Embryos derived by somatic cell nuclear transfer (SCNT) produce few pregnancies that result in a live, healthy offspring. This has largely been attributed to the aberrant reprogramming of the somatic cell DNA used for cloning. In order to improve the efficiency of cloning there is a great deal of research needed to determine the role of proteins involved in early embryonic reprogramming. In addition, studies are needed to determine effects on somatic and embryonic cell development as a result of altering these proteins. In this study we investigate the use of RNA interference in bovine somatic cells and embryos to knock down the expression of DNA methyltransferase-1 (DNMT1), an enzyme responsible for maintenance methylation in mammalian cells. We designed our experiments to test whether or not knocking down the DNMT1 gene would lead to a decrease in global methylation. It is our hypothesis that using somatic cells with reduced methylation may be advantageous for increasing the efficiency of cloning via somatic cell nuclear transfer. To accomplish this task, we have designed an infectious non-replicating lentiviral vector capable of delivering a gene that produces a short hairpin RNA targeting the mRNA of DNMT1. The construct included a sequence coding for green fluorescent protein (GFP) that will allow us to identify cells expressing the hairpin as well as a region coding for neomycin resistance so we could select for a pure population of transgenic cells to use for analysis. Infecting bovine fetal fibroblast cells with genes encoding shRNAs that target DNMT1 was successful. Quantitative real time PCR analysis of DNMT1 mRNA suggests that our shRNAs are capable of an 80% knockdown. The protein blot of indicates up to 90% knockdown of DNMT1. Cells transduced twice with a high titer virus showed the highest knockdown of both DNMT1 mRNA and the protein. Analysis of immunolabeled cytosine methylaiton showed a global decrease in DNA methylation as a result of the DNMT1 knockdown. However, double transduced cells with a high knockdown percentage of DNMT1 mRNA and protein became hypermethylated. The second experiment was conducted to determine the effect of injecting small interfering RNAs (siRNAs) targeting DNMT1 into oocytes prior to parthenogenic activation. This experiment was designed to give us information on the survivability and epigenetic profile of early embryos with decreased DNMT1. Oocytes injected with siRNA targeting DNMT1 had little development past the 8-cell stage as compared to the sham injected oocytes. This treatment group also had decreased DNA methylation as determined by immunolabeling of methylated cytosine residues.

High mobility group box 1 (HMGB-1) protein is a non-histone chromosomal protein. As a DNA binding protein, HMGB-1 is involved in the maintenance of nucleosome structure, regulation of gene transcription and it is active in DNA recombination and repair. It has been known that HMGB-1 is a late mediator of endotoxemia and sepsis. HMGB-1 is released from activated macrophages, induces the release of other proinflammatory mediators, and mediates cell death when overexpressed. We speculated that the course of sepsis maybe different without the involvement of HMGB-1. The aims of this study are to investigate the role of HMGB-1 in mediating sepsis and to observe the effects by using RNAi to affect the production of HMGB-1. Lipopolysaccharide (LPS) was used to simulate sepsis in culture as well as stimulate the release of HMGB-1 from RAW 264.7 cells. Levels of HMGB-1 in the culture medium were subsequently measured by Western blot. Other proinflammatory cytokines (TNF-£\, IL-6 and TGF-£]) were measured by ELISA. HMGB-1 could not be detected in the culture medium in the absence of LPS stimuli, but after 0.5 £gg/ml LPS treatment HMGB-1 release could be detected. HMGB-1 the amount of released from LPS activated RAW 264.7 cells was in a time- and dose-dependent manner. The present study demonstrated that RNAi in the treatment of LPS-stimulated RAW264.7 cells resulted in the blockade of HMGB-1 and decreased LPS-induced inflammatory response. The results demonstrated that HMGB-1 plays a pivotal role in macrophage inflammatory responses by modulating the production of inflammatory mediators.

Heat shock factor 1 (HSF1) is the master transcriptional regulator of the heat shock response (HSR), an evolutionarily conserved cellular stress response. HSF1 promotes the expression of a variety of molecular chaperones that aid in restoring protein homeostasis upon exposure to proteoxic stress. However, all of the proteins responsible for regulating the HSR together with HSF1 are unknown. A genome-wide yeast two hybrid screen was performed to identify new S. cerevisiae Hsf1 protein interacting partners. Two GAL4 DNA binding domain-Hsf1 fusion proteins (baits) were constructed with mutations in the Hsf1 C-terminal activation domain to dampen Hsf1 mediated auto-activation of the reporter gene. Each haploid bait strain was mated with a haploid prey strain containing one of ~6,000 S. cerevisiae open reading frames fused to the GAL4 activation domain (prey). Interaction between the bait and prey reconstituted the GAL4 protein enabling it to bind to a GAL4 DNA binding site and activate the HIS3 reporter gene. The identified proteins from 4 screens were pooled generating 240 putative Hsf1 interacting partners. This list was narrowed to 38 candidates by selecting the 15 strongest interactions identified based on colony size and 33 candidates conserved in C. elegans. Hsf1 interactions with the 14 candidates in which protein expression was confirmed were then re-tested by a manual yeast two-hybrid assay. Hsf1 interactions with Sti1, Rim2 and Prp46 were repeatable in this manual assay. A study of the impact of knockdown of each of their C. elegans homolog on the HSR was performed using RNAi in an hsp70-promoter::GFP reporter strain of C. elegans. Preliminary results suggest that knockdown of Sti1 may impact the HSR in the worm. Further study of Sti1 and other potential Hsf1 interacting partners identified in this screen is warranted.

RNA interference (RNAi) potently and specifically induces gene knockdown, and its potential for reverse genetics in Caenorhabditis elegans is enormous. However, even in these nematodes, RNAi can be induced more effectively via enhanced RNAi (Eri) mutant backgrounds. With advances in small RNA sequencing, evidence has suggested that the eri pathway plays an endogenous gene regulatory role, which competes with experimentally introduced RNAi triggers for limiting resources. However, the nature, cellular location, and physiological consequences of this small RNA pathways competition remain unclear. To answer these questions, I first fully characterized the genetic phenotypes of all known Eri mutants. I discovered that different components of the eri pathway have subtle differences upon mutation, which affects more than exogenous RNAi. I then attempted to screen for novel enhanced RNAi mutants, guided by hypothetical mechanisms or tissues of expression not associated with known mutants. After these attempts, I fully characterized the genetic mechanisms that account for enhanced RNAi. Surprisingly, I discovered that the nuclear Argonaute nrde-3 and the peri-nuclear P-granule component pgl-1 are necessary and sufficient for an Eri response. Finally, I examined the impact of the competition among microRNA, endogenous siRNA, and exogenous RNAi pathways. I discovered that C. elegans develops slower upon perturbations to its normal flux of small RNA pathways. Insights from these phenotypes and genetic mechanisms shed light on the importance of small RNA biology and offer a novel suite of tools for sensitizing RNAi in broader contexts, especially given the deep evolutionary conservation of most eri-associated genes.

Acute myeloid leukemia (AML) is a clonal neoplastic disorder organized as a cellular hierarchy, with the self-renewing leukemia stem cell (LSC) at the apex. Recurrent mutations in transcription factors (TF) and epigenetic regulators suggest that AML is driven by aberrant transcriptional circuits, but these circuits have not been fully defined in an LSC model. To study transcriptional mechanisms relevant to leukemogenesis in vivo, we generated a murine serial transplantation model of MLL-AF9-driven, myelomonocytic leukemia with genetically- and phenotypically-defined LSCs. Using this model, we pursued two related lines of investigation. First, we performed an in vivo RNA interference (RNAi) screen to identify transcription factors required for LSC function. This screen highlighted the circadian rhythm TFs, Clock and Bmal1, as genes essential for the survival of murine leukemia cells, and we validated this finding with CRISPR/Cas-based genome editing and knockdown studies in AML cell lines. Utilizing luciferase reporter mice to track expression of the circadian target gene Per2, we demonstrated that both leukemic and normal hematopoietic cells have the capacity for oscillating, circadian-dependent gene expression. Importantly, using murine knockout models, we found that normal hematopoietic stem and progenitor cells (HSPC), in contrast to leukemia cells, do not depend on Bmal1. We further demonstrated that selective depletion of LSCs following circadian perturbation is mediated through enhanced myeloid differentiation. ChIP-Seq studies revealed that the circadian rhythm network is integrally connected to the LSC self-renewal circuitry and highlighted putative Clock/Bmal1 targets in leukemia, providing a mechanistic basis for our findings. Second, we performed a functional and genomic characterization of our MLL-AF9 serial transplantation model to explore mechanisms of disease evolution and clonal selection in AML. Limiting dilution studies demonstrated that serial transplantation results in a reduction in disease latency, dramatic enrichment of leukemia-initiating cells (LIC), and reconfiguration of the LSC hierarchy. While mutations in known AML-associated genes were not linked to disease progression, RNA-sequencing (RNA-Seq) demonstrated that the increase in LIC frequency in serially transplanted leukemias is driven by changes in cell cycle and differentiation. In aggregate, these studies offer insights into the biological mechanisms regulating LSC self-renewal and disease evolution in AML.

Melanoma is the most deadly form of skin cancers, with an incidence increasing more rapidly than any other malignant cancer in the past 40 years. Metastatic melanoma is resistant to conventional treatments, such as chemotherapy and radiation therapy. Our lab has previously demonstrated that Mcl-1 is a key contributor in protecting melanoma from therapy-induced cell death. RNAi therapeutics was employed as a novel way to silence the anti-apoptotic protein by using Mcl-1 mRNA sequence-specific siRNAs in vitro. In our hands, passive non-targeted delivery of RNAi therapy into melanoma tumours has been shown to be neither effective, nor selective in vitro and in vivo. Consequently, in this study, siRNA was linked to a delivery system which expressed a ligand specifically targeting melanoma cells. Previously shown, melanoma overexpresses the cell surface ganglioside GD3, thus it is my belief that the anti-GD3 R24 monoclonal antibody can function as a targeting molecule. The antibody was linked to coated cationic liposomes (CCLs) carrying siRNA molecules. Our first step was to confirm R24 ligation to CCLs. Untargeted CCLs showed insignificant values of antibody, whereas antibody-conjugated CCLs presented approximately 30 antibodies per liposome. I also confirmed that siRNA was internalized within CCLs using spectrometry, with an encapsulation efficiency of approximately 80%. Since liposomes need to be small to be effective in vitro and in vivo, CCLs were confirmed to be less than 100nm in diameter. In vitro studies using fluorescent microscopy demonstrated greater binding to melanoma cells with antibody-conjugated CCLs as compared to untargeted CCLs. In vivo experiments showed specific localization of targeted CCLs to induced subcutaneous mouse xenograft tumours. Western blotting demonstrated greater Mcl-1 knockdown using GD3-targeted CCLs. Taken together, these results suggest that anti-GD3 antibodies can serve as targeting molecules to deliver siRNA to melanoma cells and furthermore, GD3-targeted CCLs can promote siRNA-mediated gene silencing. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2010-09-02 10:29:37.944

No description available.

A major contributor to mortality in patients with plasma cell dyscrasias (PCDs); i.e., multiple myeloma, light chain deposition disease and AL amyloidosis is the deposition as insoluble aggregates of monoclonal immunoglobulin light chain proteins (LC) in the kidneys and other organs. Currently anti-plasma cell chemotherapies are used to reduce LC synthesis, and slow deposition. While effective, these treatments are toxic, non-specific, expensive, and might not be appropriate in all cases, making the identification of an alternate means of reducing toxic LC species desirable. To this end, we have investigated whether RNA interference (RNAi) could achieve these goals. Human (RPMI 8226, Bur) and transfected mouse myeloma (SP2/O-lambda 6) cells which produce measureable quantities of human LC protein were used as model systems for testing the efficacy of both synthetic small interfering RNAs (siRNAs) and short hairpin RNA (shRNA) expression vectors in reducing LC synthesis. Sequencing of LC genes provided the basis for design of siRNA duplexes targeting either the variable (V) or joining (J) regions of individual LCs, or the constant (C) region of either kappa or lambda LC isotypes. Myeloma lines were transfected with siRNAs using lipid-based transfection media. Cells receiving non-silencing siRNAs served as controls. Exposure of myeloma lines to siRNAs was well tolerated and no cytotoxicity was observed. LC mRNA expression was shown to be reduced ≥40% in 8226 and SP2/O- lambda 6 cell lines receiving siRNA treatment as compared with untreated controls. Exposure to siRNAs was also effective in significantly reducing both intracellular and secreted LC protein levels in cell lines tested as evidenced by flow cytometry or enzyme-linked immunosorbent assays (ELISAs). Effective siRNA nucleotide sequences were used to generate shRNA cassettes which were ligated into lentiviral expression vectors under the control of the RNA polymerase III promoter, U6. These expression systems were used to generate replication incompetent lentiviral particles. Exposure of 8226 to lentiviral particles resulted in significant knockdown of LC mRNA and protein both in vitro and in xenograft tumor bearing immune compromised mice. These results provide positive evidence for the ability of RNAi based approaches to reduce LC secretion in models of PCD.

Die Vorliegende Arbeit beschäftige sich mit der Etablierung eines lentiviralen Vektorsystems, mit dem es möglich ist die RNA-Interferenz experimentell zu nutzen. Dafür wurden SEC Sequenzen in den Vektor pGJ3-eGFP kloniert. Nach Optimierung der Transfektions und Transduktionsschritte wurden im Anschluss rekombinante virale Partikel hergestellt. Zur Überprüfung der Effektivität der Induzierten RNA-Interferenz erfolgte die Transduktion einer persistierend mit Masern infizierten Zelllinie (C6SSPE). Ziel der siRNA Sequenzen war dabei die mRNA des N-Proteins, welches eine zentrale Rolle im viralen Replikationszyklus spielt. Die Reduktion der mRNA wurde über quantitative real time RT-PCR nachgewiesen.

Abstract Ascoviruses (AVs) are members of the family Ascoviridae that are transmitted by female endoparasitic wasps and cause lethal infection in lepidopteran insects. AVs possess large double stranded DNA genomes ranging from 116-186 kbp. Recently, genomes of four AV species have been completely sequenced and have revealed important genes potentially needed for virus DNA replication and infection. Phylogenetic analyses of several of these genes indicate that AVs are closely related to iridoviruses and likely evolved from them. Two unique features, mode of transmission and cytopathology which involves cleavage of cells into virus-containing vesicles, make AVs different from other insect pathogenic viruses. During this decade, tremendous advancements in the study of RNA silencing mechanisms have openned a new dimension in virology. It is now evident that viruses reshape the cellular environment by reprogramming host RNA silencing machinery. The process of RNA silencing involves small non-coding RNAs, which with the help of nuclease-containing regulatory proteins bind to complementary messenger RNA (mRNA) targets, resulting in inhibition of gene expression. This sophisticated style of gene regulation has attained a fundamental status in living organisms, since RNA silencing has been revealed to be ubiquitous from viruses to prokaryotes to eukaryotes. Two main categories of small RNAs, short interfering RNA (siRNA) and microRNA (miRNA), have been defined as major players in RNA silencing. Interestingly, viral genomes like that of their hosts, encode miRNAs that can be used during virus invasion to manipulate host genes as well as miRNA biogenesis. Here, we report on the identification of the first insect virus miRNA (HvAVmiR- 1) derived from the major capsid protein (MCP) gene of Heliothis virescens ascovirus 7 (HvAV3e). HvAV-miR-1 expression was found to be strictly regulated and specifically detected from 96 h post-infection. HvAV-miR-1 expression coincides with a marked reduction of the expression of HvAV3e DNA polymerase I, which is a predicted target. Ectopic expression of the full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs degradation of DNA polymerase I transcripts and regulates replication of HvAV3e. Further, we investigated changes in the expression levels of host miRNAs upon HvAV3e infection in an insect cell line derived from Helicoverpa zea fat body and investigated the role of a host miRNA, Hz-miR24, in the hostvirus system. It was found that Hz-miR24 is differentially expressed following virus infection, with an increase in its expression levels late in infection. Functional analyses demonstrated that Hz-miR24 targets viral DNA-dependent RNA polymerase and its β subunit mRNAs. This was confirmed using ectopic expression of Hz-miR24 and a green fluorescent protein-based reporter system. Expression of the target gene was substantially enhanced in cells transfected with a synthesized inhibitor of Hz-miR24. These findings suggest that ascoviruses encode their own miRNA(s) and concurrently manipulate host miRNAs that in turn regulate the expression of their genes at specific time points after infection. In connection to RNA silencing, we characterized a ribonuclease III (RNase III) protein encoded by HvAV3e. We found that RNase III protein was functional in vivo as well as in vitro and catalyzed long and short double stranded RNAs. Expression analyses during virus infection revealed autoregulation of this protein by degradation of its RNA transcripts. Moreover, RNase III protein was found to be involved in suppression of RNA silencing and essential for virus DNA replication and infection. Finally, we studied another ascoviral 8 protein, a putative inhibitor of apoptosis (IAP), which was found to be essential for virus DNA replication and pathology. Further, despite inhibition of apoptosis by HvAV3e, the IAP-like protein was found dispensable for the inhibition of replication. In conclusion, for successful invasion and attenuation of host antiviral responses, ascoviruses seem to utilize viral encoded proteins as well as miRNAs. Since the genomes of these viruses have only recently been sequenced, the role of many of the encoded genes essential for pathogenesis and manipulation of antiviral defence mechanisms remains to be eluciated.