recombineering-homologous-recombination-constructs-in-drosophila

Recombineering Homologous Recombination Constructs in <em>Drosophila</em>

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

The purpose of this study was to profile gene expression changes in colorectal tumors to identify new targets and strategies for the management of this disease.

Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNA interference.

Tumor necrosis factor alpha (TNF alpha) is a proinflammatory cytokine with important roles in regulating inflammatory responses as well as cell cycle proliferation and apoptosis. Although TNFalpha stimulates apoptosis, it also activates the transcription factor NF-kappa B, and studies have shown that inhibition of NF-kappa B potentiates the cytotoxicity of TNFalpha. Since several chemotherapy agents act like TNFalpha to both promote apoptosis and activate NF-kappa B, understanding the role of NF-kappa B in suppressing apoptosis may have significant clinical applications. To understand the effects of stimulation with TNFalpha and the role of NF-kappa B in regulating this response, a 23k human cDNA microarray was used to screen TNFalpha-inducible genes in HeLa cells. Real-time PCR verified expression changes in 16 of these genes and revealed three distinct temporal patterns of expression after TNFalpha stimulation. Using RNA interference to disrupt expression of the p65 subunit of NF-kappa B, all but two of the genes were shown to depend on this transcription factor for their expression, which correlated well with the existence of NF-kappa B binding sites in most of their promoters. Inflammatory, proapoptotic, and antiapoptotic genes were all shown to be regulated by NF-kappa B, demonstrating the wide variety of targets activated by NF-kappa B signaling and the necessity of differentiating among these genes for therapeutic purposes.

Identification of NF-kappa B-regulated genes induced by TNFalpha utilizing expression profiling and RNA interference.

The epidermal growth factor receptor (EGFR) gene is commonly amplified and rearranged in glioblastoma multiforme leading to overexpression of wild-type and mutant EGFRs. Expression of wild-type EGFR ligands, such as transforming growth factor-alpha (TGF-alpha) or heparin-binding EGF (HB-EGF), is also often increased in gliomas resulting in an autocrine loop that contributes to the growth autonomy of glioma cells. Glioblastoma multiformes express a characteristic EGFR mutant (EGFRvIII, de 2-7) that does not bind ligand, signals constitutively, and is more tumorigenic than the wild-type receptor. However, the downstream signals that mediate this increased tumorigenicity are not well understood. We hypothesized that signals induced specifically by EGFRvIII and not the wild-type receptor are more likely to mediate its increased tumorigenic activity and examined the gene expression profiles resulting from inducible expression of comparable levels of either wild-type EGFR or EGFRvIII in a U251-MG glioma cell line. Expression of EGFRvIII resulted in specific up-regulation of a small group of genes. Remarkably, all these genes, which include TGFA, HB-EGF, EPHA2, IL8, MAP4K4, FOSL1, EMP1, and DUSP6, influence signaling pathways known to play a key role in oncogenesis and function in interconnected networks. Increased expression of EGFRvIII-induced genes was validated by real-time PCR. The mutant receptor does not bind ligand, and EGFRvIII-induced expression of TGF-alpha and HB-EGF suggests that EGFRvIII plays a role in generating an autocrine loop using the wild-type EGFR in glioma. It also raises the possibility that EGFRvIII may signal, at least in part, through the wild-type receptor. Indeed, we show that inhibiting the activity of HB-EGF, a potent mitogen, with neutralizing antibodies reduces cell proliferation induced by expression of EGFRvIII. This suggests that the EGFRvIII-HB-EGF-wild-type EGFR autocrine loop plays an important role in signal transduction by EGFRvIII in glioma cells. We also show by immunohistochemistry that HB-EGF expression correlates with the presence of EGFRvIII in glioblastoma multiforme. Thus, our study provides a new insight into oncogenic signaling by EGFRvIII and improves our understanding of how autocrine loops are generated in glioma.

Differential gene expression analysis reveals generation of an autocrine loop by a mutant epidermal growth factor receptor in glioma cells.

The Notch pathway regulates the development of many tissues and cell types and is involved in a variety of human diseases, making it an attractive potential therapeutic target. This promise has been limited by the absence of potent inhibitors or agonists that are specific for individual human Notch receptors (NOTCH1-4). Using an unbiased functional screening, we identified monoclonal antibodies that specifically inhibit or induce activating proteolytic cleavages in NOTCH3. Remarkably, the most potent inhibitory and activating antibodies bind to overlapping epitopes within a juxtamembrane negative regulatory region that protects NOTCH3 from proteolysis and activation in its resting autoinhibited state. The inhibitory antibodies revert phenotypes conveyed on 293T cells by NOTCH3 signaling, such as increased cellular proliferation, survival, and motility, whereas the activating antibody mimics some of the effects of ligand-induced Notch activation. These findings provide insights into the mechanisms of Notch autoinhibition and activation and pave the way for the further development of specific antibody-based modulators of the Notch receptors, which are likely to be of utility in a wide range of experimental and therapeutic settings.

Modulation of Notch signaling by antibodies specific for the extracellular negative regulatory region of NOTCH3.

Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells, and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization, and function of Rab GTPases in an organism with a brain.

Systematic discovery of Rab GTPases with synaptic functions in Drosophila.

Neuronen erfordern hoch spezialisierte intrazellul&auml;ren Membran, Menschenhandel, vor allem an Synapsen. Rab GTPases gelten als Meister Regulierungsbeh&ouml;rden Membran Menschenhandel in allen Zellen, und nur sehr wenige Rabs Neuron-spezifische Funktionen bekannt haben. Hier pr&auml;sentieren wir die erste systematische Charakterisierung der neuronalen Ausdruck, subzellulare Lokalisation und Funktion der Rab GTPases im Organismus mit einem Gehirn.

Systematische Entdeckung der Rab GTPases mit synaptischen Funktionen in Drosophila.

Evaluating how an individual gene contributes to a particular biological process benefits greatly from a comprehensive understanding of all members of its gene family. Such knowledge is ideally obtained using multicellular model organisms, which provide rapid and decisive platforms for determining gene function. We recently established a novel transgenesis platform in Drosophila to systematically knock out all members of the Rab small GTPase family of membrane regulators. This platform combines BAC transgenesis/recombineering with ends-out homologous recombinations and Gateway(TM) technologies and provides a new rapid and scalable method that eases the manipulation of endogenous loci. This method not only allows for the generation of molecularly defined lesions, but also the precise replacement or tagging of genes in their endogenous loci. Using this method, we found that up to half of all Rab GTPases exhibit enriched expression at synapses in the nervous system. Here we provide critical details about the underlying recombineering and transgenesis method, new cassettes for tagging endogenous loci and information on important parameters that will allow Drosophila researchers to target members of other gene families.

Article	Year
Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNA interference. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 12631590	2003
Identification of NF-kappa B-regulated genes induced by TNFalpha utilizing expression profiling and RNA interference. Oncogene\| PubMed ID: 12673210	2003
Differential gene expression analysis reveals generation of an autocrine loop by a mutant epidermal growth factor receptor in glioma cells. Cancer research\| PubMed ID: 16424019	2006
Modulation of Notch signaling by antibodies specific for the extracellular negative regulatory region of NOTCH3. The Journal of biological chemistry\| PubMed ID: 18182388	2008
Systematic discovery of Rab GTPases with synaptic functions in Drosophila. Current biology : CB\| PubMed ID: 22000105	2011
Combining recombineering and ends-out homologous recombination to systematically characterize Drosophila gene families: Rab GTPases as a case study. Communicative & integrative biology\| PubMed ID: 22808327	2012

Shane Scoggin

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