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  • 1
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    E-TALEN: a web tool to design TALENs for genome engineering (2013)
    Oxford University Press
    Details
    Publication Date: 2013-11-02
    Description: Use of transcription activator-like effector nucleases (TALENs) is a promising new technique in the field of targeted genome engineering, editing and reverse genetics. Its applications span from introducing knockout mutations to endogenous tagging of proteins and targeted excision repair. Owing to this wide range of possible applications, there is a need for fast and user-friendly TALEN design tools. We developed E-TALEN ( http://www.e-talen.org ), a web-based tool to design TALENs for experiments of varying scale. E-TALEN enables the design of TALENs against a single target or a large number of target genes. We significantly extended previously published design concepts to consider genomic context and different applications. E-TALEN guides the user through an end-to-end design process of de novo TALEN pairs, which are specific to a certain sequence or genomic locus. Furthermore, E-TALEN offers a functionality to predict targeting and specificity for existing TALENs. Owing to the computational complexity of many of the steps in the design of TALENs, particular emphasis has been put on the implementation of fast yet accurate algorithms. We implemented a user-friendly interface, from the input parameters to the presentation of results. An additional feature of E-TALEN is the in-built sequence and annotation database available for many organisms, including human, mouse, zebrafish, Drosophila and Arabidopsis , which can be extended in the future.
    Keywords: Synthetic Biology and Assembly Cloning, Mutagenesis, Computational Methods, Targeted inhibition of gene function, Miscellaneous/other
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 2
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    Signaling specificity by Frizzled receptors in Drosophila (2000)
    American Association for the Advancement of Science (AAAS)
    Details
    Publication Date: 2000-06-10
    Description: Wnt-Frizzled (Fz) signaling pathways play recurring important roles during the development and homeostasis of vertebrates and invertebrates. Fz receptors can signal through beta-catenin-dependent and -independent pathways. In Drosophila, Fz and Fz2 are redundant receptors for Wg. In addition, Fz conveys signals through a distinct pathway to organize planar polarization of epithelial structures. We demonstrate that the cytoplasmic sequences of Fz2 and Fz preferentially activate the beta-catenin and planar polarity cascade, respectively. Both receptors activate either pathway, but with different efficiencies. Intrinsic differences in signaling efficiency in closely related receptors might be a general mechanism for generating signaling specificity in vivo.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Boutros, M -- Mihaly, J -- Bouwmeester, T -- Mlodzik, M -- New York, N.Y. -- Science. 2000 Jun 9;288(5472):1825-8.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉European Molecular Biology Laboratory, Developmental Biology Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/10846164" target="_blank"〉PubMed〈/a〉
    Keywords: Adaptor Proteins, Signal Transducing ; Animals ; Armadillo Domain Proteins ; *Body Patterning ; Cytoskeletal Proteins/metabolism ; Drosophila/genetics/growth & development/*metabolism ; *Drosophila Proteins ; Eye/growth & development/metabolism ; Frizzled Receptors ; Insect Proteins ; Larva/growth & development/metabolism ; Ligands ; Membrane Proteins/chemistry/genetics/*metabolism ; Mutation ; Phenotype ; Phosphoproteins/metabolism ; Photoreceptor Cells, Invertebrate/growth & development/metabolism ; Protein Structure, Tertiary ; Proto-Oncogene Proteins/*metabolism ; Receptors, G-Protein-Coupled ; Receptors, Neurotransmitter/chemistry/genetics/*metabolism ; Recombinant Fusion Proteins/chemistry/metabolism ; *Signal Transduction ; *Trans-Activators ; Transcription Factors ; Wings, Animal/growth & development/metabolism ; Wnt Proteins ; Wnt1 Protein ; Xenopus ; Xenopus Proteins ; *Zebrafish Proteins ; beta Catenin
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 3
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    The promise and perils of Wnt signaling through beta-catenin (2002)
    American Association for the Advancement of Science (AAAS)
    Details
    Publication Date: 2002-06-01
    Description: Wnt pathways are involved in the control of gene expression, cell behavior, cell adhesion, and cell polarity. In addition, they often operate in combination with other signaling pathways. The Wnt/beta-catenin pathway is the best studied of the Wnt pathways and is highly conserved through evolution. In this pathway, Wnt signaling inhibits the degradation of beta-catenin, which can regulate transcription of a number of genes. Some of the genes regulated are those associated with cancer and other diseases (for example, colorectal cancer and melanomas). As a result, components of the Wnt/beta-catenin pathway are promising targets in the search for therapeutic agents. Information about Wnt pathways is available both in canonical terms and at the species level. In addition to the canonical Wnt/beta-catenin pathway, information is now available for Drosophila, Caenorhabditis elegans, and Xenopus. The STKE Connections Maps for these pathways provide an important tool in accessing this large body of complex information.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Moon, Randall T -- Bowerman, Bruce -- Boutros, Michael -- Perrimon, Norbert -- New York, N.Y. -- Science. 2002 May 31;296(5573):1644-6.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Howard Hughes Medical Institute, Department of Pharmacology, and Center for Developmental Biology, University of Washington School of Medicine, Seattle, WA 98195, USA. rtmoon@u.washington.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/12040179" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Cell Nucleus/physiology ; Cell Polarity ; Cytoskeletal Proteins/*metabolism ; Cytoskeleton/physiology ; *Drosophila Proteins ; Embryonic Development ; Embryonic and Fetal Development ; Gene Expression Regulation ; Humans ; Models, Biological ; Neoplasms ; Proto-Oncogene Proteins/*metabolism ; *Signal Transduction ; *Trans-Activators ; Transcription, Genetic ; Wnt Proteins ; Wnt1 Protein ; *Zebrafish Proteins ; beta Catenin
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 4
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    Requirement of prorenin receptor and vacuolar H+-ATPase-mediated acidification for Wnt signaling (2010)
    American Association for the Advancement of Science (AAAS)
    Details
    Publication Date: 2010-01-23
    Description: Wnt/beta-catenin signaling is important in stem cell biology, embryonic development, and disease, including cancer. However, the mechanism of Wnt signal transmission, notably how the receptors are activated, remains incompletely understood. We found that the prorenin receptor (PRR) is a component of the Wnt receptor complex. PRR functions in a renin-independent manner as an adaptor between Wnt receptors and the vacuolar H+-adenosine triphosphatase (V-ATPase) complex. Moreover, PRR and V-ATPase were required to mediate Wnt signaling during antero-posterior patterning of Xenopus early central nervous system development. The results reveal an unsuspected role for the prorenin receptor, V-ATPase activity, and acidification during Wnt/beta-catenin signaling.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Cruciat, Cristina-Maria -- Ohkawara, Bisei -- Acebron, Sergio P -- Karaulanov, Emil -- Reinhard, Carmen -- Ingelfinger, Dierk -- Boutros, Michael -- Niehrs, Christof -- New York, N.Y. -- Science. 2010 Jan 22;327(5964):459-63. doi: 10.1126/science.1179802.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Division of Molecular Embryology, DKFZ-ZMBH Alliance, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20093472" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Body Patterning ; Cell Line ; Cell Line, Tumor ; Central Nervous System/cytology/embryology ; Embryo, Nonmammalian/metabolism ; Frizzled Receptors/metabolism ; Gene Expression Regulation, Developmental ; Homeodomain Proteins/genetics/metabolism ; Humans ; Hydrogen-Ion Concentration ; LDL-Receptor Related Proteins/metabolism ; Low Density Lipoprotein Receptor-Related Protein-6 ; Mice ; Nerve Tissue Proteins/genetics/metabolism ; Phosphorylation ; RNA, Small Interfering ; Receptors, Cell Surface/genetics/*metabolism ; *Signal Transduction ; Vacuolar Proton-Translocating ATPases/antagonists & inhibitors/*metabolism ; Wnt Proteins/*metabolism ; Wnt3 Protein ; Xenopus/embryology/metabolism ; Xenopus Proteins/genetics/*metabolism ; beta Catenin/metabolism
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 5
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    Genome-wide RNAi analysis of growth and viability in Drosophila cells (2004)
    American Association for the Advancement of Science (AAAS)
    Details
    Publication Date: 2004-02-07
    Description: A crucial aim upon completion of whole genome sequences is the functional analysis of all predicted genes. We have applied a high-throughput RNA-interference (RNAi) screen of 19,470 double-stranded (ds) RNAs in cultured cells to characterize the function of nearly all (91%) predicted Drosophila genes in cell growth and viability. We found 438 dsRNAs that identified essential genes, among which 80% lacked mutant alleles. A quantitative assay of cell number was applied to identify genes of known and uncharacterized functions. In particular, we demonstrate a role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival. Such a systematic screen for cell phenotypes, such as cell viability, can thus be effective in characterizing functionally related genes on a genome-wide scale.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Boutros, Michael -- Kiger, Amy A -- Armknecht, Susan -- Kerr, Kim -- Hild, Marc -- Koch, Britta -- Haas, Stefan A -- Paro, Renato -- Perrimon, Norbert -- Heidelberg Fly Array Consortium -- R01 GM078176/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 2004 Feb 6;303(5659):832-5.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/14764878" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Apoptosis ; Cell Cycle ; Cell Survival ; Cells, Cultured ; Computational Biology ; Core Binding Factor Alpha 2 Subunit ; DNA-Binding Proteins/genetics ; Drosophila Proteins/genetics/metabolism/physiology ; Drosophila melanogaster/*genetics/*growth & development ; Genes, Essential ; *Genes, Insect ; *Genome ; Humans ; Inhibitor of Apoptosis Proteins ; Phenotype ; Proteome ; Proto-Oncogene Proteins/genetics ; *RNA Interference ; RNA, Double-Stranded/genetics ; Reproducibility of Results ; Sequence Homology ; Transcription Factors/genetics/metabolism
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
    Published by American Association for the Advancement of Science (AAAS)
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  • 6
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    Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge (2013)
    National Academy of Sciences
    Details
    Publication Date: 2013-06-07
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
    Published by National Academy of Sciences
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  • 7
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    Insect virokine suppress the IMD pathway [Immunology and Inflammation] (2016)
    National Academy of Sciences
    Details
    Publication Date: 2016-01-21
    Description: Viruses are obligatory intracellular parasites that suffer strong evolutionary pressure from the host immune system. Rapidly evolving viral genomes can adapt to this pressure by acquiring genes that counteract host defense mechanisms. For example, many vertebrate DNA viruses have hijacked cellular genes encoding cytokines or cytokine receptors to disrupt host...
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
    Published by National Academy of Sciences
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  • 8
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    caRpools: an R package for exploratory data analysis and documentation of pooled CRISPR/Cas9 screens (2016)
    Oxford University Press
    Details
    Publication Date: 2016-02-06
    Description: Motivation: Genetic screens by CRISPR/Cas9-mediated genome engineering have become a powerful tool for functional genomics. However, there is currently a lack of end-to-end software pipelines to analyze CRISPR/Cas9 screens based on next generation sequencing. Results: The CRISPR-AnalyzeR for pooled screens (caRpools) is an R package for exploratory data analysis that provides a complete workflow to analyze CRISPR/Cas9 screens. To further support the analysis of large-scale screens, caRpools integrates screening documentation and generation of standardized analysis reports. Availability and implementation: caRpools, manuals and an open virtual appliance are available at http://github.com/boutroslab/caRpools . Contact: m.boutros@dkfz.de
    Print ISSN: 1367-4803
    Electronic ISSN: 1460-2059
    Topics: Biology , Computer Science , Medicine
    Published by Oxford University Press
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  • 9
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    Sin3a regulates STAT activity [Cell Biology] (2012)
    National Academy of Sciences
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    Publication Date: 2012-07-25
    Description: Tyrosine phosphorylation is a hallmark for activation of STAT proteins, but their transcriptional activity also depends on other secondary modifications. Type I IFNs can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide RNAi screen, we identified the SIN3 transcription regulator homolog A (Sin3a) as an important mediator of this STAT3-targeted transcriptional repression. Sin3a directly interacts with STAT3 and promotes its deacetylation. SIN3A silencing results in a prolonged nuclear retention of activated STAT3 and enhances its recruitment to the SOCS3 promoter, concomitant with histone hyperacetylation and enhanced STAT3-dependent transcription. Conversely, Sin3a is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. The Sin3a complex therefore acts as a context-dependent ISGF3/STAT3 transcriptional switch.
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
    Published by National Academy of Sciences
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  • 10
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    Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines (2013)
    Oxford University Press
    Details
    Publication Date: 2013-11-21
    Description: As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.
    Keywords: Targeted inhibition of gene function
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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