The Carnegie Protein Trap Library: A Versatile Tool for Drosophila Developmental Studies

Howard Hughes Medical Institute Research Laboratories, Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21218, USA.
Genetics (Impact Factor: 5.96). 04/2007; 175(3):1505-31. DOI: 10.1534/genetics.106.065961
Source: PubMed


Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

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    • "Drosophila melanogaster wild-type (Oregon R + ) and mutant stocks were reared on standard cornmeal-agar food medium at 23+1 0 C. Three GFP tagged hnRNP protein-trap homozygous viable stocks (Morin et al., 2001; Buszczak et al., 2007) viz. "
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    ABSTRACT: The nucleus limited long-noncoding hsrω-n transcripts, hnRNPs, and some other RNA processing proteins organize nucleoplasmic omega speckles in Drosophila. Unlike other nuclear speckles, omega speckles rapidly disappear following cell stress, while hnRNPs and other associated proteins move away from chromosome sites, nucleoplasm, and the disappearing speckles to get uniquely sequestered at hsrω locus. Omega speckles reappear and hnRNPs get redistributed to normal locations during recovery from stress. With a view to understand the dynamics of omega speckles and their associated proteins, we used live imaging of GFP tagged hnRNPs (Hrb87F, Hrb98DE, or Squid) in unstressed and stressed Drosophila cells. Omega speckles display size dependent mobility in nucleoplasmic domains with significant colocalization with nuclear matrix Tpr/Megator and SAFB proteins, which also accumulate at hsrω gene site after stress. Instead of moving towards the nuclear periphery located hsrω locus following heat shock or colchicine treatment, omega speckles rapidly disappear within nucleoplasm while chromosomal and nucleoplasmic hnRNPs move, stochastically or, more likely, by nuclear matrix-mediated transport to hsrω locus in non-particulate form. Continuing transcription of hsrω during cell stress is essential for sequestering incoming hnRNPs at the site. While recovering from stress, the sequestered hnRNPs are released as omega speckles in ISWI-dependent manner. Photobleaching studies reveal hnRNPs to freely move between nucleoplasm, omega speckles, chromosome regions, and hsrω gene site although their residence periods at chromosomes and hsrω locus are longer. A model for regulation of exchange of hnRNPs between nuclear compartments by hsrω-n transcripts is presented.
    Chromosoma 08/2015; 124(3):367-383. DOI:10.1007/s00412-015-0506-0 · 4.60 Impact Factor
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    • "These tagged proteins are extremely useful as they permit determination of protein localization in vivo as well as conditional, tissue specific, temporal and reversible removal of the tagged proteins (Nagarkar-Jaiswal et al., 2015). However, previous methods for generating protein trap alleles in Drosophila have allowed only about 800 genes to be successfully tagged (Kelso et al., 2004; Buszczak et al., 2007; Quinones-Coello et al., 2007; Aleksic et al., 2009; Lowe et al., 2014). "
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    ABSTRACT: Previously we described a large collection of MiMICs that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC (Minos Mediated Integration Cassette) is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. Here we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.
    eLife Sciences 06/2015; 4. DOI:10.7554/eLife.08469 · 9.32 Impact Factor
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    • "Baz has been reported to localize at the hub-GSC interface along with E-cadherin following overexpression in the germline (nos > Baz-GFP) (Leatherman and Dinardo, 2010), which was confirmed by using independent UAS- Baz-YFP construct (see below). However, closer inspection using antibody staining and Flytrap Baz- GFP that expresses endogenous levels of Baz [CC01941 (Kelso et al., 2004; Buszczak et al., 2007)] revealed that Baz forms foci at the hub-GSC interface (referred to as the 'Baz patch' hereafter), instead of entirely colocalizing with E-cadherin (Figure 1B,C). The Baz patch is a small structure, with a size of approximately 1.5 μm, and this patch is considerably smaller than the GSC-hub interface that is marked by E-cadherin (4–6 μm) (Figure 1C). "
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    ABSTRACT: Many stem cells divide asymmetrically in order to balance self-renewal with differentiation. The essence of asymmetric cell division (ACD) is the polarization of cells and subsequent division, leading to unequal compartmentalization of cellular/extracellular components that confer distinct cell fates to daughter cells. Because precocious cell division before establishing cell polarity would lead to failure in ACD, these two processes must be tightly coupled; however, the underlying mechanism is poorly understood. In Drosophila male germline stem cells, ACD is prepared by stereotypical centrosome positioning. The centrosome orientation checkpoint (COC) further serves to ensure ACD by preventing mitosis upon centrosome misorientation. Here, we show that Bazooka (Baz) provides a platform for the correct centrosome orientation and that Baz-centrosome association is the key event that is monitored by the COC. Our work provides a foundation for understanding how the correct cell polarity may be recognized by the cell to ensure productive ACD.
    eLife Sciences 03/2015; 4(4). DOI:10.7554/eLife.04960 · 9.32 Impact Factor
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