using B6C3F1 mice. We identified a locus on Chr 10 (31.87 Mb) that contained a pair of overexpressed sulfotransferases that were inversely correlated with genotoxicity. The genetically diverse DO mice provided a reproducible response to benzene exposure. The DO mice display interindividual variation in toxicity response and, as such, may more accurately reflect the range of response that is observed in human populations. Studies using DO mice can localize genetic associations with high precision. The identification of sulfotransferases as candidate genes suggests that DO mice may provide additional insight into benzene-induced genotoxicity.

  12. Mitochondria-Targeted Antioxidant SkQ1 Improves Dermal Wound Healing in Genetically Diabetic Mice.

    PubMed

    Demyanenko, Ilya A; Zakharova, Vlada V; Ilyinskaya, Olga P; Vasilieva, Tamara V; Fedorov, Artem V; Manskikh, Vasily N; Zinovkin, Roman A; Pletjushkina, Olga Yu; Chernyak, Boris V; Skulachev, Vladimir P; Popova, Ekaterina N

    2017-01-01

    Oxidative stress is widely recognized as an important factor in the delayed wound healing in diabetes. However, the role of mitochondrial reactive oxygen species in this process is unknown. It was assumed that mitochondrial reactive oxygen species are involved in many wound-healing processes in both diabetic humans and animals. We have applied the mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) to explore the role of mitochondrial reactive oxygen species in the wound healing of genetically diabetic mice. Healing of full-thickness excisional dermal wounds in diabetic C57BL/KsJ-db(-)/db(-) mice was significantly enhanced after long-term (12 weeks) administration of SkQ1. SkQ1 accelerated wound closure and stimulated epithelization, granulation tissue formation, and vascularization. On the 7th day after wounding, SkQ1 treatment increased the number of α-smooth muscle actin-positive cells (myofibroblasts), reduced the number of neutrophils, and increased macrophage infiltration. SkQ1 lowered lipid peroxidation level but did not change the level of the circulatory IL-6 and TNF. SkQ1 pretreatment also stimulated cell migration in a scratch-wound assay in vitro under hyperglycemic condition. Thus, a mitochondria-targeted antioxidant normalized both inflammatory and regenerative phases of wound healing in diabetic mice. Our results pointed to nearly all the major steps of wound healing as the target of excessive mitochondrial reactive oxygen species production in type II diabetes.

  13. Genetic ablation of lymphocytes and cytokine signaling in nonobese diabetic mice prevents diet-induced obesity and insulin resistance.

    PubMed

    Friedline, Randall H; Ko, Hwi Jin; Jung, Dae Young; Lee, Yongjin; Bortell, Rita; Dagdeviren, Sezin; Patel, Payal R; Hu, Xiaodi; Inashima, Kunikazu; Kearns, Caitlyn; Tsitsilianos, Nicholas; Shafiq, Umber; Shultz, Leonard D; Lee, Ki Won; Greiner, Dale L; Kim, Jason K

    2016-03-01

    Obesity is characterized by a dysregulated immune system, which may causally associate with insulin resistance and type 2 diabetes. Despite widespread use of nonobese diabetic (NOD) mice, NOD with severe combined immunodeficiency (scid) mutation (SCID) mice, and SCID bearing a null mutation in the IL-2 common γ chain receptor (NSG) mice as animal models of human diseases including type 1 diabetes, the underlying metabolic effects of a genetically altered immune system are poorly understood. For this, we performed a comprehensive metabolic characterization of these mice fed chow or after 6 wk of a high-fat diet. We found that NOD mice had ∼50% less fat mass and were 2-fold more insulin sensitive, as measured by hyperinsulinemic-euglycemic clamp, than C57BL/6 wild-type mice. SCID mice were also more insulin sensitive with increased muscle glucose metabolism and resistant to diet-induced obesity due to increased energy expenditure (∼10%) and physical activity (∼40%) as measured by metabolic cages. NSG mice were completely protected from diet-induced obesity and insulin resistance with significant increases in glucose metabolism in peripheral organs. Our findings demonstrate an important role of genetic background, lymphocytes, and cytokine signaling in diet-induced obesity and insulin resistance. © FASEB.

  14. Visualizing the enteric nervous system using genetically engineered double reporter mice: Comparison with immunofluorescence

    PubMed Central

    Jiang, Yanfen; Dong, Hui; Eckmann, Lars; Hanson, Elaine M.; Ihn, Katherine C.; Mittal, Ravinder K.

    2017-01-01

    Background and aims The enteric nervous system (ENS) plays a crucial role in the control of gastrointestinal motility, secretion and absorption functions. Immunohistochemistry has been widely used to visualize neurons of the ENS for more than two decades. Genetically engineered mice that report specific proteins can also be used to visualize neurons of the ENS. The goal of our study was to develop a mouse that expresses fluorescent neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT), the two proteins expressed in 95% of the ENS neurons. We compared ENS neurons visualized in the reporter mouse with the wild type mouse stained using classical immunostaining techniques. Methods Mice hemizygous for ChAT-ChR2-YFP BAC transgene with expression of the mhChR2:YFP fusion protein directed by ChAT promoter/enhancer regions on the BAC transgene were purchased commercially. The Cre/LoxP technique of somatic recombination was used to construct mice with nNOS positive neurons. The two mice were crossbred and tissues were harvested and examined using fluorescent microscopy. Immunostaining was performed in the wild type mice, using antibodies to nNOS, ChAT, Hu and PGP 9.5. Results Greater than 95% of the ENS neurons were positive for either nNOS or ChAT or both. The nNOS and ChAT neurons and their processes in the ENS were well visualized in all the regions of the GI tract, i.e., esophagus, small intestine and colon. The number of nNOS and ChAT neurons was approximately same in the reporter mouse and immunostaining method in the wild type mouse. The nNOS fluorescence in the reporter mouse was seen in both cytoplasm as well as nucleus but in the immunostained specimens it was seen only in the cytoplasm. Conclusion We propose that the genetically engineered double reporter mouse for ChAT and nNOS proteins is a powerful tool to study of the effects of various diseases on the ENS without the need for immunostaining. PMID:28158225

  15. Efficient genetic manipulation of the NOD-Rag1-/-IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology.

    PubMed

    Li, Feng; Cowley, Dale O; Banner, Debra; Holle, Eric; Zhang, Liguo; Su, Lishan

    2014-06-17

    Humanized mouse models have become increasingly important and widely used in modeling human diseases in biomedical research. Immunodeficient mice such as NOD-Rag1-/-IL2RgammaC-null (NRG) or NOD-SCID-IL2RgammaC-null (NSG) mice are critical for efficient engraftment of human cells or tissues. However, their genetic modification remains challenging due to a lack of embryonic stem cells and difficulty in the collection of timed embryos after superovulation. Here, we report the generation of gene knockout NRG mice by combining in vitro fertilization (IVF) and CRISPR/Cas9 technology. Sufficient numbers of fertilized embryos were produced through IVF, and a high rate of Fah gene targeting was achieved with microinjection of Cas9 mRNA, gRNA and single strand oligonucleotide DNA (ssDNA) into the embryos. The technology paves the way to construct NRG or NSG mutant mice to facilitate new humanized mouse models. The technology can also be readily adapted to introduce mutations in other species such as swine and non-human primates.

  16. Interactive effects between trichloroethylene and pesticides at metabolic and genetic level in mice.

    PubMed Central

    Hrelia, P; Maffei, F; Vigagni, F; Fimognari, C; Flori, P; Stanzani, R; Cantelli Forti, G

    1994-01-01

    A combined cytogeneticurine metabolite analysis approach was used to assess potential interactive effects between Fenarimol (FN), a fungicide, and trichloroethylene (TRI), a halogenated solvent. FN was demonstrated to selectively induce P450-2B1 isoforms in different organs of treated mice. Since the rate of metabolism and the stereospecificity of metabolism are dependent on the types and amount of P450s available, FN might drastically alter the metabolic activation of a precarcinogen, such as TRI, and its toxicological consequences. Male CD1 mice were divided into untreated, vehicle control, and experimental groups. Animals of the latter groups were treated ip with 150 mg/kg bw FN in corn oil, 457 mg/kg bw TRI in corn oil, TRI plus FN separated by different time intervals. Bone marrow cells were harvested for determination of micronuclei (MN) frequencies in polychromatic erythrocytes (PCE). The presence of the known metabolite of TRI, trichloroethanol (TCE), was quantitated in collected urine by gas chromatography using an electron-capture detector. Linear regression analysis shows that MN frequency by TRI is correlated with TCE concentration in urine. Observed potentiation of genotoxicity of TRI by FN pretreatment (1 hr before TRI treatment) apparently reflects changes in the spectra of enzymes involved in TRI metabolism, and altered toxicokinetic, as witnessed by the 20% difference in TCE excretion from combined treated mice. However, no increased genetic or metabolic effects were observed when FN was administered 3 hr before TRI. No significant interactive effects were observed at a genetic level when FN was administered 1 hr and 3 hr after TRI whereas a 33 to 47% loss in TCE excretion was recorded.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7698080

  17. The use of genetically modified mice in cancer risk assessment: Challenges and limitations*

    PubMed Central

    Eastmond, David A.; Vulimiri, Suryanarayana V.; French, John E.; Sonawane, Babasaheb

    2015-01-01

    The use of genetically modified (GM) mice to assess carcinogenicity is playing an increasingly important role in the safety evaluation of chemicals. While progress has been made in developing and evaluating mouse models such as the Trp53+/−, Tg.AC and the rasH2, the suitability of these models as replacements for the conventional rodent cancer bioassay and for assessing human health risks remains uncertain. The objective of this research was to evaluate the use of accelerated cancer bioassays with GM mice for assessing the potential health risks associated with exposure to carcinogenic agents. We compared the published results from the GM bioassays to those obtained in the National Toxicology Program’s conventional chronic mouse bioassay for their potential use in risk assessment. Our analysis indicates that the GM models are less efficient in detecting carcinogenic agents but more consistent in identifying non-carcinogenic agents. We identified several issues of concern related to the design of the accelerated bioassays (e.g., sample size, study duration, genetic stability and reproducibility) as well as pathway-dependency of effects, and different carcinogenic mechanisms operable in GM and non-GM mice. The use of the GM models for dose-response assessment is particularly problematic as these models are, at times, much more or less sensitive than the conventional rodent cancer bioassays. Thus, the existing GM mouse models may be useful for hazard identification, but will be of limited use for dose-response assessment. Hence, caution should be exercised when using GM mouse models to assess the carcinogenic risks of chemicals. PMID:23985072

  18. Genetic and epigenetic changes in fibrosis-associated hepatocarcinogenesis in mice.

    PubMed

    Chappell, Grace; Kutanzi, Kristy; Uehara, Takeki; Tryndyak, Volodymyr; Hong, Hue-Hua; Hoenerhoff, Mark; Beland, Frederick A; Rusyn, Ivan; Pogribny, Igor P

    2014-06-15

    Hepatocellular carcinoma (HCC) is one of the most prevalent cancers and is rising in incidence worldwide. The molecular mechanisms leading to the development of HCC are complex and include both genetic and epigenetic events. To determine the relative contribution of these alterations in liver tumorigenesis, we evaluated epigenetic modifications at both global and gene specific levels, as well as the mutational profile of genes commonly altered in liver tumors. A mouse model of fibrosis-associated liver cancer that was designed to emulate cirrhotic liver, a prevailing disease state observed in most humans with HCC, was used. Tumor and nontumor liver samples from B6C3F1 mice treated with N-nitrosodiethylamine (DEN; a single ip injection of 1 mg/kg at 14 days of age) and carbon tetrachloride (CCl4; 0.2 ml/kg, 2 times/week ip starting at 8 weeks of age for 14 weeks), as well as corresponding vehicle control animals, were analyzed for genetic and epigenetic alterations. H-ras, Ctnnb1 and Hnf1α genes were not mutated in tumors in mice treated with DEN+CCl4 . In contrast, the increased tumor incidence in mice treated with DEN+CCl4 was associated with marked epigenetic changes in liver tumors and nontumor liver tissue, including demethylation of genomic DNA and repetitive elements, a decrease in histone 3 lysine 9 trimethylation (H3K9me3) and promoter hypermethylation and functional downregulation of Riz1, a histone lysine methyltransferase tumor suppressor gene. Additionally, the reduction in H3K9me3 was accompanied by increased expression of long interspersed nucleotide elements 1 and short interspersed nucleotide elements B2, which is an indication of genomic instability. In summary, our results suggest that epigenetic events, rather than mutations in known cancer-related genes, play a prominent role in increased incidence of liver tumors in this mouse model of fibrosis-associated liver cancer.

  19. Comparison of tissue concentrations in male and female C57BL/6 mice

    USDA-ARS?s Scientific Manuscript database

    The tissue-specific response to dietary vitamin K (VK) manipulation has not been well studied in mice. This limits the use of genetically modified mouse models in VK studies. The objective of this study was to determine the sex-specific effects of dietary VK manipulation on serum, liver and extra-he...

  20. Laser microtreatment for genetic manipulations and DNA diagnostics by a combination of microbeam and photonic tweezers (laser microbeam trap)

    NASA Astrophysics Data System (ADS)

    Greulich, Karl-Otto; Monajembashi, Shamci; Celeda, D.; Endlich, N.; Eickhoff, Holger; Hoyer, Carsten; Leitz, G.; Weber, Gerd; Scheef, J.; Rueterjans, H.

    1994-12-01

    Genomes of higher organisms are larger than one typically expects. For example, the DNA of a single human cell is almost two meters long, the DNA in the human body covers the distance Earth-Sun approximately 140 times. This is often not considered in typical molecular biological approaches for DNA diagnostics, where usually only DNA of the length of a gene is investigated. Also, one basic aspect of sequencing the human genome is not really solved: the problem how to prepare the huge amounts of DNA required. Approaches from biomedical optics combined with new developments in single molecule biotechnology may at least contribute some parts of the puzzle. A large genome can be partitioned into portions comprising approximately 1% of the whole DNA using a laser microbeam. The single DNA fragment can be amplified by the polymerase chain reaction in order to obtain a sufficient amount of molecules for conventional DNA diagnostics or for analysis by octanucleotide hybridization. When not amplified by biotechnological processes, the individual DNA molecule can be visualized in the light microscope and can be manipulated and dissected with the laser microbeam trap. The DNA probes obtained by single molecule biotechnology can be employed for fluorescence in situ introduced into plant cells and subcellular structures even when other techniques fail. Since the laser microbeam trap allows to work in the interior of a cell without opening it, subcellular structures can be manipulated. For example, in algae, such structures can be moved out of their original position and used to study intracellular viscosities.

  1. Spontaneous bacterial and fungal infections in genetically engineered mice: Is Escherichia coli an emerging pathogen in laboratory mouse?

    PubMed

    Benga, Laurentiu; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Sager, Martin

    2015-01-01

    The impact of particular microbes on genetically engineered mice depends on the genotype and the environment. Infections resulting in clinical disease have an obvious impact on animal welfare and experimentation. In this study, we investigated the bacterial and fungal aetiology of spontaneous clinical disease of infectious origin among the genetically engineered mice from our institution in relation to their genotype. A total of 63 mice belonging to 33 different mice strains, from severe immunodeficient to wild-type, were found to display infections as the primary cause leading to their euthanasia. The necropsies revealed abscesses localized subcutaneously as well as in the kidney, preputial glands, seminal vesicles, in the uterus, umbilicus or in the lung. In addition, pneumonia, endometritis and septicaemia cases were recorded. Escherichia coli was involved in 21 of 44 (47.72%) of the lesions of bacterial origin, whereas [Pasteurella] pneumotropica was isolated from 19 of 44 (43.18%) cases. The infections with the two agents mentioned above included three cases of mixed infection with both pathogens. Staphylococcus aureus was considered responsible for five of 44 (11.36%) cases whereas Enterobacter cloacae was found to cause lesions in two of 44 (4.54%) mice. Overall, 16 of the 44 (36.36%) cases of bacterial aetiology affected genetically engineered mice without any explicit immunodeficiency or wild-type strains. The remaining 19 cases of interstitial pneumonia were caused by Pneumocystis murina. In conclusion, the susceptibility of genetically modified mice to opportunistic infections has to be regarded with precaution, regardless of the type of genetic modification performed. Beside the classical opportunists, such as [Pasteurella] pneumotropica and Staphylococcus aureus, Escherichia coli should as well be closely monitored to evaluate whether it represents an emerging pathogen in the laboratory mouse.

  2. Genetic Analysis of the Diversity and Origin of Hantaviruses in Peromyscus leucopus Mice in North America

    PubMed Central

    Morzunov, Sergey P.; Rowe, Joan E.; Ksiazek, Thomas G.; Peters, Clarence J.; St. Jeor, Stephen C.; Nichol, Stuart T.

    1998-01-01

    Nucleotide sequences were determined for the complete M genome segments of two distinct hantavirus genetic lineages which were detected in hantavirus antibody- and PCR-positive white-footed mice (Peromyscus leucopus) from Indiana and Oklahoma. Phylogenetic analyses indicated that although divergent from each other, the virus lineages in Indiana and Oklahoma were monophyletic and formed a newly identified unique ancestral branch within the clade of Sin Nombre-like viruses found in Peromyscus mice. Interestingly, P. leucopus-borne New York virus was found to be most closely related to the P. maniculatus-borne viruses, Sin Nombre and Monongahela, and monophyletic with Monongahela virus. In parallel, intraspecific phylogenetic relationships of P. leucopus were also determined, based on the amplification, sequencing, and analysis of the DNA fragment representing the replication control region of the rodent mitochondrial genome. P. leucopus mitochondrial DNA haplotypes were found to form four separate genetic clades, referred to here as Eastern, Central, Northwestern, and Southwestern groups. The distinct Indiana and Oklahoma virus lineages were detected in P. leucopus of the Eastern and Southwestern mitochondrial DNA haplotypes, respectively. Taken together, our current data suggests that both cospeciation of Peromyscus-borne hantaviruses with their specific rodent hosts and biogeographic factors (such as allopatric migrations, geographic separation, and isolation) have played important roles in establishment of the current genetic diversity and geographic distribution of Sin Nombre-like hantaviruses. In particular, the unusual position of New York virus on the virus phylogenetic tree is most consistent with an historically recent host-switching event. PMID:9420200

  3. Genetic analysis of the diversity and origin of hantaviruses in Peromyscus leucopus mice in North America.

    PubMed

    Morzunov, S P; Rowe, J E; Ksiazek, T G; Peters, C J; St Jeor, S C; Nichol, S T

    1998-01-01

    Nucleotide sequences were determined for the complete M genome segments of two distinct hantavirus genetic lineages which were detected in hantavirus antibody- and PCR-positive white-footed mice (Peromyscus leucopus) from Indiana and Oklahoma. Phylogenetic analyses indicated that although divergent from each other, the virus lineages in Indiana and Oklahoma were monophyletic and formed a newly identified unique ancestral branch within the clade of Sin Nombre-like viruses found in Peromyscus mice. Interestingly, P. leucopus-borne New York virus was found to be most closely related to the P. maniculatus-borne viruses, Sin Nombre and Monongahela, and monophyletic with Monongahela virus. In parallel, intraspecific phylogenetic relationships of P. leucopus were also determined, based on the amplification, sequencing, and analysis of the DNA fragment representing the replication control region of the rodent mitochondrial genome. P. leucopus mitochondrial DNA haplotypes were found to form four separate genetic clades, referred to here as Eastern, Central, Northwestern, and Southwestern groups. The distinct Indiana and Oklahoma virus lineages were detected in P. leucopus of the Eastern and Southwestern mitochondrial DNA haplotypes, respectively. Taken together, our current data suggests that both cospeciation of Peromyscus-borne hantaviruses with their specific rodent hosts and biogeographic factors (such as allopatric migrations, geographic separation, and isolation) have played important roles in establishment of the current genetic diversity and geographic distribution of Sin Nombre-like hantaviruses. In particular, the unusual position of New York virus on the virus phylogenetic tree is most consistent with an historically recent host-switching event.

  4. Managing major data of genetically modified mice: from scientific demands to legal obligations.

    PubMed

    Staudt, Michael; Trauth, Jürgen; Hindi, Iris El; Galuschka, Claudia; Sitek, Dagmar; Schenkel, Johannes

    2012-10-01

    The number of genetically modified mice is increasing rapidly. Several limitations when working with these animals are to be considered: small colonies, the continued danger of loss, often a limited breeding-success, the need to keep those mutants in stock, difficult and costly import-procedures, and also a major (scientific) value of those mutants often available only with major restrictions. To gather relevant information about all active and archived genetically modified mouse lines available in-house (>1.500) and to deal with a unique resource for several, quite different purposes, a data base was developed enabling optimum knowledge management and easy access. The data base covers also legal restraints and is being linked with the institutional publication repository. To identify the lines available detailed information is provided for each line, as the international designation, a short name, the characterization/description, and the genetic modification including the technique used therefore. The origin of the mutation (gene-ID# and donor organism), the origin of regulatory elements and their donors are listed as well as the genetic background, back-cross generation, phenotype, possible publications, keywords, and some in-house information. Also aspects of animal welfare, obligations to record genetically modified organisms, and technology transfer are displayed; the latter to make licenses possible (if legally permitted). Material transfer agreements, patents, or legal restrictions are listed. This data base helps to avoid double-imports, saves animals and costs since a redundant generation or import can be omitted. However, this is a contribution to the 3R principles developed by Russell and Burch.

  5. Immune resistance of semisyngeneic F1 hybrid mice to lymphoma grafts differs from natural hybrid resistance in its genetic pattern

    SciTech Connect

    Klein, G.O.; Klein, G.

    1984-07-01

    Resistance of semisyngeneic F1 hybrid mice immunized three times with irradiated tumor cells was compared to the genetic pattern of natural hybrid resistance to challenge with live tumor cells. Syngeneic mice responded equally well to immunization with all five hemopoietic tumor lines tested as the naturally much more highly resistant F1 hybrids. Natural hybrid resistance was found to be severely reduced by sublethal irradiation with 4 Gy, in contrast to hybrid resistance to parental bone marrow.

  6. Animal models of physiologic markers of male reproduction: genetically defined infertile mice

    SciTech Connect

    Chubb, C.

    1987-10-01

    The present report focuses on novel animal models of male infertility: genetically defined mice bearing single-gene mutations that induce infertility. The primary goal of the investigations was to identify the reproductive defects in these mutant mice. The phenotypic effects of the gene mutations were deciphered by comparing the mutant mice to their normal siblings. Initially testicular steroidogenesis and spermatogenesis were investigated. The physiologic markers for testicular steroidogenesis were steroid secretion by testes perifused in vitro, seminal vesicle weight, and Leydig cell histology. Spermatogenesis was evaluated by the enumeration of homogenization-resistant sperm/spermatids in testes and by morphometric analyses of germ cells in the seminiferous epithelium. If testicular function appeared normal, the authors investigated the sexual behavior of the mice. The parameters of male sexual behavior that were quantified included mount patency, mount frequency, intromission latency, thrusts per intromission, ejaculation latency, and ejaculation duration. Females of pairs breeding under normal circumstances were monitored for the presence of vaginal plugs and pregnancies. The patency of the ejaculatory process was determined by quantifying sperm in the female reproductive tract after sexual behavior tests. Sperm function was studied by quantitatively determining sperm motility during videomicroscopic observation. Also, the ability of epididymal sperm to function within the uterine environment was analyzed by determining sperm capacity to initiate pregnancy after artificial insemination. Together, the experimental results permitted the grouping of the gene mutations into three general categories. They propose that the same biological markers used in the reported studies can be implemented in the assessment of the impact that environmental toxins may have on male reproduction.

  7. Repeated ozone exposure exacerbates insulin resistance and activates innate immune response in genetically susceptible mice.

    PubMed

    Zhong, Jixin; Allen, Katryn; Rao, Xiaoquan; Ying, Zhekang; Braunstein, Zachary; Kankanala, Saumya R; Xia, Chang; Wang, Xiaoke; Bramble, Lori A; Wagner, James G; Lewandowski, Ryan; Sun, Qinghua; Harkema, Jack R; Rajagopalan, Sanjay

    2016-08-01

    Inhaled ozone (O3) has been demonstrated as a harmful pollutant and associated with chronic inflammatory diseases such as diabetes and vascular disorders. However, the underlying mechanisms by which O3 mediates harmful effects are poorly understood. To investigate the effect of O3 exposure on glucose intolerance, immune activation and underlying mechanisms in a genetically susceptible mouse model. Diabetes-prone KK mice were exposed to filtered air (FA), or O3 (0.5 ppm) for 13 consecutive weekdays (4 h/day). Insulin tolerance test (ITT) was performed following the last exposure. Plasma insulin, adiponectin, and leptin were measured by ELISA. Pathologic changes were examined by H&E and Oil-Red-O staining. Inflammatory responses were detected using flow cytometry and real-time PCR. KK mice exposed to O3 displayed an impaired insulin response. Plasma insulin and leptin levels were reduced in O3-exposed mice. Three-week exposure to O3 induced lung inflammation and increased monocytes/macrophages in both blood and visceral adipose tissue. Inflammatory monocytes/macrophages increased both systemically and locally. CD4 + T cell activation was also enhanced by the exposure of O3 although the relative percentage of CD4 + T cell decreased in blood and adipose tissue. Multiple inflammatory genes including CXCL-11, IFN-γ, TNFα, IL-12, and iNOS were up-regulated in visceral adipose tissue. Furthermore, the expression of oxidative stress-related genes such as Cox4, Cox5a, Scd1, Nrf1, and Nrf2, increased in visceral adipose tissue of O3-exposed mice. Repeated O3 inhalation induces oxidative stress, adipose inflammation and insulin resistance.

  8. Repeated Ozone Exposure Exacerbates Insulin Resistance And Activates Innate Immune Response In Genetically Susceptible Mice

    PubMed Central

    Zhong, Jixin; Allen, Katryn; Rao, Xiaoquan; Ying, Zhekang; Braunstein, Zachary; Kankanala, Saumya R.; Xia, Chang; Wang, Xiaoke; Bramble, Lori A.; Wagner, James G.; Lewandowski, Ryan; Sun, Qinghua; Harkema, Jack R.; Rajagopalan, Sanjay

    2016-01-01

    Background Inhaled ozone (O3) has been demonstrated as a harmful pollutant and associated with chronic inflammatory diseases such as diabetes and vascular disorders. However, the underlying mechanisms by which O3 mediates harmful effects are poorly understood. Objectives To investigate the effect of O3 exposure on glucose intolerance, immune activation and underlying mechanisms in a genetically susceptible mouse model. Methods Diabetes-prone KK mice were exposed to filtered air (FA), or O3 (0.5 ppm) for 13 consecutive weekdays (4 h/day). Insulin tolerance test (ITT) was performed following the last exposure. Plasma insulin, adiponectin, and leptin were measured by ELISA. Pathologic changes were examined by H&E and oil-red-o staining. Inflammatory responses were detected using flow cytometry and real-time PCR. Results KK mice exposed to O3 displayed an impaired insulin response. Plasma insulin and leptin levels were reduced in O3-exposed mice. Three-week exposure to O3 induced lung inflammation and increased monocytes/macrophages in both blood and visceral adipose tissue. Inflammatory monocytes/macrophages increased both systemically and locally. CD4+ T cell activation was also enhanced by the exposure of O3 although the relative percentage of CD4+ T cell decreased in blood and adipose tissue. Multiple inflammatory genes including CXCL-11, IFN-γ, TNFα, IL-12, and iNOS were up-regulated in visceral adipose tissue. Furthermore, the expression of oxidative stress-related genes such as Cox4, Cox5a, Scd1, Nrf1, and Nrf2, increased in visceral adipose tissue of O3-exposed mice. Conclusions Repeated O3 inhalation induces oxidative stress, adipose inflammation and insulin resistance. PMID:27240593

  9. RMI1 deficiency in mice protects from diet and genetic-induced obesity.

    PubMed

    Suwa, Akira; Yoshino, Masayasu; Yamazaki, Chihiro; Naitou, Masanori; Fujikawa, Rie; Matsumoto, Shun-Ichiro; Kurama, Takeshi; Shimokawa, Teruhiko; Aramori, Ichiro

    2010-02-01

    The aim of this study is to discover and characterize novel energy homeostasis-related molecules. We screened stock mouse embryonic stem cells established using the exchangeable gene trap method, and examined the effects of deficiency of the target gene on diet and genetic-induced obesity. The mutant strain 0283, which has an insertion at the recQ-mediated genome instability 1 (RMI1) locus, possesses a number of striking features that allow it to resist metabolic abnormalities. Reduced RMI1 expression, lower fasting-blood glucose and a reduced body weight (normal diet) were observed in the mutant mice. When fed a high-fat diet, the mutant mice were resistant to obesity, and also showed improved glucose intolerance and reduced abdominal fat tissue mass and food intake. In addition, the mutants were also resistant to obesity induced by the lethal yellow agouti (A(y)) gene. Endogenous RMI1 genes were found to be up-regulated in the liver and adipose tissue of KK-A(y) mice. RMI1 is a component of the Bloom's syndrome gene helicase complex that maintains genome integrity and activates cell-cycle checkpoint machinery. Interestingly, diet-induced expression of E2F8 mRNA, which is an important cell cycle-related molecule, was suppressed in the mutant mice. These results suggest that the regulation of energy balance by RMI1 is attributable to the regulation of food intake and E2F8 expression in adipose tissue. Taken together, these findings demonstrate that RMI1 is a novel molecule that regulates energy homeostasis.

  10. FKBP5 Moderates Alcohol Withdrawal Severity: Human Genetic Association and Functional Validation in Knockout Mice

    PubMed Central

    Huang, Ming-Chyi; Schwandt, Melanie L; Chester, Julia A; Kirchhoff, Aaron M; Kao, Chung-Feng; Liang, Tiebing; Tapocik, Jenica D; Ramchandani, Vijay A; George, David T; Hodgkinson, Colin A; Goldman, David; Heilig, Markus

    2014-01-01

    Alcohol withdrawal is associated with hypothalamic–pituitary–adrenal (HPA) axis dysfunction. The FKBP5 gene codes for a co-chaperone, FK506-binding protein 5, that exerts negative feedback on HPA axis function. This study aimed to examine the effects of single-nucleotide polymorphisms (SNPs) of the FKBP5 gene in humans and the effect of Fkbp5 gene deletion in mice on alcohol withdrawal severity. We genotyped six FKBP5 SNPs (rs3800373, rs9296158, rs3777747, rs9380524, rs1360780, and rs9470080) in 399 alcohol-dependent inpatients with alcohol consumption 48 h before admission and recorded scores from the Clinical Institute Withdrawal Assessment-Alcohol revised (CIWA-Ar). Fkbp5 gene knockout (KO) and wild-type (WT) mice were assessed for alcohol withdrawal using handling-induced convulsions (HICs) following both acute and chronic alcohol exposure. We found the minor alleles of rs3800373 (G), rs9296158 (A), rs1360780 (T), and rs9470080 (T) were significantly associated with lower CIWA-Ar scores whereas the minor alleles of rs3777747 (G) and rs9380524 (A) were associated with higher scores. The haplotype-based analyses also showed an association with alcohol withdrawal severity. Fkbp5 KO mice showed significantly greater HICs during withdrawal from chronic alcohol exposure compared with WT controls. This study is the first to show a genetic effect of FKBP5 on the severity of alcohol withdrawal syndrome. In mice, the absence of the Fkbp5 gene enhances sensitivity to alcohol withdrawal. We suggest that FKBP5 variants may trigger different adaptive changes in HPA axis regulation during alcohol withdrawal with concomitant effects on withdrawal severity. PMID:24603855

  11. Effect of sitagliptin treatment on metabolism and cardiac function in genetic diabetic mice.

    PubMed

    Hemmeryckx, Bianca; Swinnen, Melissa; Gallacher, David J; Rong Lu, Hua; Roger Lijnen, H

    2014-01-15

    To investigate the chronic effect of sitagliptin (7-[(3R)-3-amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-tetrahydro-(3-(trifluoromethyl)-1,2,4-triazolo[4,3-a]pyrazine phosphate (1:1) monohydrate, SIT) on metabolism and cardiac function in genetic diabetic Akita mice, 10 weeks old Akita mice were either exposed for 4 months to a high fat and high cholesterol (HF-HC) diet, with or without 10mg/kg/day SIT, or were fed for 3 months with the same diet with or without 50mg/kg/day SIT. SIT treatment of Akita mice at either a low or high dose did not affect body or liver weight. A significant increase in subcutaneous and gonadal fat mass was only observed for the 50mg/kg/day dose of SIT. Furthermore, only the 50mg/kg/day SIT dose resulted in an improvement of glycemic control, as evidenced by a decrease in fasting blood HbA1c levels and an increase in plasma adiponectin levels. Echocardiographic analysis revealed that Akita mice kept on the HF-HC diet with 10mg/kg/day of SIT for 4 months showed an increase in ejection fraction and fractional shortening, whereas the higher dose (50mg/kg/day) had no effect on these parameters, but instead induced left ventricular (LV) hypertrophy as evidenced by an enlarged LV internal diameter, volume and mass. Thus, in the diabetic Akita mouse SIT is cardioprotective at a low dose (10mg/kg/day), whereas improvement of glycemic control requires a higher dose (50mg/kg/day) which, however, induces LV hypertrophy. This mouse model may thus be useful to study the safety of anti-diabetic drugs. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Animal models of physiologic markers of male reproduction: genetically defined infertile mice.

    PubMed Central

    Chubb, C

    1987-01-01

    The present report focuses on novel animal models of male infertility: genetically defined mice bearing single-gene mutations that induce infertility. The primary goal of our investigations was to identify the reproductive defects in these mutant mice. The phenotypic effects of the gene mutations were deciphered by comparing the mutant mice to their normal siblings. Initially testicular steroidogenesis and spermatogenesis were investigated. The physiologic markers for testicular steroidogenesis were steroid secretion by testes perifused in vitro, seminal vesicle weight, and Leydig cell histology. Spermatogenesis was evaluated by the enumeration of homogenization-resistant sperm/spermatids in testes and by morphometric analyses of germ cells in the seminiferous epithelium. If testicular function appeared normal, we investigated the sexual behavior of the mice. The parameters of male sexual behavior that were quantified included mount patency, mount frequency, intromission latency, thrusts per intromission, ejaculation latency, and ejaculation duration. Females of pairs breeding under normal circumstances were monitored for the presence of vaginal plugs and pregnancies. The patency of the ejaculatory process was determined by quantifying sperm in the female reproductive tract after sexual behavior tests. Sperm function was studied by quantitatively determining sperm motility during videomicroscopic observation. Also, the ability of epididymal sperm to function within the uterine environment was analyzed by determining sperm capacity to initiate pregnancy after artificial insemination. Together, the experimental results permitted the grouping of the gene mutations into three general categories. We propose that the same biological markers used in the reported studies can be implemented in the assessment of the impact that environmental toxins may have on male reproduction. PMID:3319549

  13. Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice

    PubMed Central

    Sastre, Cristina; Fernández-Laso, Valvanera; Madrigal-Matute, Julio; Muñoz-García, Begoña; Moreno, Juan A; Pastor-Vargas, Carlos; Llamas-Granda, Patricia; Burkly, Linda C; Egido, Jesús; Martín-Ventura, Jose L; Blanco-Colio, Luis M

    2014-01-01

    Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12−/−ApoE−/−) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12−/−ApoE−/− or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12+/+ApoE−/− control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12−/−ApoE−/−. A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12+/+ApoE−/− mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12−/−ApoE−/− or anti-TWEAK treatment in TNFSF12+/+ApoE−/− mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish

  14. Fine-Mapping and Genetic Analysis of the Loci Affecting Hepatic Iron Overload in Mice

    PubMed Central

    Guo, Xin; Zhang, Zhuzhen; Zhang, Fan; Tao, Yunlong; An, Peng; Wu, Qian; Wang, Chia-Yu; Knutson, Mitchell D.; Wang, Fudi

    2013-01-01

    The liver, as the major organ for iron storage and production of hepcidin, plays pivotal roles in maintaining mammalian iron homeostasis. A previous study showed that Quantitative Trait Loci (QTLs) on chromosome 7 (Chr7) and 16 (Chr16) may control hepatic non-heme iron overload in an F2 intercross derived from C57BL/6J (B6) and SWR/J (SWR) mice. In this study, we aimed to validate the existence of these loci and identify the genes responsible for the phenotypic variations by generating congenic mice carrying SWR chromosome segments expanding these QTLs (D7Mit68-D7Mit71 and D16Mit125-D16Mit185, respectively). We excluded involvement of Chr7 based on the lack of iron accumulation in congenic mice. In contrast, liver iron accumulation was observed in Chr16 congenic mice. Through use of a series of subcongenic murine lines the interval on Chr16 was further fine-mapped to a 0.8 Mb segment spanning 11 genes. We found that the mRNA expression pattern in the liver remained unchanged for all 11 genes tested. Most importantly, we detected 4 missense mutations in 3 candidate genes including Sidt1 (P172R), Spice1(R708S), Boc (Q1051R) and Boc (S450-insertion in B6 allele) in the liver of SWR homozygous congenic mice. To further delineate potential modifier gene(s), we reconstituted seven candidate genes, Sidt1, Boc, Zdhhc23, Gramd1c, Atp6v1a, Naa50 and Gtpbp8, in mouse liver through hydrodynamic transfection. However, we were unable to detect significant changes in liver iron levels upon reconstitution of these candidate genes. Taken together, our work provides strong genetic evidence of the existence of iron modifiers on Chr16. Moreover, we were able to delineate the phenotypically responsible region to a 0.8 Mb region containing 11 coding genes, 3 of which harbor missense mutations, using a series of congenic mice. PMID:23675470

  15. Genetic Restoration of Plasma ApoE Improves Cognition and Partially Restores Synaptic Defects in ApoE-Deficient Mice.

    PubMed

    Lane-Donovan, Courtney; Wong, Wen Mai; Durakoglugil, Murat S; Wasser, Catherine R; Jiang, Shan; Xian, Xunde; Herz, Joachim

    2016-09-28

    Alzheimer's disease (AD) is the most common form of dementia in individuals over the age of 65 years. The most prevalent genetic risk factor for AD is the ε4 allele of apolipoprotein E (ApoE4), and novel AD treatments that target ApoE are being considered. One unresolved question in ApoE biology is whether ApoE is necessary for healthy brain function. ApoE knock-out (KO) mice have synaptic loss and cognitive dysfunction; however, these findings are complicated by the fact that ApoE knock-out mice have highly elevated plasma lipid levels, which may independently affect brain function. To bypass the effect of ApoE loss on plasma lipids, we generated a novel mouse model that expresses ApoE normally in peripheral tissues, but has severely reduced ApoE in the brain, allowing us to study brain ApoE loss in the context of a normal plasma lipid profile. We found that these brain ApoE knock-out (bEKO) mice had synaptic loss and dysfunction similar to that of ApoE KO mice; however, the bEKO mice did not have the learning and memory impairment observed in ApoE KO mice. Moreover, we found that the memory deficit in the ApoE KO mice was specific to female mice and was fully rescued in female bEKO mice. Furthermore, while the AMPA/NMDA ratio was reduced in ApoE KO mice, it was unchanged in bEKO mice compared with controls. These findings suggest that plasma lipid levels can influence cognition and synaptic function independent of ApoE expression in the brain. One proposed treatment strategy for Alzheimer's disease (AD) is the reduction of ApoE, whose ε4 isoform is the most common genetic risk factor for the disease. A major concern of this strategy is that an animal model of ApoE deficiency, the ApoE knock-out (KO) mouse, has reduced synapses and cognitive impairment; however, these mice also develop dyslipidemia and severe atherosclerosis. Here, we have shown that genetic restoration of plasma ApoE to wild-type levels normalizes plasma lipids in ApoE KO mice. While this does

  16. Blood pressure, heart rate and tubuloglomerular feedback in A1AR-deficient mice with different genetic backgrounds.

    PubMed

    Kim, S M; Mizel, D; Qin, Y; Huang, Y; Schnermann, J

    2015-01-01

    Differences in genetic background between control mice and mice with targeted gene mutations have been recognized as a potential cause for phenotypic differences. In this study, we have used A1AR-deficient mice in a C57Bl/6 and SWR/J congenic background to assess the influence of background on the effect of A1AR-deficiency on cardiovascular and renal functional parameters. In A1AR+/+ and A1AR-/- mice in C57Bl/6 and SWR/J congenic backgrounds, we assessed blood pressure and heart rate using radio-telemetry, plasma renin concentrations and tubuloglomerular feedback. We did not detect significant differences in arterial blood pressure (MAP) and heart rates (HR) between A1AR+/+ and A1AR-/- mice in either C57Bl/6, SWR/J or mixed backgrounds. MAP and HR were significantly higher in SWR/J than in C57Bl/6 mice. A high NaCl intake increased MAP in A1AR-/- mice on C57Bl/6 background while there was less or no salt sensitivity in the SWR/J background. No significant differences in plasma renin concentration were detected between A1AR-/- and A1AR+/+ mice in any of the strains. Tubuloglomerular feedback was found to be absent in A1AR-/- mice with SWR/J genetic background. While this study confirmed important differences between inbred mouse strains, we did not identify phenotypic modifications of A1AR-related effects on blood pressure, heart rate and plasma renin by differences in genetic background. © 2014 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  17. Genetic ablation of lymphocytes and cytokine signaling in nonobese diabetic mice prevents diet-induced obesity and insulin resistance

    PubMed Central

    Friedline, Randall H.; Ko, Hwi Jin; Jung, Dae Young; Lee, Yongjin; Bortell, Rita; Dagdeviren, Sezin; Patel, Payal R.; Hu, Xiaodi; Inashima, Kunikazu; Kearns, Caitlyn; Tsitsilianos, Nicholas; Shafiq, Umber; Shultz, Leonard D.; Lee, Ki Won; Greiner, Dale L.; Kim, Jason K.

    2016-01-01

    Obesity is characterized by a dysregulated immune system, which may causally associate with insulin resistance and type 2 diabetes. Despite widespread use of nonobese diabetic (NOD) mice, NOD with severe combined immunodeficiency (scid) mutation (SCID) mice, and SCID bearing a null mutation in the IL-2 common γ chain receptor (NSG) mice as animal models of human diseases including type 1 diabetes, the underlying metabolic effects of a genetically altered immune system are poorly understood. For this, we performed a comprehensive metabolic characterization of these mice fed chow or after 6 wk of a high-fat diet. We found that NOD mice had ∼50% less fat mass and were 2-fold more insulin sensitive, as measured by hyperinsulinemic-euglycemic clamp, than C57BL/6 wild-type mice. SCID mice were also more insulin sensitive with increased muscle glucose metabolism and resistant to diet-induced obesity due to increased energy expenditure (∼10%) and physical activity (∼40%) as measured by metabolic cages. NSG mice were completely protected from diet-induced obesity and insulin resistance with significant increases in glucose metabolism in peripheral organs. Our findings demonstrate an important role of genetic background, lymphocytes, and cytokine signaling in diet-induced obesity and insulin resistance.—Friedline, R. H., Ko, H. J., Jung, D. Y., Lee, Y., Bortell, R., Dagdeviren, S., Patel, P. R., Hu, X., Inashima, K., Kearns, C., Tsitsilianos, N., Shafiq, U., Shultz, L. D., Lee, K. W., Greiner, D. L., Kim, J. K. Genetic ablation of lymphocytes and cytokine signaling in nonobese diabetic mice prevents diet-induced obesity and insulin resistance. PMID:26644351

  18. Genetic polymorphisms and their association with brain and behavioural measures in heterogeneous stock mice.

    PubMed

    Janecka, Magdalena; Marzi, Sarah J; Parsons, Michael J; Liu, Lin; Paya-Cano, Jose L; Smith, Rebecca G; Fernandes, Cathy; Schalkwyk, Leonard C

    2017-02-01

    Although the search for quantitative trait loci for behaviour remains a considerable challenge, the complicated genetic architecture of quantitative traits is beginning to be understood. The current project utilised heterogeneous stock (HS) male mice (n = 580) to investigate the genetic basis for brain weights, activity, anxiety and cognitive phenotypes. We identified 126 single nucleotide polymorphisms (SNPs) in genes involved in regulation of neurotransmitter systems, nerve growth/death and gene expression, and subsequently investigated their associations with changes in behaviour and/or brain weights in our sample. We found significant associations between four SNP-phenotype pairs, after controlling for multiple testing. Specificity protein 2 (Sp2, rs3708840), tryptophan hydroxylase 1 (Tph1, rs262731280) and serotonin receptor 3A (Htr3a, rs50670893) were associated with activity/anxiety behaviours, and microtubule-associated protein 2 (Map2, rs13475902) was associated with cognitive performance. All these genes except for Tph1 were expressed in the brain above the array median, and remained significantly associated with relevant behaviours after controlling for the family structure. Additionally, we found evidence for a correlation between Htr3a expression and activity. We discuss our findings in the light of the advantages and limitations of currently available mouse genetic tools, suggesting further directions for association studies in rodents.

  19. Systems genetic analysis of hippocampal neuroanatomy and spatial learning in mice.

    PubMed

    Delprato, A; Bonheur, B; Algéo, M-P; Rosay, P; Lu, L; Williams, R W; Crusio, W E

    2015-11-01

    Variation in hippocampal neuroanatomy correlates well with spatial learning ability in mice. Here, we have studied both hippocampal neuroanatomy and behavior in 53 isogenic BXD recombinant strains derived from C57BL/6J and DBA/2J parents. A combination of experimental, neuroinformatic and systems genetics methods was used to test the genetic bases of variation and covariation among traits. Data were collected on seven hippocampal subregions in CA3 and CA4 after testing spatial memory in an eight-arm radial maze task. Quantitative trait loci were identified for hippocampal structure, including the areas of the intra- and infrapyramidal mossy fibers (IIPMFs), stratum radiatum and stratum pyramidale, and for a spatial learning parameter, error rate. We identified multiple loci and gene variants linked to either structural differences or behavior. Gpc4 and Tenm2 are strong candidate genes that may modulate IIPMF areas. Analysis of gene expression networks and trait correlations highlight several processes influencing morphometrical variation and spatial learning. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  20. Genetic polymorphisms and their association with brain and behavioural measures in heterogeneous stock mice

    PubMed Central

    Janecka, Magdalena; Marzi, Sarah J.; Parsons, Michael J.; Liu, Lin; Paya-Cano, Jose L.; Smith, Rebecca G.; Fernandes, Cathy; Schalkwyk, Leonard C.

    2017-01-01

    Although the search for quantitative trait loci for behaviour remains a considerable challenge, the complicated genetic architecture of quantitative traits is beginning to be understood. The current project utilised heterogeneous stock (HS) male mice (n = 580) to investigate the genetic basis for brain weights, activity, anxiety and cognitive phenotypes. We identified 126 single nucleotide polymorphisms (SNPs) in genes involved in regulation of neurotransmitter systems, nerve growth/death and gene expression, and subsequently investigated their associations with changes in behaviour and/or brain weights in our sample. We found significant associations between four SNP-phenotype pairs, after controlling for multiple testing. Specificity protein 2 (Sp2, rs3708840), tryptophan hydroxylase 1 (Tph1, rs262731280) and serotonin receptor 3A (Htr3a, rs50670893) were associated with activity/anxiety behaviours, and microtubule-associated protein 2 (Map2, rs13475902) was associated with cognitive performance. All these genes except for Tph1 were expressed in the brain above the array median, and remained significantly associated with relevant behaviours after controlling for the family structure. Additionally, we found evidence for a correlation between Htr3a expression and activity. We discuss our findings in the light of the advantages and limitations of currently available mouse genetic tools, suggesting further directions for association studies in rodents. PMID:28145470

  1. Genetic manipulation of HSP26 and YHR087W stress genes may improve fermentative behaviour in wine yeasts under vinification conditions.

    PubMed

    Jiménez-Martí, E; Zuzuarregui, A; Ridaura, I; Lozano, N; del Olmo, M

    2009-03-31

    Throughout wine production yeast cells are affected by a plethora of stress conditions that compromise their ability to carry out the whole process. In recent years important knowledge about the mechanisms involved in stress response in both laboratory and wine yeast strains has been obtained. Several studies have indicated that a correlation exists between stress resistance, expression of stress response genes and fermentative behaviour. In this work we introduce several genetic manipulations in two genes induced by several stress conditions: HSP26 (which encodes a heat shock protein) and YHR087W (encoding a protein of unknown function) in two different wine yeasts, ICV16 and ICV27. These manipulations include expression in multicopy and centromeric plasmids, and substitution of the promoter in one of the genomic copies of these genes for that of the SPI1 gene, encoding for a cell wall protein of unknown function, or the PGK1 gene, which encodes the phosphoglycerate kinase glycolytic enzyme. Our results indicate that some of these modifications result in strains with higher expression of these genes, better resistance to certain stress conditions, and even improved fermentative behaviour. The modifications of the YHR087W gene are particularly interesting, and suggest an important role of this gene in the vinification process.

  2. Combined Pharmacological and Genetic Manipulations Unlock Unprecedented Temporal Elasticity and Reveal Phase-Specific Modulation of the Molecular Circadian Clock of the Mouse Suprachiasmatic Nucleus

    PubMed Central

    Patton, Andrew P.; Chesham, Johanna E.

    2016-01-01

    The suprachiasmatic nucleus (SCN) is the master circadian oscillator encoding time-of-day information. SCN timekeeping is sustained by a cell-autonomous transcriptional–translational feedback loop, whereby expression of the Period and Cryptochrome genes is negatively regulated by their protein products. This loop in turn drives circadian oscillations in gene expression that direct SCN electrical activity and thence behavior. The robustness of SCN timekeeping is further enhanced by interneuronal, circuit-level coupling. The aim of this study was to combine pharmacological and genetic manipulations to push the SCN clockwork toward its limits and, by doing so, probe cell-autonomous and emergent, circuit-level properties. Circadian oscillation of mouse SCN organotypic slice cultures was monitored as PER2::LUC bioluminescence. SCN of three genetic backgrounds—wild-type, short-period CK1εTau/Tau mutant, and long-period Fbxl3Afh/Afh mutant—all responded reversibly to pharmacological manipulation with period-altering compounds: picrotoxin, PF-670462 (4-[1-Cyclohexyl-4-(4-fluorophenyl)-1H-imidazol-5-yl]-2-pyrimidinamine dihydrochloride), and KNK437 (N-Formyl-3,4-methylenedioxy-benzylidine-gamma-butyrolactam). This revealed a remarkably wide operating range of sustained periods extending across 25 h, from ≤17 h to >42 h. Moreover, this range was maintained at network and single-cell levels. Development of a new technique for formal analysis of circadian waveform, first derivative analysis (FDA), revealed internal phase patterning to the circadian oscillation at these extreme periods and differential phase sensitivity of the SCN to genetic and pharmacological manipulations. For example, FDA of the CK1εTau/Tau mutant SCN treated with the CK1ε-specific inhibitor PF-4800567 (3-[(3-Chlorophenoxy)methyl]-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine hydrochloride) revealed that period acceleration in the mutant is due to inappropriately phased

  3. GENETIC FACTORS CONTROLLING ANTI-SHEEP ERYTHROCYTE ANTIBODY RESPONSE AND IMMUNOGLOBULIN SYNTHESIS IN BACKCROSS AND F2 PROGENY OF MICE GENETICALLY SELECTED FOR "HIGH" OR "LOW" ANTIBODY SYNTHESIS

    PubMed Central

    Lieberman, R.; Stiffel, C.; Asofsky, R.; Mouton, D.; Biozzi, G.; Benacerraf, B.

    1972-01-01

    Agglutinin responses to sheep erythrocytes and immunoglobulin heavy chain phenotypes determined in F1, F2, and backcross progeny of mice genetically selected for high and low antibody synthesis indicated that an immune response gene for sheep erythrocytes is linked to the immunoglobulin heavy chain allotype. Mice homozygous for the phenotype of the high line had significantly higher titers than mice homozygous for the phenotype of the low line. An association was also observed in some progeny of the backcross of the F1 generation with the low line. However, the control of the immune response was clearly multigenic since heterozygous mice of the same phenotype (2/3, 5) resulting from the two backcrosses (high and low) had very different immune responses. Immunoglobulin levels in the same progeny showed no linkage to the immunoglobulin allotype but a rather simple pattern of inheritance. PMID:4115709

  4. Genetic deficiency of Syk protects mice from autoantibody-induced arthritis

    PubMed Central

    Jakus, Zoltán; Simon, Edina; Balázs, Bálint; Mócsai, Attila

    2010-01-01

    Objective The Syk tyrosine kinase plays an important role in diverse functions in hematopoietic lineage cells. Although previous in vitro and pharmacologic analyses suggested Syk to be a possible player in the development of autoimmune arthritis, no in vivo genetic studies addressing that issue have yet been reported. The aim of the present study was to test whether genetic deficiency of Syk affects autoantibody-induced experimental arthritis in the K/BxN serum–transfer model. Methods Syk−/− bone marrow chimeras carrying a Syk-deficient hematopoietic system were generated by transplanting Syk−/− fetal liver cells into lethally irradiated wild-type recipients. After complete repopulation of the hematopoietic compartment, autoantibody-mediated arthritis was induced by injection of arthritogenic K/BxN serum. Arthritis development was monitored by macroscopic and microscopic observation of the ankle joints, micro–computed tomography of bone morphology, as well as a joint function assay. Results Genetic deficiency of Syk in the hematopoietic compartment completely blocked the development of all macroscopic and microscopic signs of arthritis. The Syk−/− mutation also prevented the appearance of periarticular bone erosions. Finally, Syk−/− bone marrow chimeras were completely protected from arthritis-induced loss of articular function. Conclusion Our results indicate that Syk is critically involved in the development of all clinically relevant aspects of autoantibody-mediated K/BxN serum–transfer arthritis in experimental mice. These results provide the first in vivo genetic evidence of the role of Syk in the development of autoimmune arthritis. PMID:20201079

  5. Identifying genetic loci and spleen gene coexpression networks underlying immunophenotypes in BXD recombinant inbred mice

    PubMed Central

    Lynch, Rachel M.; Naswa, Sudhir; Rogers, Gary L.; Kania, Stephen A.; Das, Suchita; Chesler, Elissa J.; Saxton, Arnold M.; Langston, Michael A.

    2010-01-01

    The immune system plays a pivotal role in the susceptibility to and progression of a variety of diseases. Due to a strong genetic basis, heritable differences in immune function may contribute to differential disease susceptibility between individuals. Genetic reference populations, such as the BXD (C57BL/6J × DBA/2J) panel of recombinant inbred (RI) mouse strains, provide unique models through which to integrate baseline phenotypes in healthy individuals with heritable risk for disease because of the ability to combine data collected from these populations across both multiple studies and time. We performed basic immunophenotyping (e.g., percentage of circulating B and T lymphocytes and CD4+ and CD8+ T cell subpopulations) in peripheral blood of healthy mice from 41 BXD RI strains to define the immunophenotypic variation in this strain panel and to characterize the genetic architecture that underlies these traits. Significant QTL models that explained the majority (50–77%) of phenotypic variance were derived for each trait and for the T:B cell and CD4+:CD8+ ratios. Combining QTL mapping with spleen gene expression data uncovered two quantitative trait transcripts, Ptprk and Acp1, as candidates for heritable differences in the relative abundance of helper and cytotoxic T cells. These data will be valuable in extracting genetic correlates of the immune system in the BXD panel. In addition, they will be a useful resource for prospective, phenotype-driven model selection to test hypotheses about differential disease or environmental susceptibility between individuals with baseline differences in the composition of the immune system. PMID:20179155

  6. Genetic manipulation of Prochlorococcus strain MIT9313: green fluorescent protein expression from an RSF1010 plasmid and Tn5 transposition.

    PubMed

    Tolonen, Andrew C; Liszt, Gregory B; Hess, Wolfgang R

    2006-12-01

    Prochlorococcus is the smallest oxygenic phototroph yet described. It numerically dominates the phytoplankton community in the mid-latitude oceanic gyres, where it has an important role in the global carbon cycle. The complete genomes of several Prochlorococcus strains have been sequenced, revealing that nearly half of the genes in each genome are of unknown function. Genetic methods, such as reporter gene assays and tagged mutagenesis, are critical to unveiling the functions of these genes. Here, we describe conditions for the transfer of plasmid DNA into Prochlorococcus strain MIT9313 by interspecific conjugation with Escherichia coli. Following conjugation, E. coli bacteria were removed from the Prochlorococcus cultures by infection with E. coli phage T7. We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus strain MIT9313. When this plasmid was modified to contain green fluorescent protein, we detected its expression in Prochlorococcus by Western blotting and cellular fluorescence. Further, we applied these conjugation methods to show that a mini-Tn5 transposon will transpose in vivo in Prochlorococcus. These genetic advances provide a basis for future genetic studies with Prochlorococcus, a microbe of ecological importance in the world's oceans.

  7. Genetic Manipulation of Prochlorococcus Strain MIT9313: Green Fluorescent Protein Expression from an RSF1010 Plasmid and Tn5 Transposition▿

    PubMed Central

    Tolonen, Andrew C.; Liszt, Gregory B.; Hess, Wolfgang R.

    2006-01-01

    Prochlorococcus is the smallest oxygenic phototroph yet described. It numerically dominates the phytoplankton community in the mid-latitude oceanic gyres, where it has an important role in the global carbon cycle. The complete genomes of several Prochlorococcus strains have been sequenced, revealing that nearly half of the genes in each genome are of unknown function. Genetic methods, such as reporter gene assays and tagged mutagenesis, are critical to unveiling the functions of these genes. Here, we describe conditions for the transfer of plasmid DNA into Prochlorococcus strain MIT9313 by interspecific conjugation with Escherichia coli. Following conjugation, E. coli bacteria were removed from the Prochlorococcus cultures by infection with E. coli phage T7. We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus strain MIT9313. When this plasmid was modified to contain green fluorescent protein, we detected its expression in Prochlorococcus by Western blotting and cellular fluorescence. Further, we applied these conjugation methods to show that a mini-Tn5 transposon will transpose in vivo in Prochlorococcus. These genetic advances provide a basis for future genetic studies with Prochlorococcus, a microbe of ecological importance in the world's oceans. PMID:17041154

  8. Populations at risk: conservation genetics of kangaroo mice (Microdipodops) of the Great Basin Desert.

    PubMed

    Andersen, John J; Portnoy, David S; Hafner, John C; Light, Jessica E

    2013-08-01

    The Great Basin Desert of western North America has experienced frequent habitat alterations due to a complex biogeographic history and recent anthropogenic impacts, with the more recent alterations likely resulting in the decline of native fauna and flora. Dark (Microdipodops megacephalus) and pallid (M. pallidus) kangaroo mice are ecological specialists found within the Great Basin Desert and are potentially ideal organisms for assessing ecosystem health and inferring the biogeographic history of this vulnerable region. Herein, newly acquired nuclear-encoded microsatellite loci were utilized to assess patterns of variation within and among spatially discrete groups of kangaroo mice and to evaluate gene flow, demographic trends, and genetic integrity. Results confirm that there are at least three genetically distinct units within M. megacephalus and two such units within M. pallidus. The three units of M. megacephalus appear to have different demographic histories, with effectively no gene flow among them since their divergence. Similarly, the two units of M. pallidus also appear to have experienced different demographic histories, with effectively no gene exchange. Contemporary effective population sizes of all groups within Microdipodops appear to be low (<500), suggesting that each genetic lineage may have difficulty coping with changing environmental pressures and hence may be at risk of extirpation. Results of this study indicate that each Microdipodops group should be recognized, and therefore managed, as a separate unit in an effort to conserve these highly specialized taxa that contribute to the diversity of the Great Basin Desert ecosystem. The Great Basin Desert of western North America has experienced frequent habitat alterations due to a complex biogeographic history and recent anthropogenic impacts, with the more recent alterations likely resulting in the decline of native fauna and flora. Herein, newly acquired nuclear-encoded microsatellite

  9. Populations at risk: conservation genetics of kangaroo mice (Microdipodops) of the Great Basin Desert

    PubMed Central

    Andersen, John J; Portnoy, David S; Hafner, John C; Light, Jessica E

    2013-01-01

    Abstract The Great Basin Desert of western North America has experienced frequent habitat alterations due to a complex biogeographic history and recent anthropogenic impacts, with the more recent alterations likely resulting in the decline of native fauna and flora. Dark (Microdipodops megacephalus) and pallid (M. pallidus) kangaroo mice are ecological specialists found within the Great Basin Desert and are potentially ideal organisms for assessing ecosystem health and inferring the biogeographic history of this vulnerable region. Herein, newly acquired nuclear-encoded microsatellite loci were utilized to assess patterns of variation within and among spatially discrete groups of kangaroo mice and to evaluate gene flow, demographic trends, and genetic integrity. Results confirm that there are at least three genetically distinct units within M. megacephalus and two such units within M. pallidus. The three units of M. megacephalus appear to have different demographic histories, with effectively no gene flow among them since their divergence. Similarly, the two units of M. pallidus also appear to have experienced different demographic histories, with effectively no gene exchange. Contemporary effective population sizes of all groups within Microdipodops appear to be low (<500), suggesting that each genetic lineage may have difficulty coping with changing environmental pressures and hence may be at risk of extirpation. Results of this study indicate that each Microdipodops group should be recognized, and therefore managed, as a separate unit in an effort to conserve these highly specialized taxa that contribute to the diversity of the Great Basin Desert ecosystem. The Great Basin Desert of western North America has experienced frequent habitat alterations due to a complex biogeographic history and recent anthropogenic impacts, with the more recent alterations likely resulting in the decline of native fauna and flora. Herein, newly acquired nuclear

  10. Rabies virus immunity in genetically selected high- and low-responder lines of mice.

    PubMed Central

    Nilsson, M R; Sant'anna, O A; Siqueira, M; Nilsson, T T; Gennari, M

    1979-01-01

    The antibody responsiveness to and the specific vaccination effect of rabies virus infection were investigated in high- and low-responder lines of mice produced by two-way selective breedings for quantitative production of antibodies to flagellar (H/f and L/f lines) or somatic (H/s and L/s lines) antigens of salmonellae. After specific immunization, both high lines were more resistant to rabies virus infection than were the low lines, and the protector effect was related to the level of antibody produced, as demonstrated by neutralizing serum activity. The present findings confirm the nonspecific genetic modification of the general antibody responsiveness induced in high- and low-responder lines selected for quantitative antibody production. PMID:478636

  11. Modeling resilience to schizophrenia in genetically modified mice: a novel approach to drug discovery

    PubMed Central

    Mihali, Andra; Subramani, Shreya; Kaunitz, Genevieve; Rayport, Stephen; Gaisler-Salomon, Inna

    2012-01-01

    Complex psychiatric disorders, such as schizophrenia, arise from a combination of genetic, developmental, environmental and social factors. These vulnerabilities can be mitigated by adaptive factors in each of these domains engendering resilience. Modeling resilience in mice using transgenic approaches offers a direct path to intervention, as resilience mutations point directly to therapeutic targets. As prototypes for this approach, we discuss the three mouse models of schizophrenia resilience, all based on modulating glutamatergic synaptic transmission. This motivates the broader development of schizophrenia resilience mouse models independent of specific pathophysiological hypotheses as a strategy for drug discovery. Three guiding validation criteria are presented. A resilience-oriented approach should identify pharmacologically tractable targets and in turn offer new insights into pathophysiological mechanisms. PMID:22853787

  12. Anti-diabetic activity of a mineraloid isolate, in vitro and in genetically diabetic mice.

    PubMed

    Deneau, Joel; Ahmed, Taufeeq; Blotsky, Roger; Bojanowski, Krzysztof

    2011-01-01

    Type II diabetes is a metabolic disease mediated through multiple molecular pathways. Here, we report anti-diabetic effect of a standardized isolate from a fossil material - a mineraloid leonardite - in in vitro tests and in genetically diabetic mice. The mineraloid isolate stimulated mitochondrial metabolism in human fibroblasts and this stimulation correlated with enhanced expression of genes coding for mitochondrial proteins such as ATP synthases and ribosomal protein precursors, as measured by DNA microarrays. In the diabetic animal model, consumption of the Totala isolate resulted in decreased weight gain, blood glucose, and glycated hemoglobin. To our best knowledge, this is the first description ever of a fossil material having anti-diabetic activity in pre-clinical models.

  13. Lentivectors encoding immunosuppressive proteins genetically engineer pancreatic beta-cells to correct diabetes in allogeneic mice.

    PubMed

    Kojaoghlanian, T; Joseph, A; Follenzi, A; Zheng, J H; Leiser, M; Fleischer, N; Horwitz, M S; DiLorenzo, T P; Goldstein, H

    2009-03-01

    The effectiveness of genetic engineering with lentivectors to protect transplanted cells from allogeneic rejection was examined using, as a model, type 1 diabetes treatment with beta-cell transplantation, whose widespread use has been limited by the requirement for sustained immunosuppressive treatment to prevent graft rejection. We examined whether lentivectors expressing select immunosuppressive proteins encoded by the adenoviral genome early region 3 (AdE3) would protect transplanted beta-cells from an alloimmune attack. The insulin-producing beta-cell line beta TC-tet (C3HeB/FeJ-derived) was transduced with lentiviruses encoding the AdE3 proteins gp19K and RID alpha/beta. The efficiency of lentiviral transduction of beta TC-tet cells exceeded 85%. Lentivector expression of gp19K decreased surface class I major histocompatibility complex expression by over 90%, whereas RID alpha/beta expression inhibited cytokine-induced Fas upregulation by over 75%. beta TC-tet cells transduced with gp19K and RID alpha/beta lentivectors, but not with a control lentivector, provided prolonged correction of hyperglycemia after transplantation into diabetic BALB/c severe combined immunodeficient mice reconstituted with allogeneic immune effector cells or into diabetic allogeneic BALB/c mice. Thus, genetic engineering of beta-cells using gp19K- and RID alpha/beta-expressing lentiviral vectors may provide an alternative that has the potential to eliminate or reduce treatment with the potent immunosuppressive agents necessary at present for prolonged engraftment with transplanted islets.

  14. Characterization and genetic manipulation of primed stem cells into a functional naïve state with ESRRB

    PubMed Central

    Rossello, Ricardo Antonio; Pfenning, Andreas; Howard, Jason T; Hochgeschwender, Ute

    2016-01-01

    AIM To identify differences between primed mouse embryonic stem cells (ESCs) and fully functional naive ESCs; to manipulate primed cells into a naive state. METHODS We have cultured 3 lines of cells from different mouse strains that have been shown to be naive or primed as determined by generating germline-transmitting chimeras. Cells were put through a battery of tests to measure the different features. RNA from cells was analyzed using microarrays, to determine a priority list of the differentially expressed genes. These were later validated by quantificational real-time polymerase chain reaction. Viral cassettes were created to induce expression of differentially expressed genes in the primed cells through lentiviral transduction. Primed reprogrammed cells were subjected to in-vivo incorporation studies. RESULTS Most results show that both primed and naive cells have similar features (morphology, proliferation rates, stem cell genes expressed). However, there were some genes that were differentially expressed in the naïve cells relative to the primed cells. Key upregulated genes in naïve cells include ESRRB, ERAS, ATRX, RNF17, KLF-5, and MYC. After over-expressing some of these genes the primed cells were able to incorporate into embryos in-vivo, re-acquiring a feature previously absent in these cells. CONCLUSION Although there are no notable phenotypic differences, there are key differences in gene expression between these naïve and primed stem cells. These differences can be overcome through overexpression. PMID:27822342

  15. Lin28a transgenic mice manifest size and puberty phenotypes identified in human genetic association studies.

    PubMed

    Zhu, Hao; Shah, Samar; Shyh-Chang, Ng; Shinoda, Gen; Einhorn, William S; Viswanathan, Srinivas R; Takeuchi, Ayumu; Grasemann, Corinna; Rinn, John L; Lopez, Mary F; Hirschhorn, Joel N; Palmert, Mark R; Daley, George Q

    2010-07-01

    Recently, genome-wide association studies have implicated the human LIN28B locus in regulating height and the timing of menarche. LIN28B and its homolog LIN28A are functionally redundant RNA-binding proteins that block biogenesis of let-7 microRNAs. lin-28 and let-7 were discovered in Caenorhabditis elegans as heterochronic regulators of larval and vulval development but have recently been implicated in cancer, stem cell aging and pluripotency. The let-7 targets Myc, Kras, Igf2bp1 and Hmga2 are known regulators of mammalian body size and metabolism. To explore the function of the Lin28-Let-7 pathway in vivo, we engineered transgenic mice to express Lin28a and observed in them increased body size, crown-rump length and delayed onset of puberty. Investigation of metabolic and endocrine mechanisms of overgrowth in these transgenic mice revealed increased glucose metabolism and insulin sensitivity. Here we report a mouse that models the human phenotypes associated with genetic variation in the Lin28-Let-7 pathway.

  16. Platelet-activating factor (PAF) receptor and genetically engineered PAF receptor mutant mice.

    PubMed

    Ishii, S; Shimizu, T

    2000-01-01

    Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid mediator. Although PAF was initially recognized for its potential to induce platelet aggregation and secretion, intense investigations have elucidated potent biological actions of PAF in a broad range of cell types and tissues, many of which also produce the molecule. PAF acts by binding to a unique G-protein-coupled seven transmembrane receptor. PAF receptor is linked to intracellular signal transduction pathways, including turnover of phosphatidylinositol, elevation in intracellular calcium concentration, and activation of kinases, resulting in versatile bioactions. On the basis of numerous pharmacological reports, PAF is thought to have many pathophysiological and physiological functions. Recently advanced molecular technics enable us not only to clone PAF receptor cDNAs and genes, but also generate PAF receptor mutant animals, i.e., PAF receptor-overexpressing mouse and PAF receptor-deficient mouse. These mutant mice gave us a novel and specific approach for identifying the pathophysiological and physiological functions of PAF. This review also describes the phenotypes of these mutant mice and discusses them by referring to previously reported pharmacological and genetical data.

  17. Effects of Genetically Modified Milk Containing Human Beta-Defensin-3 on Gastrointestinal Health of Mice

    PubMed Central

    Yang, Yange; Shi, Zhaopeng; Gao, Ming-Qing; Zhang, Yong

    2016-01-01

    This study was performed to investigate the effects of genetically modified (GM) milk containing human beta-defensin-3 (HBD3) on mice by a 90-day feeding study. The examined parameters included the digestibility of GM milk, general physical examination, gastric emptying function, intestinal permeability, intestinal microflora composition of mice, and the possibility of horizontal gene transfer (HGT). The emphasis was placed on the effects on gastrointestinal (GI) tract due to the fact that GI tract was the first site contacting with food and played crucial roles in metabolic reactions, nutrition absorption and immunity regulation in the host. However, the traditional methods for analyzing the potential toxicological risk of GM product pay little attention on GI health. In this study, the results showed GM milk was easy to be digested in simulated gastric fluid, and it did not have adverse effects on general and GI health compared to conventional milk. And there is little possibility of HGT. This study may enrich the safety assessment of GM product on GI health. PMID:27438026

  18. Effects of Genetically Modified Milk Containing Human Beta-Defensin-3 on Gastrointestinal Health of Mice.

    PubMed

    Chen, Xin; Yang, Yange; Shi, Zhaopeng; Gao, Ming-Qing; Zhang, Yong

    2016-01-01

    This study was performed to investigate the effects of genetically modified (GM) milk containing human beta-defensin-3 (HBD3) on mice by a 90-day feeding study. The examined parameters included the digestibility of GM milk, general physical examination, gastric emptying function, intestinal permeability, intestinal microflora composition of mice, and the possibility of horizontal gene transfer (HGT). The emphasis was placed on the effects on gastrointestinal (GI) tract due to the fact that GI tract was the first site contacting with food and played crucial roles in metabolic reactions, nutrition absorption and immunity regulation in the host. However, the traditional methods for analyzing the potential toxicological risk of GM product pay little attention on GI health. In this study, the results showed GM milk was easy to be digested in simulated gastric fluid, and it did not have adverse effects on general and GI health compared to conventional milk. And there is little possibility of HGT. This study may enrich the safety assessment of GM product on GI health.

  19. Divergence and inheritance of neocortical heterotopia in inbred and genetically-engineered mice.

    PubMed

    Toia, Alyssa R; Cuoco, Joshua A; Esposito, Anthony W; Ahsan, Jawad; Joshi, Alok; Herron, Bruce J; Torres, German; Bolivar, Valerie J; Ramos, Raddy L

    2017-01-18

    Cortical function emerges from the intrinsic properties of neocortical neurons and their synaptic connections within and across lamina. Neurodevelopmental disorders affecting migration and lamination of the neocortex result in cognitive delay/disability and epilepsy. Molecular layer heterotopia (MLH), a dysplasia characterized by over-migration of neurons into layer I, are associated with cognitive deficits and neuronal hyperexcitability in humans and mice. The breadth of different inbred mouse strains that exhibit MLH and inheritance patterns of heterotopia remain unknown. A neuroanatomical survey of numerous different inbred mouse strains, 2 first filial generation (F1) hybrids, and one consomic strain (C57BL/6J-Chr 1(A/J)/NaJ) revealed MLH only in C57BL/6 mice and the consomic strain. Heterotopia were observed in numerous genetically-engineered mouse lines on a congenic C57BL/6 background. These data indicate that heterotopia formation is a weakly penetrant trait requiring homozygosity of one or more C57BL/6 alleles outside of chromosome 1. These data are relevant toward understanding neocortical development and disorders affecting neocortical lamination.

  20. A genetically engineered live attenuated vaccine of Coccidioides posadasii protects BALB/c mice against coccidioidomycosis.

    PubMed

    Xue, Jianmin; Chen, Xia; Selby, Dale; Hung, Chiung-Yu; Yu, Jieh-Juen; Cole, Garry T

    2009-08-01

    Coccidioidomycosis (also known as San Joaquin Valley fever) is an occupational disease. Workers exposed to outdoor dust which contains spores of the soil-inhabiting fungus have a significantly increased risk of respiratory infection. In addition, people with compromised T-cell immunity, the elderly, and certain racial groups, particularly African-Americans and Filipinos, who live in regions of endemicity in the southwestern United States have an elevated incidence of symptomatic infection caused by inhalation of spores of Coccidioides posadasii or Coccidioides immitis. Recurring epidemics and escalation of medical costs have helped to motivate production of a vaccine against valley fever. The major focus has been the development of a defined, T-cell-reactive, recombinant protein vaccine. However, none of the products described to date have provided full protection to coccidioidal disease-susceptible BALB/c mice. Here we describe the first genetically engineered, live, attenuated vaccine that protects both BALB/c and C57BL/6 mice against coccidioidomycosis. Two chitinase genes (CTS2 and CTS3) were disrupted to yield the attenuated strain, which was unable to endosporulate and was no longer infectious. Vaccinated survivors mounted an immune response characterized by production of both T-helper-1- and T-helper-2-type cytokines. Histology revealed well-formed granulomas and markedly diminished inflammation. Significantly fewer organisms were observed in the lungs of survivors than in those of nonvaccinated mice. Additional investigations are required to further define the nature of the live, attenuated vaccine-induced immunity against Coccidioides infection.

  1. Ontogenic and morphological study of gonadal formation in genetically-modified sex reversal XYPOS mice

    PubMed Central

    UMEMURA, Yuria; MIYAMOTO, Ryosuke; HASHIMOTO, Rie; KINOSHITA, Kyoko; OMOTEHARA, Takuya; NAGAHARA, Daichi; HIRANO, Tetsushi; KUBOTA, Naoto; MINAMI, Kiichi; YANAI, Shogo; MASUDA, Natsumi; YUASA, Hideto; MANTANI, Youhei; MATSUO, Eiko; YOKOYAMA, Toshifumi; KITAGAWA, Hiroshi; HOSHI, Nobuhiko

    2015-01-01

    Mammalian sexual fate is determined by the presence or absence of sex determining region of the Y chromosome (Sry) in the “bipotential” gonads. Recent studies have demonstrated that both male and female sexual development are induced by distinct and active genetic pathways. Breeding the Y chromosome from Mus m. domesticus poschiavinus (POS) strains into C57BL/6J (B6J) mice (B6J-XYPOS) has been shown to induce sex reversal (75%: bilateral ovary, 25%: true hermaphrodites). However, our B6N-XYPOS mice, which were generated by backcrossing of B6J-XYPOS on an inbred B6N-XX, develop as males (36%: bilateral testis with fertility as well as bilateral ovary (34%), and the remainder develop as true hermaphrodites. Here, we investigated in detail the expressions of essential sex-related genes and histological features in B6N-XYPOS mice from the fetal period to adulthood. The onsets of both Sry and SRY-box 9 (Sox9) expressions as determined spatiotemporally by whole-mount immunohistochemistry in the B6N-XYPOS gonads occurred 2–3 tail somites later than those in B6N-XYB6 gonads, but earlier than those in B6J-XYPOS, respectively. It is possible that such a small difference in timing of the Sry expression underlies testicular development in our B6N-XYPOS. Our study is the first to histologically show the expression and ectopic localization of a female-related gene in the XYPOS testes and a male-related gene in the XYPOS ovaries. The results from these and previous experiments indicate that the interplay between genome variants, epigenetics and developmental gene regulation is crucial for testis development. PMID:26194606

  2. Mice genetically depleted of brain serotonin do not display a depression-like behavioral phenotype.

    PubMed

    Angoa-Pérez, Mariana; Kane, Michael J; Briggs, Denise I; Herrera-Mundo, Nieves; Sykes, Catherine E; Francescutti, Dina M; Kuhn, Donald M

    2014-10-15

    Reductions in function within the serotonin (5HT) neuronal system have long been proposed as etiological factors in depression. Selective serotonin reuptake inhibitors (SSRIs) are the most common treatment for depression, and their therapeutic effect is generally attributed to their ability to increase the synaptic levels of 5HT. Tryptophan hydroxylase 2 (TPH2) is the initial and rate-limiting enzyme in the biosynthetic pathway of 5HT in the CNS, and losses in its catalytic activity lead to reductions in 5HT production and release. The time differential between the onset of 5HT reuptake inhibition by SSRIs (minutes) and onset of their antidepressant efficacy (weeks to months), when considered with their overall poor therapeutic effectiveness, has cast some doubt on the role of 5HT in depression. Mice lacking the gene for TPH2 are genetically depleted of brain 5HT and were tested for a depression-like behavioral phenotype using a battery of valid tests for affective-like disorders in animals. The behavior of TPH2(-/-) mice on the sucrose preference test, tail suspension test, and forced swim test and their responses in the unpredictable chronic mild stress and learned helplessness paradigms was the same as wild-type controls. While TPH2(-/-) mice as a group were not responsive to SSRIs, a subset responded to treatment with SSRIs in the same manner as wild-type controls with significant reductions in immobility time on the tail suspension test, indicative of antidepressant drug effects. The behavioral phenotype of the TPH2(-/-) mouse questions the role of 5HT in depression. Furthermore, the TPH2(-/-) mouse may serve as a useful model in the search for new medications that have therapeutic targets for depression that are outside of the 5HT neuronal system.

  3. Mice Genetically Depleted of Brain Serotonin do not Display a Depression-like Behavioral Phenotype

    PubMed Central

    Angoa-Pérez, Mariana; Kane, Michael J.; Briggs, Denise I.; Herrera-Mundo, Nieves; Sykes, Catherine E.; Francescutti, Dina M.; Kuhn, Donald M.

    2016-01-01

    Reductions in function within the serotonin (5HT) neuronal system have long been proposed as etiological factors in depression. Serotonin selective reuptake inhibitors (SSRIs) are the most common treatment for depression and their therapeutic effect is generally attributed to their ability to increase the synaptic levels of 5HT. Tryptophan hydroxylase 2 (TPH2) is the initial and rate-limiting enzyme in the biosynthetic pathway of 5HT in the CNS and losses in its catalytic activity lead to reductions in 5HT production and release. The time differential between the onset of 5HT reuptake inhibition by SSRIs (minutes) and onset of their anti-depressant efficacy (weeks to months), when considered with their overall poor therapeutic effectiveness, has cast some doubt on the role of 5HT in depression. Mice lacking the gene for TPH2 are genetically depleted of brain 5HT and were tested for a depression-like behavioral phenotype using a battery of valid tests for affective-like disorders in animals. The behavior of TPH2−/− mice on the sucrose preference test, tail suspension test and forced swim test and their responses in the unpredictable chronic mild stress and learned helplessness paradigms was the same as wild-type controls. While TPH2−/− mice as a group were not responsive to SSRIs, a subset responded to treatment with SSRIs in the same manner as wild-type controls with significant reductions in immobility time on the tail suspension test, indicative of antidepressant drug effects. The behavioral phenotype of the TPH2−/− mouse questions the role of 5HT in depression. Furthermore, the TPH2−/− mouse may serve as a useful model in the search for new medications that have therapeutic targets for depression that are outside of the 5HT neuronal system. PMID:25089765

  4. Facilitation of Direct Conditional Knockout of Essential Genes in Bacillus licheniformis DSM13 by Comparative Genetic Analysis and Manipulation of Genetic Competence▿ †

    PubMed Central

    Hoffmann, Kerstin; Wollherr, Antje; Larsen, Michael; Rachinger, Michael; Liesegang, Heiko; Ehrenreich, Armin; Meinhardt, Friedhelm

    2010-01-01

    The genetic manageability of the biotechnologically important Bacillus licheniformis is hampered due to its poor transformability, whereas Bacillus subtilis efficiently takes up DNA during genetic competence, a quorum-sensing-dependent process. Since the sensor histidine kinase ComP, encoded by a gene of the quorum-sensing module comQXPA of B. licheniformis DSM13, was found to be inactive due to an insertion element within comP, the coding region was exchanged with a functional copy. Quorum sensing was restored, but the already-poor genetic competence dropped further. The inducible expression of the key regulator for the transcription of competence genes, ComK, in trans resulted in highly competent strains and facilitated the direct disruption of genes, as well as the conditional knockout of an essential operon. As ComK is inhibited at low cell densities by a proteolytic complex in which MecA binds ComK and such inhibition is antagonized by the interaction of MecA with ComS (the expression of the latter is controlled by cell density in B. subtilis), we performed an in silico analysis of MecA and the hitherto unidentified ComS, which revealed differences for competent and noncompetent strains, indicating that the reduced competence possibly is due to a nonfunctional coupling of the comQXPA-encoded quorum module and ComK. The obtained increased genetic tractability of this industrial workhorse should improve a wide array of scientific investigations. PMID:20543043

  5. Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice.

    PubMed

    Ichise, Hirotake; Hori, Akiko; Shiozawa, Seiji; Kondo, Saki; Kanegae, Yumi; Saito, Izumu; Ichise, Taeko; Yoshida, Nobuaki

    2016-07-29

    Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes.

  6. Genetic and epigenetic changes in fibrosis-associated hepatocarcinogenesis in mice

    PubMed Central

    Chappell, Grace; Kutanzi, Kristy; Uehara, Takeki; Tryndyak, Volodymyr; Hong, Hue-Hua; Hoenerhoff, Mark; Beland, Frederick A.; Rusyn, Ivan; Pogribny, Igor P.

    2014-01-01

    Hepatocellular carcinoma (HCC) is one of the most prevalent cancers and is rising in incidence worldwide. The molecular mechanisms leading to the development of HCC are complex and include both genetic and epigenetic events. To determine the relative contribution of these alterations in liver tumorigenesis, we evaluated epigenetic modifications at both global and gene specific levels, as well as the mutational profile of genes commonly altered in liver tumors. A mouse model of fibrosis-associated liver cancer that was designed to emulate cirrhotic liver, a prevailing disease state observed in most humans with HCC, was used. Tumor and non-tumor liver samples from B6C3F1 mice treated with N-nitrosodiethylamine (DEN; a single ip injection of 1 mg/kg at 14 days of age) and carbon tetrachloride (CCl4; 0.2 ml/kg, 2 times/week ip starting at 8 weeks of age for 14 weeks), as well as corresponding vehicle control animals, were analyzed for genetic and epigenetic alterations. H-ras, Ctnnb1, and Hnf1α genes were not mutated in tumors in mice treated with DEN+CCl4. In contrast, the increased tumor incidence in mice treated with DEN+CCl4 was associated with marked epigenetic changes in liver tumors and non-tumor liver tissue, including demethylation of genomic DNA and repetitive elements, a decrease in histone 3 lysine 9 trimethylation (H3K9me3), and promoter hypermethylation and functional down-regulation of Riz1, a histone lysine methyltransferase tumor suppressor gene. Additionally, the reduction in H3K9me3 was accompanied by increased expression of long interspersed nucleotide elements (LINE) 1 and short interspersed nucleotide elements (SINE) B2, which is an indication of genomic instability. In summary, our results suggest that epigenetic events, rather than mutations in known cancer-related genes, play a prominent role in increased incidence of liver tumors in this mouse model of fibrosis-associated liver cancer. PMID:24242335

  7. Variability of the Intestinal Uptake of Lipids Is Genetically Determined in Mice

    PubMed Central

    Keelan, M.; Hui, D.Y.; Wild, G.; Clandinin, M.T.

    2008-01-01

    The response of the plasma cholesterol concentration to changes in dietary lipids varies widely in humans and animals. There are variations in the in vivo absorption of cholesterol between different strains of mice. This study was undertaken in three strains of inbred mice to test the hypotheses that: (i) there are strain differences in the in vitro uptake of fatty acids and cholesterol and (ii) the adaptability of the intestine to respond to variations in dietary lipids is genetically determined. An in vitro intestinal ring technique was used to assess the uptake of medium- and long-chain fatty acids and cholesterol into jejunum and ileum of adult DBA/2, C57BL6, and C57L/J mice. The jejunal uptake of cholesterol was similar in C57L/J, DBA/2, or C57BL6 fed ad libitum a low-fat (5.7% fat, no cholesterol) chow diet. This is in contrast to a previous demonstration that in vivo cholesterol absorption was lower in C57L/J than in the other murine strains. The jejunal uptake of several long-chain fatty acids was greater in DBA/2 fed for 4 wk the high-fat (15.8% fat and 1.25% cholesterol) as compared with the low-fat diet. Furthermore, on the high-fat diet, the uptake of many long-chain fatty acids was higher in DBA/2 than in C57BL6 or C57L/J. The differences in cholesterol and fatty acid uptake were not explained by variations in food uptake, body weight gain, or the weight of the intestine. In summary: (i) there are strain differences in the in vitro intestinal uptake of fatty acids but not of cholesterol; (ii) a high-fat diet enhances the uptake of long-chain fatty acids in only one of the three strains examined in this study; and (iii) the pattern of strain- and diet-associated alterations in the in vivo absorption of cholesterol differs from the pattern of changes observed in vitro. We speculate that genetic differences in cholesterol and fatty acid uptake are explained by variations in the expression of protein-mediated components of lipid uptake. PMID:10984106

  8. Genetic manipulation of porcine epidemic diarrhoea virus recovered from a full-length infectious cDNA clone.

    PubMed

    Jengarn, Juggragarn; Wongthida, Phonphimon; Wanasen, Nanchaya; Frantz, Phanramphoei Namprachan; Wanitchang, Asawin; Jongkaewwattana, Anan

    2015-08-01

    Porcine epidemic diarrhoea virus (PEDV) causes acute diarrhoea and dehydration in swine of all ages, with significant mortality in neonatal pigs. The recent rise of PEDV outbreaks in Asia and North America warrants an urgent search for effective vaccines. However, PEDV vaccine research has been hampered by difficulties in isolating and propagating the virus in mammalian cells, thereby complicating the recovery of infectious PEDV using a full-length infectious clone. Here, we engineered VeroE6 cells to stably express porcine aminopeptidase N (pAPN) and used them as a platform to obtain a high-growth variant of PEDV, termed PEDVAVCT12. Subsequently, the full-length cDNA clone was constructed by assembling contiguous cDNA fragments encompassing the complete genome of PEDVAVCT12 in a bacterial artificial chromosome. Infectious PEDV could be recovered, and the rescued virus displayed phenotypic properties identical to the parental virus. Interestingly, we found that PEDVAVCT12 contained a C-terminal deletion of the spike gene, resulting in disruption of the ORF3 start codon. When a functional ORF3 gene was restored, the recombinant virus could not be rescued, suggesting that ORF3 could suppress PEDV replication in vitro. In addition, a high-growth and genetically stable recombinant PEDV expressing a foreign protein could be rescued by replacing the ORF3 gene with the mCherry gene. Together, the results of this study provide a means to generate genetically defined PEDV as a promising vaccine candidate.

  9. Genetic Contributors to Intergenerational CAG Repeat Instability in Huntington’s Disease Knock-In Mice

    PubMed Central

    Neto, João Luís; Lee, Jong-Min; Afridi, Ali; Gillis, Tammy; Guide, Jolene R.; Dempsey, Stephani; Lager, Brenda; Alonso, Isabel; Wheeler, Vanessa C.; Pinto, Ricardo Mouro

    2017-01-01

    Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in exon 1 of the HTT gene. Longer repeat sizes are associated with increased disease penetrance and earlier ages of onset. Intergenerationally unstable transmissions are common in HD families, partly underlying the genetic anticipation seen in this disorder. HD CAG knock-in mouse models also exhibit a propensity for intergenerational repeat size changes. In this work, we examine intergenerational instability of the CAG repeat in over 20,000 transmissions in the largest HD knock-in mouse model breeding datasets reported to date. We confirmed previous observations that parental sex drives the relative ratio of expansions and contractions. The large datasets further allowed us to distinguish effects of paternal CAG repeat length on the magnitude and frequency of expansions and contractions, as well as the identification of large repeat size jumps in the knock-in models. Distinct degrees of intergenerational instability were observed between knock-in mice of six background strains, indicating the occurrence of trans-acting genetic modifiers. We also found that lines harboring a neomycin resistance cassette upstream of Htt showed reduced expansion frequency, indicative of a contributing role for sequences in cis, with the expanded repeat as modifiers of intergenerational instability. These results provide a basis for further understanding of the mechanisms underlying intergenerational repeat instability. PMID:27913616

  10. Genetic Contributors to Intergenerational CAG Repeat Instability in Huntington's Disease Knock-In Mice.

    PubMed

    Neto, João Luís; Lee, Jong-Min; Afridi, Ali; Gillis, Tammy; Guide, Jolene R; Dempsey, Stephani; Lager, Brenda; Alonso, Isabel; Wheeler, Vanessa C; Pinto, Ricardo Mouro

    2017-02-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in exon 1 of the HTT gene. Longer repeat sizes are associated with increased disease penetrance and earlier ages of onset. Intergenerationally unstable transmissions are common in HD families, partly underlying the genetic anticipation seen in this disorder. HD CAG knock-in mouse models also exhibit a propensity for intergenerational repeat size changes. In this work, we examine intergenerational instability of the CAG repeat in over 20,000 transmissions in the largest HD knock-in mouse model breeding datasets reported to date. We confirmed previous observations that parental sex drives the relative ratio of expansions and contractions. The large datasets further allowed us to distinguish effects of paternal CAG repeat length on the magnitude and frequency of expansions and contractions, as well as the identification of large repeat size jumps in the knock-in models. Distinct degrees of intergenerational instability were observed between knock-in mice of six background strains, indicating the occurrence of trans-acting genetic modifiers. We also found that lines harboring a neomycin resistance cassette upstream of Htt showed reduced expansion frequency, indicative of a contributing role for sequences in cis, with the expanded repeat as modifiers of intergenerational instability. These results provide a basis for further understanding of the mechanisms underlying intergenerational repeat instability.

  11. Implantation of Genetically Engineered Fibroblasts into Mice: Implications for Gene Therapy

    NASA Astrophysics Data System (ADS)

    Selden, Richard F.; Skoskiewicz, Marek J.; Burke Howie, Kathleen; Russell, Paul S.; Goodman, Howard M.

    1987-05-01

    In a variety of human genetic diseases, replacement of the absent or defective protein provides significant therapeutic benefits. As a model for a somatic cell gene therapy system, cultured murine fibroblasts were transfected with a human growth hormone (hGH) fusion gene and cells from one of the resulting clonal lines were subsequently implanted into various locations in mice. Such implants synthesized and secreted hGH, which was detectable in the serum. The function of the implants depended on their location and size, and on the histocompatibility of the donor cells with their recipients. The expression of hGH could be modified by addition of regulatory effectors, and, with appropriate immunosuppression, the implants survived for more than 3 months. This approach to gene therapy, here termed ``transkaryotic implantation,'' is potentially applicable to many genetic diseases in that (i) the transfected cell line can be extensively characterized prior to implantation, (ii) several anatomical sites are suitable for implantation, and (iii) regulated expression of the gene of therapeutic interest can be obtained.

  12. Genetics of carbon tetrachloride-induced liver injury in mice. II. Multigenic regulation.

    PubMed Central

    Biesel, K. W.; Ehrinpreis, M. N.; Bhathal, P. S.; Mackay, I. R.; Rose, N. R.

    1984-01-01

    Mice from many different congenic inbred strains were given an intramuscular injection of carbon tetrachloride (CCl4) in olive oil to determine the genetic influences on induction of, and recovery from, liver damage. Liver and blood samples were taken at days 1, 4 and 7. The degree of necrosis and lymphoid infiltration appeared to be controlled, qualitatively and quantitatively, by both H-2-linked and Ah-linked genes. Strain differences were noted in the patterns of hepatocellular necrosis and the proportions of lymphoid, monocytic and other inflammatory cells which characterized the infiltrating population. Kinetic studies of F1 hybrids from matings between the susceptible BALB/cJ male parent and the resistant SJL/J female parent suggested that two dominant genetic influences play a major role in CCl4-induced liver damage. The gene contributed from the BALB/cJ parent affects the extent of liver necrosis and a second gene from SJL/J augments recovery. These results suggest, therefore, that the CCl4-induced liver damage and subsequent recovery are under multigenic control by H-2, Ah and possibly other genes. PMID:6696829

  13. Epistasis contributes to the genetic buffering of plasma HDL cholesterol in mice

    PubMed Central

    Churchill, Gary A.

    2010-01-01

    Stressful environmental factors, such as a high-fat diet, can induce responses in the expression of genes that act to maintain physiological homeostasis. We observed variation in plasma concentrations of high-density lipoprotein (HDL) cholesterol across inbred mouse strains in response to high dietary fat intake. Several strains, including C57BL/6J, have stable levels of plasma HDL independent of diet, whereas other strains, including DBA2/J, show marked changes in plasma HDL. To explore this phenomenon further, we used publicly available data from a C57BL/6J × DBA/2J intercross to identify genetic factors that associate with HDL under high-fat diet conditions. Our analysis identified an epistatic interaction that plays a role in the buffering of HDL levels in C57BL/6J mice, and we have identified Arl4d as a candidate gene that mediates this effect. Structural modeling further elucidates the interaction of genetic factors that contribute to the robustness of HDL in response to high-fat diet in the C57BL/6J strain. PMID:20858711

  14. Exercise, weight loss, and changes in body composition in mice: phenotypic relationships and genetic architecture.

    PubMed

    Kelly, Scott A; Nehrenberg, Derrick L; Hua, Kunjie; Garland, Theodore; Pomp, Daniel

    2011-02-24

    The regulation of body weight and composition is complex, simultaneously affected by genetic architecture, the environment, and their interactions. We sought to analyze the complex phenotypic relationships between voluntary exercise, food consumption, and changes in body weight and composition and simultaneously localize quantitative trait loci (QTL) controlling these traits. A large (n = 815) murine advanced intercross line (G(4)) was created from a reciprocal cross between a high-running line and the inbred strain C57BL/6J. Body weight and composition (% fat, % lean) were measured at 4, 6, and 8 wk of age. After measurements at 8 wk of age, mice were given access to running wheels, during which food consumption was quantified and after which body weight and composition were assessed to evaluate exercise-induced changes. Phenotypic correlations indicated that the relationship between exercise and overall change in weight and adiposity depended on body composition before the initiation of exercise. Interval mapping revealed QTL for body weight, % fat, and % lean at 4, 6, and 8 wk of age. Furthermore, QTL were observed for food consumption and changes in weight, % fat, and % lean in response to short-term exercise. Here we provide some clarity for the relationship between weight loss, reduction in adiposity, food consumption, and exercise. Simultaneously, we reinforce the genetic basis for body weight and composition with some independent loci controlling growth at different ages. Finally, we present unique QTL providing insight regarding variation in weight loss and reduction in adiposity in response to exercise.

  15. Genetic tracking of mice and other bioproxies to infer human history.

    PubMed

    Jones, Eleanor P; Eager, Heidi M; Gabriel, Sofia I; Jóhannesdóttir, Fríða; Searle, Jeremy B

    2013-05-01

    The long-distance movements made by humans through history are quickly erased by time but can be reconstructed by studying the genetic make-up of organisms that travelled with them. The phylogeography of the western house mouse (Mus musculus domesticus), whose current widespread distribution around the world has been caused directly by the movements of (primarily) European people, has proved particularly informative in a series of recent studies. The geographic distributions of genetic lineages in this commensal have been linked to the Iron Age movements within the Mediterranean region and Western Europe, the extensive maritime activities of the Vikings in the 9th to 11th centuries, and the colonisation of distant landmasses and islands by the Western European nations starting in the 15th century. We review here recent insights into human history based on phylogeographic studies of mice and other species that have travelled with humans, and discuss how emerging genomic methodologies will increase the precision of these inferences. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Systems genetic analysis of hippocampal neuroanatomy and spatial learning in mice

    PubMed Central

    Delprato, Anna; Bonheur, Brice; Algéo, Marie-Paule; Rosay, Philippe; Lu, Lu; Williams, Robert W.

    2016-01-01

    Variation in hippocampal neuroanatomy correlates well with spatial learning ability in mice. Here we have studied both hippocampal neuroanatomy and behavior in 53 isogenic BXD recombinant strains derived from C57BL/6J and DBA/2J parents. A combination of experimental, neuroinformatic, and systems genetics methods were used to test the genetic bases of variation and covariation among traits. Data were collected on seven hippocampal subregions in CA3 and CA4 after testing spatial memory in an 8-arm radial maze task. Quantitative trait loci (QTLs) were identified for hippocampal structure, including the areas of the intra- and infrapyramidal mossy fibers, stratum radiatum, and stratum pyramidale, and for a spatial learning parameter, error rate. We identified multiple loci and gene variants linked to either structural differences or behavior. Gpc4 and Tenm2 are strong candidate genes that may modulate intra- and infrapyramidal mossy fiber areas. Analysis of gene-expression networks and trait correlations highlight several processes influencing morphometrical variation and spatial learning. PMID:26449520

  17. Genetic manipulation of longevity-related genes as a tool to regulate yeast life span and metabolite production during winemaking

    PubMed Central

    2013-01-01

    Background Yeast viability and vitality are essential for different industrial processes where the yeast Saccharomyces cerevisiae is used as a biotechnological tool. Therefore, the decline of yeast biological functions during aging may compromise their successful biotechnological use. Life span is controlled by a variety of molecular mechanisms, many of which are connected to stress tolerance and genomic stability, although the metabolic status of a cell has proven a main factor affecting its longevity. Acetic acid and ethanol accumulation shorten chronological life span (CLS), while glycerol extends it. Results Different age-related gene classes have been modified by deletion or overexpression to test their role in longevity and metabolism. Overexpression of histone deacetylase SIR2 extends CLS and reduces acetate production, while overexpression of SIR2 homolog HST3 shortens CLS, increases the ethanol level, and reduces acetic acid production. HST3 overexpression also enhances ethanol tolerance. Increasing tolerance to oxidative stress by superoxide dismutase SOD2 overexpression has only a moderate positive effect on CLS. CLS during grape juice fermentation has also been studied for mutants on several mRNA binding proteins that are regulators of gene expression at the posttranscriptional level; we found that NGR1 and UTH4 deletions decrease CLS, while PUF3 and PUB1 deletions increase it. Besides, the pub1Δ mutation increases glycerol production and blocks stress granule formation during grape juice fermentation. Surprisingly, factors relating to apoptosis, such as caspase Yca1 or apoptosis-inducing factor Aif1, play a positive role in yeast longevity during winemaking as their deletions shorten CLS. Conclusions Manipulation of regulators of gene expression at both transcriptional (i.e., sirtuins) and posttranscriptional (i.e., mRNA binding protein Pub1) levels allows to modulate yeast life span during its biotechnological use. Due to links between aging and

  18. The Behavioral Consequence of Phenylketonuria in Mice Depends on the Genetic Background

    PubMed Central

    Bruinenberg, Vibeke M.; van der Goot, Els; van Vliet, Danique; de Groot, Martijn J.; Mazzola, Priscila N.; Heiner-Fokkema, M. Rebecca; van Faassen, Martijn; van Spronsen, Francjan J.; van der Zee, Eddy A.

    2016-01-01

    To unravel the role of gene mutations in the healthy and the diseased state, countless studies have tried to link genotype with phenotype. However, over the years, it became clear that the strain of mice can influence these results. Nevertheless, identical gene mutations in different strains are often still considered equals. An example of this, is the research done in phenylketonuria (PKU), an inheritable metabolic disorder. In this field, a PKU mouse model (either on a BTBR or C57Bl/6 background) is often used to examine underlying mechanisms of the disease and/or new treatment strategies. Both strains have a point mutation in the gene coding for the enzyme phenylalanine hydroxylase which causes toxic concentrations of the amino acid phenylalanine in blood and brain, as found in PKU patients. Although the mutation is identical and therefore assumed to equally affect physiology and behavior in both strains, no studies directly compared the two genetic backgrounds to test this assumption. Therefore, this study compared the BTBR and C57Bl/6 wild-type and PKU mice on PKU-relevant amino acid- and neurotransmitter-levels and at a behavioral level. The behavioral paradigms were selected from previous literature on the PKU mouse model and address four domains, namely (1) activity levels, (2) motor performance, (3) anxiety and/or depression-like behavior, and (4) learning and memory. The results of this study showed comparable biochemical changes in phenylalanine and neurotransmitter concentrations. In contrast, clear differences in behavioral outcome between the strains in all four above-mentioned domains were found, most notably in the learning and memory domain. The outcome in this domain seem to be primarily due to factors inherent to the genetic background of the mouse and much less by differences in PKU-specific biochemical parameters in blood and brain. The difference in behavioral outcome between PKU of both strains emphasizes that the consequence of the PAH

  19. The Behavioral Consequence of Phenylketonuria in Mice Depends on the Genetic Background.

    PubMed

    Bruinenberg, Vibeke M; van der Goot, Els; van Vliet, Danique; de Groot, Martijn J; Mazzola, Priscila N; Heiner-Fokkema, M Rebecca; van Faassen, Martijn; van Spronsen, Francjan J; van der Zee, Eddy A

    2016-01-01

    To unravel the role of gene mutations in the healthy and the diseased state, countless studies have tried to link genotype with phenotype. However, over the years, it became clear that the strain of mice can influence these results. Nevertheless, identical gene mutations in different strains are often still considered equals. An example of this, is the research done in phenylketonuria (PKU), an inheritable metabolic disorder. In this field, a PKU mouse model (either on a BTBR or C57Bl/6 background) is often used to examine underlying mechanisms of the disease a