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  • 1
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    A versatile microsatellite instability reporter system in human cells (2013)
    Oxford University Press
    Details
    Publication Date: 2013-09-06
    Description: Here, we report the investigation of microsatellite instability (MSI) in human cells with a newly developed reporter system based on fluorescence. We composed a vector into which microsatellites of different lengths and nucleotide composition can be introduced between a functional copy of the fluorescent protein mCherry and an out-of-frame copy of EGFP; in vivo frameshifting will lead to EGFP expression, which can be quantified by fluorescence activated cell sorting (FACS). Via targeted recombineering, single copy reporters were introduced in HEK293 and MCF-7 cells. We found predominantly –1 and +1 base pair frameshifts, the levels of which are kept in tune by mismatch repair. We show that tract length and composition greatly influences MSI. In contrast, a tracts’ potential to form a G-quadruplex structure, its strand orientation or its transcriptional status is not affecting MSI. We further validated the functionality of the reporter system for screening microsatellite mutagenicity of compounds and for identifying modifiers of MSI: using a retroviral miRNA expression library, we identified miR-21, which targets MSH2, as a miRNA that induces MSI when overexpressed. Our data also provide proof of principle for the strategy of combining fluorescent reporters with next-generation sequencing technology to identify genetic factors in specific pathways.
    Keywords: Recombination
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Intellectual property in plant breeding: comparing different levels and forms of protection (2015)
    Oxford University Press
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    Publication Date: 2015-12-29
    Description: Welfare trade-offs between intellectual property (IP) protections provided by patents and by plant variety protection (PVP) are explored. PVP breeders’ exemption weakens IP protection, but may speed the transfer of research gains across firms. A model is developed assuming firms optimise research given existing IP protection. A baseline scenario supporting each system is used to perform welfare analysis, and study how the balance is altered between systems. Survey data suggest patents are more appropriate for longer-term, higher-risk research, whereas PVP is better suited for traditional breeding. A scenario where patents and licensing co-exist dominates PVP in all commercially relevant areas.
    Keywords: O31 - Innovation and Invention: Processes and Incentives, O34 - Intellectual Property Rights, Q16 - R&D ; Agricultural Technology ; Agricultural Extension Services
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
    Published by Oxford University Press
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  • 3
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    Genome engineering using a synthetic gene circuit in Bacillus subtilis (2015)
    Oxford University Press
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    Publication Date: 2015-04-02
    Description: Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters ( B. subtilis xylA (P xyl ) and spac (P spac )) and two repressor genes ( lacI and xylR ). P xyl - lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and P spac –chloramphenicol resistant genes ( cat ) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the P spac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete P xyl - lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene ( amyE ) and a 38-kb operon ( ppsABCDE ). This method will be useful to construct designer Bacillus strains for various industrial applications.
    Keywords: Recombination
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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