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  • Genetics Society of America (GSA)  (1)
  • Oxford University Press  (1)
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  • 1
    Publication Date: 2016-08-10
    Description: Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila . miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a , and the previously uncharacterized gene MESR4 , in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola) gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.
    Electronic ISSN: 2160-1836
    Topics: Biology
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  • 2
    Publication Date: 2013-10-19
    Description: Motivation:  Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) is widely used to study the in vivo binding sites of transcription factors (TFs) and their regulatory targets. Recent improvements to ChIP-seq, such as increased resolution, promise deeper insights into transcriptional regulation, yet require novel computational tools to fully leverage their advantages. Results:  To this aim, we have developed peakzilla, which can identify closely spaced TF binding sites at high resolution (i.e. resolves individual binding sites even if spaced closely), as we demonstrate using semisynthetic datasets, performing ChIP-seq for the TF Twist in Drosophila embryos with different experimental fragment sizes, and analyzing ChIP-exo datasets. We show that the increased resolution reached by peakzilla is highly relevant, as closely spaced Twist binding sites are strongly enriched in transcriptional enhancers, suggesting a signature to discriminate functional from abundant non-functional or neutral TF binding. Peakzilla is easy to use, as it estimates all the necessary parameters from the data and is freely available. Availability and implementation:  The peakzilla program is available from https://github.com/steinmann/peakzilla or http://www.starklab.org/data/peakzilla/ . Contact:   stark@starklab.org Supplementary information: Supplementary data are available at Bioinformatics online.
    Print ISSN: 1367-4803
    Electronic ISSN: 1460-2059
    Topics: Biology , Computer Science , Medicine
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