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Description
A novel clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool for simultaneous gene editing and regulation was designed and tested. This study used the CRISPR-associated protein 9 (Cas9) endonuclease in complex with a 14-nucleotide (nt) guide RNA (gRNA) to repress a gene of interest using the Krüppel associated box (KRAB) domain, while also performing a separate gene modification using a 20-nt gRNA targeted to a reporter vector. DNA Ligase IV (LIGIV) was chosen as the target for gene repression, given its role in nonhomologous end joining, a common DNA repair process that competes with the more precise homology-directed repair (HDR).
To test for gene editing, a 20-nt gRNA was designed to target a disrupted enhanced green fluorescent protein (EGFP) gene present in a reporter vector. After the gRNA introduced a double-stranded break, cells attempted to repair the cut site via HDR using a DNA template within the reporter vector. In the event of successful gene editing, the EGFP sequence was restored to a functional state and green fluorescence was detectable by flow cytometry. To achieve gene repression, a 14-nt gRNA was designed to target LIGIV. The gRNA included a com protein recruitment domain, which recruited a Com-KRAB fusion protein to facilitate gene repression via chromatin modification of LIGIV. Quantitative polymerase chain reaction was used to quantify repression.
This study expanded upon earlier advancements, offering a novel and versatile approach to genetic modification and transcriptional regulation using CRISPR/Cas9. The overall results show that both gene editing and repression were occurring, thereby providing support for a novel CRISPR/Cas system capable of simultaneous gene modification and regulation. Such a system may enhance the genome engineering capabilities of researchers, benefit disease research, and improve the precision with which gene editing is performed.
To test for gene editing, a 20-nt gRNA was designed to target a disrupted enhanced green fluorescent protein (EGFP) gene present in a reporter vector. After the gRNA introduced a double-stranded break, cells attempted to repair the cut site via HDR using a DNA template within the reporter vector. In the event of successful gene editing, the EGFP sequence was restored to a functional state and green fluorescence was detectable by flow cytometry. To achieve gene repression, a 14-nt gRNA was designed to target LIGIV. The gRNA included a com protein recruitment domain, which recruited a Com-KRAB fusion protein to facilitate gene repression via chromatin modification of LIGIV. Quantitative polymerase chain reaction was used to quantify repression.
This study expanded upon earlier advancements, offering a novel and versatile approach to genetic modification and transcriptional regulation using CRISPR/Cas9. The overall results show that both gene editing and repression were occurring, thereby providing support for a novel CRISPR/Cas system capable of simultaneous gene modification and regulation. Such a system may enhance the genome engineering capabilities of researchers, benefit disease research, and improve the precision with which gene editing is performed.
ContributorsChapman, Jennifer E (Author) / Kiani, Samira (Thesis advisor) / Ugarova, Tatiana (Thesis advisor) / Marchant, Gary (Committee member) / Arizona State University (Publisher)
Created2018
Description
Platelets are specialized blood cells that play crucial role in normal physiologic and pathologic processes such as hemostasis, inflammation, wound healing, and host defense. Activation of platelets is essential for platelet function and it includes a complex interplay of adhesion and intracellular signaling molecules. Platelets are known to be activated during vessel injury by a complex interaction of soluble agonists and once activated, they adhere to sub-endothelial matrix to aggregate and secrete granules leading to the formation of platelet aggregate that is necessary for thrombus formation. Platelet integrin plays a central role in platelet adhesive reactions by serving as a receptor for fibrinogen involved in bidirectional transmembrane signaling. In order to elucidate the interaction of integrin with cytoplasmic signaling molecules during inside-out and outside-in signaling, we have studied the kinetics of the recruitment of talin, kindling, filmin-A, skelemin, Scr and syk to the B3 cytoplasmic tails. Platelets were isolated from human blood and activated with ADP/Epinephrine for different times. The complexes of *** with signaling proteins were obtained by immunoprecipitation of platelet lysates with anit-*** monoclonal antibody and then analyzed by Western blotting using antibodies directed against selected signaling proteins. Our results show different kinetics in recruitment of signaling molecules to the B3 integrin cytoplasmic tail during inside-out and outside in signaling.
ContributorsYantas, Alexa Susan (Author) / Ugarova, Tatiana (Thesis director) / Podolnikova, Nataly (Committee member) / Turaga, Ramya (Committee member) / Barrett, The Honors College (Contributor)
Created2012-05
Description
Macrophage fusion resulting multinucleated giant cells (MGCs) formation is associated with numerous chronic inflammatory diseases including the foreign body reaction to implanted
biomaterials. Despite long-standing predictions, there have been attempts to use live-cell
imaging to investigate the morphological features initiating macrophage fusion because
macrophages do not fuse on clean glass required for most imaging techniques. Consequently,
the mechanisms of macrophage fusion remain poorly understood. The goal of this research
project was to characterize the early and late stages of macrophage multinucleation using
fusogenic optical quality substrate. Live-cell imaging with phase-contrast and lattice-light
sheet microscopy revealed that an actin-based protrusion initiates macrophage fusion. WASpdeficient macrophages and macrophages isolated from myeloid cell-specific Cdc42-/- mice
fused at very low rates. In addition, inhibiting the Arp2/3 complex impaired both the formation
of podosomes and macrophage fusion.
Analyses of the late stages of macrophage multinucleation on biomaterials implanted into
mice revealed novel actin-based zipper-like structures (ZLSs) formed at contact sites between
MGCs. The model system that was developed for the induction of ZLSs in vitro allowed for
the characterization of protein composition using confocal and super-resolution microscopy.
Live-cell imaging demonstrated that ZLSs are dynamic formations undergoing continuous
assembly and disassembly and that podosomes are precursors of these structures. It was further
found that E-cadherin and nectin-2 are involved in ZLS formation by bridging the plasma
membranes together. ii
Macrophage fusion on implanted biomaterials inherently involves their adhesion to the
implant surface. While biomaterials rapidly acquire a layer of host proteins, a biological
substrate that is required for macrophage fusion is unknown. It was shown that mice with
fibrinogen deficiency as well as mice expressing fibrinogen incapable of fibrin polymerization
displayed a dramatic reduction of macrophage fusion on biomaterials. Furthermore, these mice
were protected from the formation of the dense collagenous capsule enveloping the implant. It
was also found that the main cell type responsible for the deposition of collagen in the capsule
were mononuclear macrophages but not myofibroblasts. Together, these findings reveal a
critical role of the actin cytoskeleton in macrophage fusion and identify potential targets to
reduce the drawbacks of macrophage fusion on implanted biomaterials.
ContributorsBalabiyev, Arnat (Author) / Ugarova, Tatiana (Thesis advisor) / Roberson, Robert (Committee member) / Chandler, Douglas (Committee member) / Baluch, Page (Committee member) / Arizona State University (Publisher)
Created2021
Description
Adsorption of fibrinogen on various surfaces, including biomaterials, dramatically reduces the adhesion of platelets and leukocytes. The mechanism by which fibrinogen renders surfaces non-adhesive is its surface-induced self-assembly leading to the formation of a nanoscale multilayer matrix. Under the applied tensile force exerted by cellular integrins, the fibrinogen matrix extends as a result of the separation of layers which prevents the transduction of strong mechanical forces, resulting in weak intracellular signaling and feeble cell adhesion. Furthermore, upon detachment of adherent cells, a weak association between fibrinogen molecules in the superficial layers of the matrix allows integrins to pull fibrinogen molecules out of the matrix. Whether the latter mechanism contributes to the anti-adhesive mechanism under the flow is unclear. In the present study, using several experimental flow systems, it has been demonstrated that various blood cells as well as model HEK293 cells expressing the fibrinogen receptors, were able to remove fibrinogen molecules from the matrix in a time- and cell concentration-dependent manner. In contrast, insignificant fibrinogen dissociation occurred in a cell-free buffer, and crosslinking fibrinogen matrix significantly reduced cell-mediated dissociation of adsorbed fibrinogen. Surprisingly, cellular integrins contributed minimally to fibrinogen dissociation since function-blocking anti-integrin antibodies did not significantly inhibit this process. In addition, erythrocytes that are not known to express functional fibrinogen receptors and naked liposomes caused fibrinogen dissociation, suggesting that the removal of fibrinogen from the matrix may be caused by nonspecific low-affinity interactions of cells with the fibrinogen matrix. These results indicate that the peeling effect exerted by flowing cells upon their contact with the fibrinogen matrix is involved in the anti-adhesive mechanism.
ContributorsMursalimov, Aibek (Author) / Ugarova, Tatiana (Thesis advisor) / Chandler, Douglas (Committee member) / Podolnikova, Nataly (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2022