Browsing by Author "Wagner, Daniel S"

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    A Rice CRISPy Treat: Improving CRISPR-Cas9 gene editing in the zebrafish to facilitate analysis of genes implicated in neural angiogenesis in an F0 screen
    (2018-04-16) Clements, Thomas P; Wagner, Daniel S
    Scientists are eager for novel mutants to study gene function, evolutionary relationships, and even perform drug screens. Zebrafish are a well-established model for scientific research (Kimmel et al., 1995) and have been a premiere model for both forward and reverse genetic research. However, each current method to produce novel gene knockouts (KO) is not without their drawbacks. In order to address this need, I have developed a novel Cas9 fusion (ExoCas9) to enhance the efficiency of CRISPR-Cas based gene knockouts in the zebrafish. This fusion increases the efficiency of gene KOs as well as the average size of deletions produced. I used this fusion to screen for F0 neural angiogenic specific defects (brain hemorrhages) in genes implicated in TGF-β pathway response. I tested 70 single guide (sgRNAs) representing 26 individual genes using ExoCas9 and confirmed most targeted genes produced an observable brain hemorrhage phenotype greater than 10% for at least one sgRNA. I also looked for malformations in zebrafish vasculature hallmarks in the Casper KDR transgenic line, which has GFP-labeled vasculature, on a subset of genes from the ExoCas9 F0 screen. I prioritized additional experiments in genes also implicated in WNT neural angiogenesis (Hupe et al., 2017). I hypothesize that TGF-β and Wnt- pathways coordinately regulate a set of genes essential for brain angiogenesis and blood brain barrier (BBB) formation.
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    A two-pronged approach towards the development of novel therapeutics for advanced endometrial cancer
    (2015-11-24) Engel, Brian Joseph; Carson, Daniel D; Jacot, Jeffrey G; McCrea, Pierre D; Stern, Michael; Wagner, Daniel S
    Endometrial cancer is the fourth most common cancer among women. The standard of care involves hysterectomy with adjuvant radiation and chemotherapy for advanced disease. Despite these efforts, treatment of advanced and metastatic disease is not very effective. This body of work describes a two-pronged approach to address lack of treatments for advanced endometrial cancer. The first approach was an in-depth study of the mechanism and physiological effect of mucin 1 (MUC1)-driven epidermal growth factor receptor (EGFR) expression and signaling. MUC1 is a large, heavily glycosylated transmembrane protein that functions to lubricate surfaces, provides protection from external insult and plays an important role in embryo implantation. EGFR is a receptor tyrosine kinase that influences cellular proliferation, migration and apoptosis. MUC1 increases EGFR gene expression, mRNA levels, protein levels and signaling in endometrial cancer cell lines. Consequently, MUC1 expression is associated with increased EGF-dependent cellular proliferation, survival and resistance to EGFR inhibitors. In addition, MUC1 and EGFR co-expression is associated with increased cellular proliferation in endometrial tumors. The second approach involved the development and characterization of an advanced three dimensional (3D) hyaluronic acid (HA)-based culture model that is compatible with existing high throughput drug screening methodologies. This system incorporates three layers: an acellular cushion layer; an encapsulated cancer cell layer for growth in 3D; and a collagen-containing layer that supports the growth of stromal cells on top of the hydrogel (2.5D). The robustness of this system was evaluated by incorporating endometrial or prostate cancer cells with associated stromal cells. Both culture systems provided high cancer and stromal cell viability and facilitated paracrine interactions. The response to cytotoxic drugs from cells cultured in 3D HA better matched clinical data than cells grown in 2D and 3D-alginate. These studies provide mechanistic evidence for regulation that occurs in advanced endometrial cancer, as well as an improved platform to screen for effective therapeutics. The 3D culture system could be leveraged to evaluate novel therapeutics for the treatment of advanced endometrial cancer which may include MUC1 and EGF-directed therapies.
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    Magnetic nanomaterials for applications in magnetic resonance imaging and cancer stem cell biology
    (2014-11-04) Jebb, Meghan Helen; Wilson, Lon J.; Lewis, Michael T; Martí, Angel A; Wagner, Daniel S
    Magnetic nanomaterials are uniquely suited for applications in biology and medicine. With size compatibility, tunable physical properties, and the capacity for external magnetic control, nanoscale magnetic particles have been exploited for drug delivery, hyperthermia-mediated cancer therapy, cell separation and isolation, and magnetic resonance imaging (MRI). This work explores several different nano-based materials for MRI and cancer cell isolation applications. First, Gd3+-loaded carbon nanotube capsules, or gadonanotubes (GNTs), have been analyzed by X-ray absorption spectroscopy to account for the structural contributions to their high MRI contrast enhancement properties. This work revealed the existence of small [Gd-O9] sites in the GNTs with short Gd-O (and thus Gd-H) bond lengths, which contribute to their high performance. Secondly, two new nanomaterials were developed, by loading paramagnetic Mn2+ ions into or onto ultra-short single-walled carbon nanotubes (US-tubes) or GNTs (manganonanotubes and manganogadonanotubes, MNTs and MGTs, respectively). With relaxivity (r1) values of 65 (MNT), 74 (MGT), and 110 (GNT) mM-1s-1 per ion and approximately 13-fold contrast enhancements in all cases over the free ions, US-tubes have been further confirmed as a universal platform for the enhancement of MRI contrast agent properties with the GNTs being the best r1 agents developed to date. Finally, a method for the isolation of quiescent breast cancer cells has been developed, using iron oxide nanoparticles (IONs) as intracellular labels. Once isolated, functional assays were employed to characterize the drug resistance and stem-like nature of the quiescent subpopulation. The project has thus demonstrated how a magnetic nanomaterial-facilitated procedure can be exploited to probe fundamental questions in cancer stem cell biology.
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    Multiplexed Spatial Analyses in situ and in living cells
    (2015-12-04) Zimak, Jan; Diehl, Michael R; Qutub, Amina A; Wagner, Daniel S
    High-content spatial analyses are critical to understanding the structural organization and dynamics of many complex biological processes. Increasing the number of cellular components that can be visualized will help delineate the functions of many interacting and competing cellular pathways. However, the physical limitations of spectral bandwidth and the experimental difficulty of genomic manipulation have hampered traditional approaches to multiplex molecular analyses in both fixed samples and live cells. The programmable and predictable nature of the DNA molecule makes it a tantalizing candidate for an engineering tool to help alleviate some of these limitations. This thesis seeks to harness both the chemical and biological utility of DNA as a building block to multiplex the color and control the number and location of fluorescent reporters in biological samples. First in the context of in situ immunofluorescence imaging of fixed cells or tissues, And second in the context of live-cell imaging of genomically engineered cells. In the first case, by utilizing the stand displacement chemical reaction between dynamic DNA complexes and DNA-conjugated antibodies we selectively couple fluorophores to, and then remove them, from their protein targets. We leverage this mechanism to facilitate multiple sequential round of fluorescence microscopy where the same color dye molecules are used reiteratively to visualize different antibody-tagged markers. By optimizing the DNA-antibody conjugation chemistry and incubation protocol we now routinely perform 9 marker analyses of paraffin-fixed tissue sections with these DNA probes. Then automating the sequence design process enabled more complex probe designs to be use for balancing marker levels appropriately for hyperspectral imaging experiments. Here, discrete and reconfigurable control over amplification gains, greatly improved the spectral un-mixing of different antibody signals. Secondly, we focus on dissecting network-level functions of cytoskeletal regulatory proteins during epithelial cell polarization and morphogenesis. DNA-based STORM microscopy revealed that a scaffold protein, IQGAP1, associates with specialized actin filaments within cell-cell junctions and with basket-like structures in the basal actin cortex of normal epithelial cells. This work uses IQGAP1 as a platform, as it lies at the nexus of cell signaling and cytoskeletal regulatory networks. We construct multi-gene systems that simultaneously sense and control intracellular expression levels of IQGAP1 and track the actin cytoskeleton. By combining novel molecular biology techniques to manipulate the DNA in live cells. We use a barcoded self-assembly technique to construct large vectors that contain several transcriptional elements. These multi-gene systems are then stably incorporated into cells engineered with genomic ‘landing pads’ using locus-specific integration. Finally, we demonstrate functional circuits by linearly controlling intracellular IQGAP1 levels. These results will support future single-cell and multiplexed population-level analyses of IQGAP1 functions in epithelial cells, allow us to study IQGAP1 recruitment to epithelial cell-cell junctions and to examine how it influences cellular transitions.