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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 12 June 2012, Vol.109(24), pp.9354-9
    Description: Gene targeting by zinc-finger nucleases in one-cell embryos provides an expedite mutagenesis approach in mice, rats, and rabbits. This technology has been recently used to create knockout and knockin mutants through the deletion or insertion of nucleotides. Here we apply zinc-finger nucleases in one-cell mouse embryos to generate disease-related mutants harboring single nucleotide or codon replacements. Using a gene-targeting vector or a synthetic oligodesoxynucleotide as template for homologous recombination, we introduced missense and silent mutations into the Rab38 gene, encoding a small GTPase that regulates intracellular vesicle trafficking. These results demonstrate the feasibility of seamless gene editing in one-cell embryos to create genetic disease models and establish synthetic oligodesoxynucleotides as a simplified mutagenesis tool.
    Keywords: Embryo, Mammalian ; Gene Targeting ; Mutation
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 05 March 2013, Vol.110(10), pp.3782-7
    Description: The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.
    Keywords: Disease Models, Animal ; Germ-Line Mutation ; Genetic Diseases, Inborn -- Genetics ; Oligodeoxyribonucleotides -- Administration & Dosage
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States, June 12, 2012, Vol.109(24), p.9354(6)
    Description: Gene targeting by zinc-finger nucleases in one-cell embryos provides an expedite mutagenesis approach in mice, rats, and rabbits. This technology has been recently used to create knockout and knock in mutants through the deletion or insertion of nucleotides. Here we apply zinc-finger nucleases in one-cell mouse embryos to generate disease-related mutants harboring single nucleotide or codon replacements. Using a gene-targeting vector or a synthetic oligodesoxynucleotide as template for homologous recombination, we introduced missense and silent mutations into the Rab38 gene, encoding a small GTPase that regulates intracellular vesicle trafficking. These results demonstrate the feasibility of seamless gene editing in one-cell embryos to create genetic disease models and establish synthetic oligodesoxynucleotides as a simplified mutagenesis tool. doi/ 10.1073/pnas.1121203109
    Keywords: Gene Mutation -- Research ; Embryonic Development -- Physiological Aspects ; Embryonic Development -- Genetic Aspects ; Mutagenesis -- Research
    ISSN: 0027-8424
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  • 4
    In: Nature Biotechnology, 2010, Vol.28(7), p.684
    Keywords: Membrane Proteins ; Genetic Aspects ; Animal Genetics ; Research ; Medicine ; Engineering ; Biology;
    ISSN: 1087-0156
    E-ISSN: 15461696
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  • 5
    Language: English
    In: PLoS ONE, 01 January 2014, Vol.9(10), p.e111239
    Description: A significant challenge facing high-throughput phenotyping of in-vivo knockout mice is ensuring phenotype calls are robust and reliable. Central to this problem is selecting an appropriate statistical analysis that models both the experimental design (the workflow and the way control mice are selected for comparison with knockout animals) and the sources of variation. Recently we proposed a mixed model suitable for small batch-oriented studies, where controls are not phenotyped concurrently with mutants. Here we evaluate this method both for its sensitivity to detect phenotypic effects and to control false positives, across a range of workflows used at mouse phenotyping centers. We found the sensitivity and control of false positives depend on the workflow. We show that the phenotypes in control mice fluctuate unexpectedly between batches and this can cause the false positive rate of phenotype calls to be inflated when only a small number of batches are tested, when the effect of knockout becomes confounded with temporal fluctuations in control mice. This effect was observed in both behavioural and physiological assays. Based on this analysis, we recommend two approaches (workflow and accompanying control strategy) and associated analyses, which would be robust, for use in high-throughput phenotyping pipelines. Our results show the importance in modelling all sources of variability in high-throughput phenotyping studies.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 6
    Language: English
    In: PLoS ONE, 01 January 2013, Vol.8(1), p.e54851
    Description: Stress, the physiological reaction to a stressor, is initiated in teleost fish by hormone cascades along the hypothalamus-pituitary-interrenal (HPI) axis. Cortisol is the major stress hormone and contributes to the appropriate stress response by regulating gene expression after binding to the glucocorticoid receptor. Cortisol is inactivated when 11β-hydroxysteroid dehydrogenase (HSD) type 2 catalyzes its oxidation to cortisone. In zebrafish, Danio rerio, cortisone can be further reduced to 20β-hydroxycortisone. This reaction is catalyzed by 20β-HSD type 2, recently discovered by us. Here, we substantiate the hypothesis that 20β-HSD type 2 is involved in cortisol catabolism and stress response. We found that hsd11b2 and hsd20b2 transcripts were up-regulated upon cortisol treatment. Moreover, a cortisol-independent, short-term physical stressor led to the up-regulation of hsd11b2 and hsd20b2 along with several HPI axis genes. The morpholino-induced knock down of hsd20b2 in zebrafish embryos revealed no developmental phenotype under normal culture conditions, but prominent effects were observed after a cortisol challenge. Reporter gene experiments demonstrated that 20β-hydroxycortisone was not a physiological ligand for the zebrafish glucocorticoid or mineralocorticoid receptor but was excreted into the fish holding water. Our experiments show that 20β-HSD type 2, together with 11β-HSD type 2, represents a short pathway in zebrafish to rapidly inactivate and excrete cortisol. Therefore, 20β-HSD type 2 is an important enzyme in stress response.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 7
    Language: English
    In: Science (New York, N.Y.), 28 May 2010, Vol.328(5982), pp.1158-61
    Description: Obesity results from chronic energy surplus and excess lipid storage in white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) efficiently burns lipids through adaptive thermogenesis. Studying mouse models, we show that cyclooxygenase (COX)-2, a rate-limiting enzyme in prostaglandin (PG) synthesis, is a downstream effector of beta-adrenergic signaling in WAT and is required for the induction of BAT in WAT depots. PG shifted the differentiation of defined mesenchymal progenitors toward a brown adipocyte phenotype. Overexpression of COX-2 in WAT induced de novo BAT recruitment in WAT, increased systemic energy expenditure, and protected mice against high-fat diet-induced obesity. Thus, COX-2 appears integral to de novo BAT recruitment, which suggests that the PG pathway regulates systemic energy homeostasis.
    Keywords: Energy Metabolism ; Thermogenesis ; Adipocytes, Brown -- Physiology ; Adipose Tissue, Brown -- Physiology ; Adipose Tissue, White -- Physiology ; Cyclooxygenase 2 -- Genetics ; Prostaglandins -- Metabolism
    ISSN: 00368075
    E-ISSN: 1095-9203
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  • 8
    Language: English
    In: PLoS ONE, 01 January 2015, Vol.10(5), p.e0125304
    Description: Cataracts are the major eye disorder and have been associated mainly with mutations in lens-specific genes, but cataracts are also frequently associated with complex syndromes. In a large-scale high-throughput ENU mutagenesis screen we analyzed the offspring of paternally treated C3HeB/FeJ mice for obvious dysmorphologies. We identified a mutant suffering from rough coat and small eyes only in homozygotes; homozygous females turned out to be sterile. The mutation was mapped to chromosome 7 between the markers 116J6.1 and D7Mit294;4 other markers within this interval did not show any recombination among 160 F2-mutants. The critical interval (8.6 Mb) contains 3 candidate genes (Apoe, Six5, Opa3); none of them showed a mutation. Using exome sequencing, we identified a c.2209T〉C mutation in the Xpd/Ercc2 gene leading to a Ser737Pro exchange. During embryonic development, the mutant eyes did not show major changes. Postnatal histological analyses demonstrated small cortical vacuoles; later, cortical cataracts developed. Since XPD/ERCC2 is involved in DNA repair, we checked also for the presence of the repair-associated histone γH2AX in the lens. During the time, when primary lens fiber cell nuclei are degraded, γH2AX was strongly expressed in the cell nuclei; later, it demarcates clearly the border of the lens cortex to the organelle-free zone. Moreover, we analyzed also whether seemingly healthy heterozygotes might be less efficient in repair of DNA damage induced by ionizing radiation than wild types. Peripheral lymphocytes irradiated by 1Gy Cs137 showed 6 hrs after irradiation significantly more γH2AX foci in heterozygotes than in wild types. These findings demonstrate the importance of XPD/ERCC2 not only for lens fiber cell differentiation, but also for the sensitivity to ionizing radiation. Based upon these data, we hypothesize that variations in the human XPD/ERCC2 gene might increase the susceptibility for several disorders besides Xeroderma pigmentosum in heterozygotes under particular environmental conditions.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 9
    Language: English
    In: Journal of Theoretical Biology, 07 October 2017, Vol.430, pp.32-44
    Description: During pancreas development, positive endocrine progenitors are specified by Delta/Notch (D/N) mediated lateral inhibition in the growing ducts. During neurogenesis, genes that determine the transition from the proneural state to neuronal or glial lineages are oscillating before their expression is sustained. Although the basic gene regulatory network is very similar, cycling gene expression in pancreatic development was not investigated yet, and previous simulations of lateral inhibition in pancreas development excluded by design the possibility of oscillations. To explore this possibility, we developed a dynamic model of a growing duct that results in an oscillatory phase before the determination of endocrine progenitors by lateral inhibition. The basic network (D/N + Hes1 + Neurog3) shows scattered, stable expression after displaying transient expression. Furthermore, we included the Hes1 negative feedback as previously discussed in neurogenesis and show the consequences for expression in pancreatic duct development. Interestingly, a weakened HES1 action on the promoter allows the coexistence of stable patterning and oscillations. In conclusion, cycling gene expression and lateral inhibition are not mutually exclusive. In this way, we argue for a unified mode of D/N mediated lateral inhibition in neurogenic and pancreatic progenitor specification.
    Keywords: Pancreatogenesis ; Cycling Gene Expression ; Neurog3 ; Endocrine Progenitor ; Simulation ; Biology
    ISSN: 0022-5193
    E-ISSN: 1095-8541
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  • 10
    Language: English
    In: Diabetologia, 2011, Vol.54(8), pp.2132-2142
    Description: Byline: F. W. Kiefer (1), S. Neschen (2), B. Pfau (1), B. Legerer (1), A. Neuhofer (1), M. Kahle (2), M. Hrabe de Angelis (2), M. Schlederer (3), M. Mair (3), L. Kenner (3), J. Plutzky (4), M. Zeyda (1), T. M. Stulnig (1) Keywords: Gluconeogenesis; High-fat diet; Inflammation; Insulin resistance; Non-alcoholic fatty liver disease Abstract: Aims/hypothesis Obesity is strongly associated with the development of non-alcoholic fatty liver disease (NAFLD). The cytokine osteopontin (OPN) was recently shown to be involved in obesity-induced adipose tissue inflammation and reduced insulin response. Accumulating evidence links OPN to the pathogenesis of NAFLD. Here we aimed to identify the role of OPN in obesity-associated hepatic steatosis and impaired hepatic glucose metabolism. Methods Wild-type (WT) and Opn (also known as Spp1) knockout (Opn .sup.-/-) mice were fed a high-fat or low-fat diet to study OPN effects in obesity-driven hepatic alterations. Results We show that genetic OPN deficiency protected from obesity-induced hepatic steatosis, at least in part, by downregulating hepatic triacylglycerol synthesis. Conversely, absence of OPN promoted fat storage in adipose tissue thereby preventing the obesity-induced shift to ectopic fat accumulation in the liver. Euglycaemic--hyperinsulinaemic clamp studies revealed that insulin resistance and excess hepatic glucose production in obesity were significantly attenuated in Opn .sup.-/- mice. OPN deficiency markedly improved hepatic insulin signalling as shown by enhanced insulin receptor substrate-2 phosphorylation and prevented upregulation of the major hepatic transcription factor Forkhead box O1 and its gluconeogenic target genes. In addition, obesity-driven hepatic inflammation and macrophage accumulation was blocked by OPN deficiency. Conclusions/interpretation Our data strongly emphasise OPN as mediator of obesity-associated hepatic alterations including steatosis, inflammation, insulin resistance and excess gluconeogenesis. Targeting OPN action could therefore provide a novel therapeutic strategy to prevent obesity-related complications such as NAFLD and type 2 diabetes. Author Affiliation: (1) Department of Medicine III, Clinical Division of Endocrinology and Metabolism, Medical University of Vienna, Waehringer Guertel 18-20, 1090, Vienna, Austria (2) Institute of Experimental Genetics, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany (3) Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria (4) Department of Medicine, Brigham and Women's Hospital Boston, Cardiovascular Division, Harvard Medical School, Boston, MA, USA Article History: Registration Date: 19/04/2011 Received Date: 19/12/2010 Accepted Date: 04/04/2011 Online Date: 12/05/2011 Article note: Electronic supplementary material The online version of this article (doi: 10.1007/s00125-011-2170-0) contains supplementary material, which is available to authorised users.
    Keywords: Gluconeogenesis ; High-fat diet ; Inflammation ; Insulin resistance ; Non-alcoholic fatty liver disease
    ISSN: 0012-186X
    E-ISSN: 1432-0428
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