76 research outputs found

    Efficient Culturing and Genetic Manipulation of Human Pluripotent Stem Cells

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    Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs

    A TALEN Genome-Editing System for Generating Human Stem Cell-Based Disease Models

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    SummaryTranscription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease

    Space charge in ionization detectors and the NA48 electromagnetic calorimeter

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    The subject of space charge due to positive ions slowly moving in parallel plate ionization chambers is considered. A model for the degradation of the detector response is developed, with particular emphasis on electromagnetic calorimeters. The topics discussed include: (a) the stationary and (b) the time dependent cases; (c) the limit of very large space charge; (d) the electric field dependence of the electron drift velocity; (e) the effect of longitudinal development of showers; (f) the behavior of the average reductions of response and (g) the non-uniformity of response for different positions of the shower axis inside the cell defined by the electrodes. The NA48 calorimeter is used as application and for comparison of results

    Addendum 2 to P253: a high sensitivity investigation of KsK_{s} and neutral hyperon decays using a modified KsK_{s} beam

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    β-Aminoisobutyric Acid Induces Browning of White Fat and Hepatic β-Oxidation and Is Inversely Correlated with Cardiometabolic Risk Factors

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    The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) regulates metabolic genes in skeletal muscle and contributes to the response of muscle to exercise. Muscle PGC-1α transgenic expression and exercise both increase the expression of thermogenic genes within white adipose. How the PGC-1α-mediated response to exercise in muscle conveys signals to other tissues remains incompletely defined. We employed a metabolomic approach to examine metabolites secreted from myocytes with forced expression of PGC-1α, and identified β-aminoisobutyric acid (BAIBA) as a small molecule myokine. BAIBA increases the expression of brown adipocyte-specific genes in white adipocytes and β-oxidation in hepatocytes both in vitro and in vivo through a PPARα-mediated mechanism, induces a brown adipose-like phenotype in human pluripotent stem cells, and improves glucose homeostasis in mice. In humans, plasma BAIBA concentrations are increased with exercise and inversely associated with metabolic risk factors. BAIBA may thus contribute to exercise-induced protection from metabolic diseases

    Direct search for light gluinos

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    We present the results for a direct search for light gluinos through the appearance of η3π0\eta\rightarrow 3\pi^{0} with high transverse momentum in the vacuum tank of the NA48 experiment at CERN. We find one event within a lifetime range of 10910310^{-9}-10^{-3}s and another one between 101010910^{-10}-10^{-9}s. Both events are consistent with the expected background from neutrons in the beam, produced by 450 GeV protons impinging on the Be targets, which interact with the residual air in the tank. From these data we give limits on the production of the hypothetical gg~g\widetilde{g} bound state, the R0R^0 hadron, and its R0ηγ~R^0\rightarrow\eta\widetilde{\gamma} decay in the R0R^0 mass range between 1 and 5~GeV
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