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211.
Osiak A Radecke F Guhl E Radecke S Dannemann N Lütge F Glage S Rudolph C Cantz T Schwarz K Heilbronn R Cathomen T 《PloS one》2011,6(12):e28911
Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells. 相似文献
212.
Pütz MM Bouche FB de Swart RL Muller CP 《International journal for parasitology》2003,33(5-6):525-545
Vaccination with the current live attenuated measles vaccine is one of the most successful and cost-effective medical interventions. However, as a result of persisting maternal antibodies and immaturity of the infant immune system, this vaccine is poorly immunogenic in children <9 months old. Immunity against the live vaccine is less robust than natural immunity and protection less durable. There may also be some concern about (vaccine) virus spread during the final stage of an eventual measles eradication program. Opinions may differ with respect to the potential threat that some of these concerns may be to the World Health Organisation goal of measles elimination, but there is a consensus that the development of new measles vaccines cannot wait. Candidate vaccines are based on viral or bacterial vectors expressing recombinant viral proteins, naked DNA, immune stimulating complexes or synthetic peptides mimicking neutralising epitopes. While some of these candidate vaccines have proven their efficacy in monkey studies, aerosol formulated live attenuated measles vaccine are evaluated in clinical trials. 相似文献
213.
Wouter Coppieters Juliette Riquet Juan-José Arranz Paulette Berzi Nadine Cambisano Bernard Grisart Latifa Karim Fabienne Marcq Laurence Moreau Carine Nezer Patricia Simon Pascal Vanmanshoven Danny Wagenaar Michel Georges 《Mammalian genome》1998,9(7):540-544
A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing
milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome
(Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified
using the grand2-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons
and confirming the predicted QTL allele substitution effect.
Received: 30 December 1997 / Accepted: 21 February 1998 相似文献
214.
Sterpenich V Albouy G Boly M Vandewalle G Darsaud A Balteau E Dang-Vu TT Desseilles M D'Argembeau A Gais S Rauchs G Schabus M Degueldre C Luxen A Collette F Maquet P 《PLoS biology》2007,5(11):e282
Emotional events are usually better remembered than neutral ones. This effect is mediated in part by a modulation of the hippocampus by the amygdala. Sleep plays a role in the consolidation of declarative memory. We examined the impact of sleep and lack of sleep on the consolidation of emotional (negative and positive) memories at the macroscopic systems level. Using functional MRI (fMRI), we compared the neural correlates of successful recollection by humans of emotional and neutral stimuli, 72 h after encoding, with or without total sleep deprivation during the first post-encoding night. In contrast to recollection of neutral and positive stimuli, which was deteriorated by sleep deprivation, similar recollection levels were achieved for negative stimuli in both groups. Successful recollection of emotional stimuli elicited larger responses in the hippocampus and various cortical areas, including the medial prefrontal cortex, in the sleep group than in the sleep deprived group. This effect was consistent across subjects for negative items but depended linearly on individual memory performance for positive items. In addition, the hippocampus and medial prefrontal cortex were functionally more connected during recollection of either negative or positive than neutral items, and more so in sleeping than in sleep-deprived subjects. In the sleep-deprived group, recollection of negative items elicited larger responses in the amygdala and an occipital area than in the sleep group. In contrast, no such difference in brain responses between groups was associated with recollection of positive stimuli. The results suggest that the emotional significance of memories influences their sleep-dependent systems-level consolidation. The recruitment of hippocampo-neocortical networks during recollection is enhanced after sleep and is hindered by sleep deprivation. After sleep deprivation, recollection of negative, potentially dangerous, memories recruits an alternate amygdalo-cortical network, which would keep track of emotional information despite sleep deprivation. 相似文献
215.
Marion Lagune Vincent Le Moigne Matt D. Johansen Flor Vsquez Sotomayor Wassim Daher Ccile Petit Gina Cosentino Laura Paulowski Thomas Gutsmann Matthias Wilmanns Florian P. Maurer Jean-Louis Herrmann Fabienne Girard-Misguich Laurent Kremer 《PLoS pathogens》2022,18(8)
ESX type VII secretion systems are complex secretion machineries spanning across the mycobacterial membrane and play an important role in pathogenicity, nutrient uptake and conjugation. We previously reported the role of ESX-4 in modulating Mycobacterium abscessus intracellular survival. The loss of EccB4 was associated with limited secretion of two effector proteins belonging to the WXG-100 family, EsxU and EsxT, and encoded by the esx-4 locus. This prompted us to investigate the function of M. abscessus EsxU and EsxT in vitro and in vivo. Herein, we show that EsxU and EsxT are substrates of ESX-4 and form a stable 1:1 heterodimer that permeabilizes artificial membranes. While expression of esxU and esxT was up-regulated in M. abscessus-infected macrophages, their absence in an esxUT deletion mutant prevented phagosomal membrane disruption while maintaining M. abscessus in an unacidified phagosome. Unexpectedly, the esxUT deletion was associated with a hyper-virulent phenotype, characterised by increased bacterial loads and mortality in mouse and zebrafish infection models. Collectively, these results demonstrate that the presence of EsxU and EsxT dampens survival and persistence of M. abscessus during infection. 相似文献
216.
According to portfolio theory applied to fisheries management, economic returns are stabilised by harvesting in a portfolio stocks of species whose returns are negatively correlated and for which the portfolio economic return variance is smaller than the sum of stock specific return variances. Also, variability is expected to decrease with portfolio width. Using a range of indicators, these predictions were tested for the French fishing fleets in the Bay of Biscay (Northeast Atlantic) during the period 2001–2009. For this, vessels were grouped into eight fishing fleets based on the gears used and exploited species were grouped into five functional groups. The portfolio width of fleets ranged from 1–3 functional groups, or 4–19 species. Economic fleet returns (sale revenues minus fishing costs) varied strongly between years; the interannual variability was independent of portfolio width (species or functional groups). Energy ratio expressed by the ratio between fuel energy used for fishing and energy contained in landings varied from 0.3 for purse seines to 9.7 for trawlers using bottom trawls alone or in combination with pelagic trawls independent of portfolio width. Interannual variability in total sale revenues was larger than the sum of species specific sales revenue variability, except for fleets using hooks and pelagic trawlers; it increased with the number of species exploited. In conclusion, the interannual variability of economic returns or energy ratios of French fisheries in the Bay of Biscay did not decrease with the number of species or functional groups exploited, though it varied between fleets. 相似文献
217.
218.
Fabienne Defrise-Quertain Pierre Chatelain Jean-Marie Ruysschaert Michel Delmelle 《生物化学与生物物理学报:生物膜》1982,688(1):116-122
Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature. 相似文献
219.
Marianne Goyer Alicia Loiselet Fabienne Bon Coralie L’Ollivier Michael Laue Gudrun Holland Alain Bonnin Frederic Dalle 《PloS one》2016,11(3)
C. albicans is a commensal yeast of the mucous membranes in healthy humans that can also cause disseminated candidiasis, mainly originating from the digestive tract, in vulnerable patients. It is necessary to understand the cellular and molecular mechanisms of the interaction of C. albicans with enterocytes to better understand the basis of commensalism and pathogenicity of the yeast and to improve the management of disseminated candidiasis. In this study, we investigated the kinetics of tight junction (TJ) formation in parallel with the invasion of C. albicans into the Caco-2 intestinal cell line. Using invasiveness assays on Caco-2 cells displaying pharmacologically altered TJ (i.e. differentiated epithelial cells treated with EGTA or patulin), we were able to demonstrate that TJ protect enterocytes against invasion of C. albicans. Moreover, treatment with a pharmacological inhibitor of endocytosis decreased invasion of the fungus into Caco-2 cells displaying altered TJ, suggesting that facilitating access of the yeast to the basolateral side of intestinal cells promotes endocytosis of C. albicans in its hyphal form. These data were supported by SEM observations of differentiated Caco-2 cells displaying altered TJ, which highlighted membrane protrusions engulfing C. albicans hyphae. We furthermore demonstrated that Als3, a hypha-specific C. albicans invasin, facilitates internalization of the fungus by active penetration and induced endocytosis by differentiated Caco-2 cells displaying altered TJ. However, our observations failed to demonstrate binding of Als3 to E-cadherin as the trigger mechanism of endocytosis of C. albicans into differentiated Caco-2 cells displaying altered TJ. 相似文献
220.
Knut Eichler Fabienne Bourgis Anne Buchet Hans-Peter Kleber Marie-Andrée Mandrand-Berthelot 《Molecular microbiology》1994,13(5):775-786
The sequence encompassing the cai genes of Escherichia coli, which encode the carnitine pathway, has been determined. Apart from the already identified caiB gene coding for the carnitine dehydratase, five additional open reading frames were identified. They belong to the caiTABCDE operon, which was shown to be located at the first minute on the chromosome and transcribed during anaerobic growth in the presence of carnitine. The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a functional H-NS protein, in relation to the consensus sequences detected in the promoter region of the cai operon. In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicted molecular masses of 56 613 Da (CaiT), 42 564 Da (CaiA), 59311 Da (CaiC), 32 329 Da (CaiD) and 21 930 Da (CaiE). Amino acid sequence similarity or enzymatic analysis supported the function assigned to each protein. CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/carnitine CoA ligase. CaiD bears strong homology with enoyl hydratases/isomerases. Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the basal level of carnitine dehydratase activity. It is inferred that CaiE is an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities. Taken together, these data suggest that the carnitine pathway in E. coli resembles that found in a strain situated between Agrobacterium and Rhizobium. 相似文献