Relevant bibliographies by topics / Episomal

Academic literature on the topic 'Episomal'

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Published: 4 June 2021
Last updated: 26 January 2023

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Journal articles on the topic "Episomal"

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Habel, Marie-Eve, Mathieu Drouin, and Daniel Jung. "Maintenance of Epstein–Barr virus-derived episomal vectors in the murine Sp2/0 myeloma cell line is dependent upon exogenous expression of human EBP2." Biochemistry and Cell Biology 82, no. 3 (June 1, 2004): 375–80. http://dx.doi.org/10.1139/o04-037.

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Vectors carrying the origin of replication (oriP) and driving expression of the EBNA-1 protein from Epstein–Barr virus (EBV) replicate as extrachromosomal episomes in human cells. Whether these vectors can be maintained as episomes in murine cells is still controversial. Here we demonstrate that EBNA-1 expression alone was unable to maintain episomal expression of an EBV-based vector in the murine Sp2/0 cell line. However, we were able to obtain long-term episome maintenance in Sp2/0 cells after exogenously expressing human EBP2 by genetic engineering. Our results provide further evidence for the fundamental role of human EBP2 in episomal maintenance of EBV-based vectors. Moreover, we demonstrate that EBV-based vectors can be successfully used in cells presumably incompetent for episomal maintenance.Key words: EBV vector, EBNA-1, EBP2, episomal maintenance, mouse cell, Sp2/0 myeloma cell line.
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Griffiths, Rhoswyn, Sally M. Harrison, Stuart Macnab, and Adrian Whitehouse. "Mapping the minimal regions within the ORF73 protein required for herpesvirus saimiri episomal persistence." Journal of General Virology 89, no. 11 (November 1, 2008): 2843–50. http://dx.doi.org/10.1099/vir.0.2008/002725-0.

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Herpesvirus saimiri (HVS) establishes a persistent infection in which the viral genome persists as a circular non-integrated episome. ORF73 tethers HVS episomes to host mitotic chromosomes, allowing episomal persistence via an interaction with the chromosome-associated protein, MeCP2. Here we demonstrate that ORF73 also interacts with the linker histone H1 via its C terminus, suggesting it associates with multiple chromosome-associated proteins. In addition, we show that the C terminus is also required for the ability of ORF73 to bind the terminal repeat region of the HVS genome. These results suggest that the ORF73 C terminus contains all the necessary elements required for HVS episomal persistence. Using a range of ORF73 C terminus deletions to rescue the episomal maintenance properties of a HVSΔ73 recombinant virus, we show that a C terminus region comprising residues 285–407 is sufficient to maintain the HVS episome in a dividing cell population.
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Angeletti, Peter C., Kitai Kim, Fiona J. Fernandes, and Paul F. Lambert. "Stable Replication of Papillomavirus Genomes in Saccharomyces cerevisiae." Journal of Virology 76, no. 7 (April 1, 2002): 3350–58. http://dx.doi.org/10.1128/jvi.76.7.3350-3358.2002.

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ABSTRACT Papillomaviruses normally replicate in stratified squamous epithelial tissues of their mammalian hosts, in which the viral genome is found as a nuclear plasmid. Two viral proteins, E1, a helicase, and E2, a transcriptional activator and plasmid maintenance factor, are known to contribute to the episomal replication of the viral genome. Recently, our laboratory discovered that papillomaviruses can also replicate in an E1-independent manner in mammalian cells (K. Kim and P. F. Lambert, Virology, in press; K. Kim and P. F. Lambert, submitted for publication). In this study, we describe experiments investigating the capacity of the human papillomavirus type 16 (HPV16) genome to replicate in yeast (Saccharomyces cerevisiae). The full-length HPV16 genome, when linked in cis to a selectable yeast marker gene, either TRP1 or URA3, could replicate stably as an episome in yeast. The replication of papillomavirus genomes in yeast is not limited to HPV16. Bovine papillomavirus type 1 and HPV6b, -11, -16, -18, and -31 were all capable of replicating in short-term assays over a period of 20 cell doublings. The long-term persistence of viral episomes did not require any one viral gene, as mutant genomes defective in single genes also replicated episomally. These results indicate that the viral episome can replicate in the absence of the E1 DNA helicase. Similarly, E2 was also not required for replication in yeast, and E2 mutant viral genomes were stably maintained in the absence of selection, indicating the existence of an E2-independent mechanism for plasmid maintenance. The episomal replication of papillomavirus genomes in yeast provides a genetically manipulatable system in which to investigate cellular factors required for episomal replication and may provide a novel means for generating infectious papillomavirus.
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Sharkey, Mark, Karine Triques, Daniel R. Kuritzkes, and Mario Stevenson. "In Vivo Evidence for Instability of Episomal Human Immunodeficiency Virus Type 1 cDNA." Journal of Virology 79, no. 8 (April 15, 2005): 5203–10. http://dx.doi.org/10.1128/jvi.79.8.5203-5210.2005.

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ABSTRACT Current regimens for the management of human immunodeficiency virus type 1 (HIV-1) infection suppress plasma viremia to below detectable levels for prolonged intervals. Nevertheless, there is a rapid resumption in plasma viremia if therapy is interrupted. Attempts to characterize the extent of viral replication under conditions of potent suppression and undetectable plasma viremia have been hampered by a lack of convenient assays that can distinguish latent from ongoing viral replication. Using episomal viral cDNA as a surrogate for ongoing replication, we previously presented evidence that viral replication persists in the majority of infected individuals with a sustained aviremic status. The labile nature of viral episomes and hence their validity as surrogate markers of ongoing replication in individuals with long-term-suppressed HIV-1 infection have been analyzed in short-term in vitro experiments with conflicting results. Since these in vitro experiments do not shed light on the long-term in vivo dynamics of episomal cDNA or recapitulate the natural targets of infection in vivo, we have analyzed the dynamics of episomal cDNA turnover in vivo by following the emergence of an M184V polymorphism in plasma viral RNA, in episomal cDNA, and in proviral DNA in patients on suboptimal therapies. We demonstrate that during acquisition of drug resistance, wild-type episomal cDNAs are replaced by M184V-harboring episomes. Importantly, a complete replacement of wild-type episomes with M184V-containing episomes occurred while proviruses remained wild type. This indicates that episomal cDNAs are turned over by degradation rather than through death or tissue redistribution of the infected cell itself. Therefore, evolution of episomal viral cDNAs is a valid surrogate of ongoing viral replication in HIV-1-infected individuals.
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Wang, Xiao-Yin, Xi Zhang, Tian-Yun Wang, Yan-Long Jia, Dan-Hua Xu, and Dan-Dan Yi. "Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells." Molecular Biology of the Cell 30, no. 22 (October 15, 2019): 2761–70. http://dx.doi.org/10.1091/mbc.e19-02-0108.

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Matrix attachment regions (MARs) can mediate the replication of vector episomes in mammalian cells; however, the molecular mode of action remains unclear. Here, we assessed the characteristics of MARs and the mechanism that mediates episomal vector replication in mammalian cells. Five shortened subfragments of β-interferon MAR fragments were cloned and transferred into CHO cells, and transgene expression levels, presence of the gene, and the episomal maintenance mechanism were determined. Three shortened MAR derivatives (position 781–1320, 1201–1740, and 1621–2201) retained full MAR activity and mediated episomal vector replication. Moreover, the three shortened MARs showed higher transgene expression levels, greater efficiency in colony formation, and more persistent transgene expression compared with those of the original pEPI-1 plasmid, and three functional truncated MARs can bind to SAF-A MAR-binding protein. These results suggest that shortened MARs are sufficient for replication and maintenance of episomes in CHO cells.
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Madireddy, Advaitha, Pravinkumar Purushothaman, Christopher P. Loosbroock, Erle S. Robertson, Carl L. Schildkraut, and Subhash C. Verma. "G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV." Nucleic Acids Research 44, no. 8 (February 2, 2016): 3675–94. http://dx.doi.org/10.1093/nar/gkw038.

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Abstract Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases.
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Sears, John, John Kolman, Geoffrey M. Wahl, and Ashok Aiyar. "Metaphase Chromosome Tethering Is Necessary for the DNA Synthesis and Maintenance of oriP Plasmids but Is Insufficient for Transcription Activation by Epstein-Barr Nuclear Antigen 1." Journal of Virology 77, no. 21 (November 1, 2003): 11767–80. http://dx.doi.org/10.1128/jvi.77.21.11767-11780.2003.

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ABSTRACT Epstein-Barr Virus (EBV) infects resting B cells, within which it establishes latency as a stable, circular episome with only two EBV components, the cis element oriP and the latently expressed protein EBNA1. It is believed that EBNA1's ability to tether oriP episomes to metaphase chromosomes is required for its stable replication. We created fusions between the DNA-binding domain (DBD) of EBNA1 and the cellular chromatin-binding proteins HMGA1a and HMG1 to determine the minimal requirements for stable maintenance of an oriP-based episome. These two proteins differ in that HMGA1a can associate with metaphase chromosomes but HMG1 cannot. Interestingly, coinciding with metaphase chromosome association, HMGA1a-DBD but not HMG1-DBD supported both the transient replication and stable maintenance of oriP plasmids, with efficiencies quantitatively similar to that of EBNA1. However, HMGA1a-DBD activated transcription from EBNA1-dependent episomal reporter to only 20% of the level of EBNA1. Furthermore, EBNA1 but not HMGA1a-DBD activated transcription from a chromosomally integrated EBNA1-dependent transcription reporter. This indicates that EBNA1 possesses functional domains that support transcription activation independent of its ability to tether episomal oriP plasmids to cellular chromosomes. We provide evidence that metaphase chromosome tethering is a fundamental requirement for maintenance of an oriP plasmid but is insufficient for EBNA1 to activate transcription.
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Calderwood, Michael, Robert E. White, Rhoswyn A. Griffiths, and Adrian Whitehouse. "Open reading frame 73 is required for herpesvirus saimiri A11-S4 episomal persistence." Journal of General Virology 86, no. 10 (October 1, 2005): 2703–8. http://dx.doi.org/10.1099/vir.0.81230-0.

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Herpesvirus saimiri (HVS) establishes a latent infection in which the viral genome persists as a non-integrated episome. Analysis has shown that only open reading frames (ORFs) 71–73 are transcribed in an in vitro model of HVS latency. ORF73 also colocalizes with HVS genomic DNA on host mitotic chromosomes and maintains the stability of HVS terminal-repeat-containing plasmids. However, it is not known whether ORF73 is the only HVS-encoded protein required for episomal maintenance. In this study, the elements required for episomal maintenance in the context of a full-length HVS genome were examined by mutational analysis. A recombinant virus, HVS-BACΔ71-73, lacking the latency-associated genes was unable to persist in a dividing cell population. However, retrofitting an ORF73 expression cassette into the recombinant virus rescued episomal maintenance. This indicates that ORF73 is the key trans-acting factor for episomal persistence and efficient establishment of a latent infection.
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Duensing, Stefan, Anette Duensing, Elsa R. Flores, Anh Do, Paul F. Lambert, and Karl Münger. "Centrosome Abnormalities and Genomic Instability by Episomal Expression of Human Papillomavirus Type 16 in Raft Cultures of Human Keratinocytes." Journal of Virology 75, no. 16 (August 15, 2001): 7712–16. http://dx.doi.org/10.1128/jvi.75.16.7712-7716.2001.

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ABSTRACT Primary human keratinocytes with ectopic expression of high-risk human papillomavirus (HPV) E6 and E7 oncoproteins display abnormal centrosome numbers, multipolar mitoses, and aneusomy. However, it has not been explored whether these abnormalities can occur in cells containing HPV episomes where E6 and E7 expression is under viral transcriptional control. Here, we demonstrate that centrosome abnormalities and genomic instability occur in organotypic raft cultures of human keratinocytes with episomal HPV-16 even at low copy numbers. We conclude that HPV-16 DNA, when maintained as an episome, can disturb centrosome homeostasis and subvert genomic integrity of the host cell during early stages of the viral infection.
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Oh, Stephen T., Michelle S. Longworth, and Laimonis A. Laimins. "Roles of the E6 and E7 Proteins in the Life Cycle of Low-Risk Human Papillomavirus Type 11." Journal of Virology 78, no. 5 (March 1, 2004): 2620–26. http://dx.doi.org/10.1128/jvi.78.5.2620-2626.2004.

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ABSTRACT Many important functions have been attributed to the high-risk human papillomavirus (HPV) E6 and E7 proteins, including binding and degradation of p53 as well as interacting with Rb proteins. In contrast, the physiological roles of the low-risk E6 and E7 proteins remain unclear. Previous studies demonstrated that the high-risk E6 and E7 proteins also play roles in the productive life cycle by facilitating the maintenance of viral episomes (J. T. Thomas, W. G. Hubert, M. N. Ruesch, and L. A. Laimins, Proc. Natl. Acad. Sci. USA 96:8449-8454, 1999). In order to determine whether low-risk E6 or E7 is similarly necessary for the stable maintenance of episomes, HPV type 11 (HPV-11) genomes that contained translation termination mutations in E6 or E7 were constructed. Upon transfection into normal human keratinocytes, genomes in which E6 function was abolished were unable to be maintained episomally. Transfection of genomes containing substitution mutations in amino acids conserved in high- and low-risk HPV types suggested that multiple protein domains are involved in this process. Examination of cells transfected with HPV-11 genomes in which E7 function was inhibited were found to exhibit a more complex phenotype. At the second passage following transfection, mutant genomes were maintained as episomes but at significantly reduced levels than in cells transfected with the wild-type HPV-11 genome. Upon further passage in culture, however, the episomal forms of these E7 mutant genomes quickly disappeared. These findings identify important new functions for the low-risk E6 and E7 proteins in the episomal maintenance of low-risk HPV-11 genomes and suggest that they may act in a manner similar to that observed for the high-risk proteins.
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Dissertations / Theses on the topic "Episomal"

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Cheung, Wing Tung. "Development of large episomal constructs for gene therapy." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431229.

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Falcon, Alaric Antonio. "Building an episomal model of aging in saccharomyces cerevesiae." [Gainesville, Fla.] : University of Florida, 2004. http://wwwlib.umi.com/cr/ufl/fullcit?p3136937.

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Thesis (Ph.D.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 117 pages. Includes Vita. Includes bibliographical references.
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Griffiths, Rhoswyn Ann. "Virus - host cell interactions required for herpesvirus saimiri episomal persistence." Thesis, University of Leeds, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445379.

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Kymalainen, Hanna. "Development of viral & non-viral episomal vectors for gene therapy applications." Thesis, University of London, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.589000.

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Gene therapy consists of methods which attempt to repair or replace defective genes responsible for disease, or to add genes to a therapeutic effect. To achieve this, two episomally maintained recombinant viral vectors have shown promising results: integration-deficient lentiviral vectors (IDLVs), and adeno-associated virus (AA V) vectors. The non-integrating nature of these vectors improves their safety profile but also limits transgene retention as nuclear episomes generally get lost during cell division. In the present study, the establishment of stable replicating episomes via transduction with AA V and IDL V gene therapy vectors was examined in CHO cells. Different DNA elements and cell culture conditions were evaluated, and in particular the effects of (i) DNA elements called S/MARs (scaffold/matrix attachment regions) which are involved in chromatin organisation, transcription and replication, and (ii) induction of transient cell cycle arrest in transfected and transduced cell populations. In the case of both AA V and IDL V vectors, the incorporation of S/MAR elements into vector transcription units had only marginal effects on the establishment of stable transgene- positive cell populations, either with or without induction oftransient cell cycle arrest. However, a striking general result was observed in cell populations transduced with IDL Vs and subjected to a transient cell cycle arrest soon after transduction. Under these conditions, following release from cell cycle arrest and in the absence of any selection pressure, substantial populations (10-25%) of proliferating and stably transduced cells emerged and were maintained over at least 100 population doublings. This establishment of stable transduction was seen only with IDLVs, was crucially . dependent on the induction of a period of transient cell cycle arrest, occurred independently of the presence of S/MAR elements, and resulted in transgene-positive cell populations which could be isolated and propagated as stable clonal cell lines. In these polyclonal and clonal IDL V -transduced cell lines, the existence of non-integrated vector genomes in the form of multi-copy nuclear episomes was confirmed by evidence from linear amplification -mediated PCR, deep sequencing, Southern blotting and FISH (fluorescent in situ hybridisation). 2 The cumulative evidence suggests that transduction of eHO cells with IDL Vs followed by a short period of induced cell cycle arrest leads to the establishment of stable IDL V- based nuclear episomes which are transcriptionally active and undergo replication and segregation during cell division without the need for antibiotic-based or other positive selection pressure. Preliminary investigations were also done to test the capacity of combined IDL V transduction and transient cell cycle arrest to establish stable episome Hel.a cells and murine haematopoietic stem cells. However, further experiments are required either to optimise the protocol in these cells or to find other clinically relevant cell types in which the protocol can be implemented. The transfer of this technology to a variety of clinically relevant human stem or progenitor cell populations could improve the safety profile of a range of gene therapy strategies currently under investigation. 3
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Kunaparaju, Raj Kumar Biotechnology &amp Biomolecular Sciences Faculty of Science UNSW. "Epi-CHO, an episomal expression system for recombinant protein production in CHO cells." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2008. http://handle.unsw.edu.au/1959.4/41499.

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The current project is to develop a transient expression system for Chinese Hamster Ovary (CHO) cells based on autonomous replication and retention of plasmid DNA. The expression system, named Epi-CHO comprises (1) a recombinant CHO-K1 cell line encoding the Polyoma (Py) virus large T-Antigen (PyLT-Ag), and (2) a DNA expression vector, pPy/EBV encoding the Py Origin (PyOri) for autonomous replication and encoding the Epstein-Barr virus (EBV), Nuclear Antigen-1 (EBNA-1) and EBV Origin of replication (OriP) for plasmid retention. The CHO-K1 cell line expressing PyLT-Ag, named CHO-T was adapted to suspension growth in serum-free media (EXCELL-302) to facilitate large scale transient transfection and recombinant (r) protein production. PyLT-Ag-expressed in CHO-T supported replication of PyOri-containing plasmids and enhanced growth and r- protein production. A scalable cationic lipid based transfection was optimised for CHO-T cells using LipofectAMINE-2000??. Destabilised Enhanced Green Fluorescence Protein (D2EGFP) and Human Growth Hormone (HGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of CHO-T cells with the vector pPy/EBV encoding D2EGFP showed prolonged and enhanced EGFP expression, and transfection with pPy/EBV encoding HGH resulted in a final concentration of 75 mg/L of HGH in culture supernatant 11 days following transfection.
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Argyros, Orestis. "Development of novel episomal non-viral vectors for stable, long-term expression for gene therapy." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497718.

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Schneider, Hauke. "Identifikation Apoptose-assoziierter Gene in B-Zellen und Charakterisierung des Genproduktes LAPTM5." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2003. http://dx.doi.org/10.18452/14833.

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Programmierter Zelltod (PCD) oder Apoptose ist ein universeller biologischer Prozess, der in multizellulären Organismen essentiell ist für die Differenzierung und Homöostase von Geweben. Bei der Entwicklung eines funktionsfähigen zellulären Immunsystems können autoreaktive Zellen entstehen. Die negative Selektion von unreifen, autoreaktiven B-Zellen erfolgt durch IgM-vermittelte Apoptose und ist von de novo Transkription abhängig. Die genauen Mechanismen der IgM-vermittelten Apoptose und die involvierten Genprodukte sind nur unzureichend charakterisiert. Zur Identifikation Apoptose-assoziierter Gene in B-Zellen wurden die Differential Display-Analyse durchgeführt und die Expressionsmuster apoptotischer und nicht-apoptotischer BL60-Zellen untersucht. Es wurden 38 differentielle Fragmente identifiziert und kloniert. Sequenzanalysen ergaben Homologien eines Fragments mit LAPTM5, einem lysosomal-assoziierten Transmembran-Protein mit vorwiegender Expression in hämatopoetischem Gewebe. Northern Blot-Analysen zeigten 2 Stunden nach Inkubation von BL-60-Zellen mit anti-IgM einen Anstieg der Genexpression von LAPTM5. Das LAPTM5-Gen liegt auf Chromosom 1 (1p34), ist evolutionär konserviert und besitzt keine Homologien zu bekannten Genen. Die Untersuchung der gewebespezifischen Expression von LAPTM5 ergab neben der hohen Expressionsrate in hämatopoetischem Gewebe eine sehr starke Expression in Skelett- und Herzmuskelgewebe. Elektronenmikroskopisch zeigte sich eine Lokalisation des Proteins in späten Endosomen und Lysosomen. Daneben konnte LAPTM5 auch auf der Oberfläche von BL60-Zellen detektiert werden. Während des apoptotischen Prozesses bleibt die Menge an LAPTM5 auf der Zelloberfläche konstant. Zur weiteren Charakterisierung von LAPTM5 und anderen Kandidaten-Genen wurde ein episomales Expressions- und Selektionssystem entwickelt. Cotransfektionsanalysen unter Verwendung eines GFP (green fluorescent protein)- Konstrukts zeigten, dass die aufgereinigten Zellen zu 80% das interessierende Gen exprimieren und die Expression länger als vier Wochen anhält.
Programmed cell death (PCD) or apoptosis is a key feature of normal development and tissue homeostasis. In the development of a functional immunesystem the occurence of autoreactive cells is tightly controlled and prevented by apoptosis. The negative selection of autoreactive immature B cells after encountering self antigen occures via surface IgM (sIgM) mediated apoptosis and depends on de novo gene transcription. The precise mechanism of this process and the possible involvement of different genes in the regulation of sIgM-mediated cell death is not understood so far. In order to identify genes associated with B cell apoptosis Differential Display RT-PCR (DD) was performed to analyze sIgM-mediated apoptosis in the human Burkitt lymphoma line BL60. The expression patterns of apoptotic and non-apoptotic cells were investigated and 38 differentially expressed gene fragments were found. Subsequent northern blot analysis showed that LAPTM5, a lysosomal associated membrane protein preferential ly expressed in adult hematopoietic tissue, is up-regulated 2 hours after induction of apoptosis. LAPTM5 is a protein with five transmembrane domains, it is conserved during evolution and the gene, mapping to chromosome 1p34, has no homology to known genes. In contrast to earlier data a very high expression of the protein was detected not only in hematopoietic tissue but also in skeletal muscle and heart muscle. Electronmicroscopy was performed to investigate the subcellular localization in detail and showed that LAPTM5 is mainly present in late endosomes and lysosomes. FACS analysis revealed a surface expression of LAPTM5 and a constant amount of LAPTM5 at the surface during IgM mediated apoptosis. To further investigate the functional role of candidate genes during apoptosis an episomal expression and selection system was established. Cotransfection analysis with GFP (green fluorescent protein) showed that 80% of the separeted cells express the gene of interest for at least four weeks.
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Chanson, Aurelie Heitiare. "Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity." Thesis, Queensland University of Technology, 2009. https://eprints.qut.edu.au/30290/1/Aurelie_Chanson_Thesis.pdf.

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Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.
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Chanson, Aurelie Heitiare. "Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity." Queensland University of Technology, 2009. http://eprints.qut.edu.au/30290/.

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Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.
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10

Groger, Richard Kevin. "Development of episomal expression systems for genetically engineering human hematopoietic cells: Model analyses of the M-CSF:M-CSF receptor pair." Case Western Reserve University School of Graduate Studies / OhioLINK, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=case1054915738.

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Books on the topic "Episomal"

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E, Fachruddin A. Ritumpanna wélenrénngé: Sebuah episoda sastra Bugis klasik Galigo. Jakarta: Ecole français d'Extrême-Orient, 1999.

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Dialog Nasional Pasaribu Saruksuk (1988 Sirau, Indonesia). Pasaribu: Hasil Dialog Nasional Pasaribu Saruksuk & kesepakatan Barus : berikut episoda dan kissah leluhur. 2nd ed. Bandung: Tarombo Pasaribu Saruksuk, 1993.

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David, Sherman. History of the revisions of the discipline of the Methodist Episcopal Church. Memphis, TN: General Books, 2010.

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Mihaela, Bogdan, and Aloman Nicolae. NEPOTRIVIT Episodul 1. Independently Published, 2017.

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Wolstenholme, G. E. W., and Maeve O'Connor. Bacterial Episomes and Plasmids. Wiley & Sons, Incorporated, John, 2009.

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Symposium, Ciba Foundation. Bacterial Episomes and Plasmids. Wiley & Sons, Incorporated, John, 2009.

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Episodal pollution caused by a barrier across Eloor branch of River Periyar. New Delhi: Central Board for the Prevention and Control of Water Pollution, 1986.

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Book chapters on the topic "Episomal"

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Yu, Junying. "Episomal Vectors." In Primary and Stem Cells, 49–83. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118147177.ch4.

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Lufino, Michele M. P., Anna R. Popplestone, Sally A. Cowley, Pauline A. H. Edser, William S. James, and Richard Wade-Martins. "Episomal Transgene Expression in Pluripotent Stem Cells." In Methods in Molecular Biology, 369–87. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-201-4_27.

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Attal, Joé, Marie Georges Stinnakre, Marie Claire Théron, Michel Terqui, and Louis Marie Houdebine. "The Use of Episomal Vectors for Transgenesis." In Transgenic Animals, 251–55. London: CRC Press, 2022. http://dx.doi.org/10.1201/9781003211099-52.

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Argyros, Orestis, Suet-Ping Wong, Charles Coutelle, and Richard P. Harbottle. "Utilizing Minicircle Vectors for the Episomal Modification of Cells." In Minicircle and Miniplasmid DNA Vectors, 93–113. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2013. http://dx.doi.org/10.1002/9783527670420.ch7.

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Garrels, Wiebke, Khursheed Iqbal, and Wilfried A. Kues. "Episomal Expression of Minicircles and Conventional Plasmids in Mammalian Embryos." In Minicircle and Miniplasmid DNA Vectors, 189–202. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2013. http://dx.doi.org/10.1002/9783527670420.ch11.

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Wen, Wei, Tao Cheng, and Xiao-Bing Zhang. "Episomal Reprogramming of Human Peripheral Blood Mononuclear Cells into Pluripotency." In Methods in Molecular Biology, 117–33. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-1084-8_8.

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Liu, Ying, Kate Judd, and Uma Lakshmipathy. "Stable Transfection Using Episomal Vectors to Create Modified Human Embryonic Stem Cells." In Methods in Molecular Biology, 263–72. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-348-0_21.

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Roux, Indra, and Yit Heng Chooi. "Heterologous Expression of Fungal Biosynthetic Pathways in Aspergillus nidulans Using Episomal Vectors." In Methods in Molecular Biology, 75–92. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2273-5_5.

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Su, Ruijun Jeanna, Amanda Neises, and Xiao-Bing Zhang. "Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors." In Methods in Molecular Biology, 57–69. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/7651_2014_139.

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Koirala, Adarsha, Shannon M. Conley, and Muna I. Naash. "Episomal Maintenance of S/MAR-Containing Non-Viral Vectors for RPE-Based Diseases." In Retinal Degenerative Diseases, 703–9. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4614-3209-8_88.

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Conference papers on the topic "Episomal"

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Jin, Chuan, Mohanraj Ramachandran, Grammatiki Fotaki, Berith Nilsson, Magnus Essand, and Di Yu. "Abstract A171: Long-term episomal gene transfer for safe engineering of T cells for adoptive cell therapy of cancer." In Abstracts: CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/2326-6074.cricimteatiaacr15-a171.

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Mazda, Osam, Tsunao Kishida, Masahiro Matsui, Hiroshi Nakano, Koichiro Yoshimoto, Taketoshi Shimada, Shigeru Nakai, Jiro Imanishr, and Yasuo Hisa. "Nonviral gene administration by means of the Epstein-Barr virus (EBV)-based episomal vectors and it application to gene therapy and regenerative medicine." In 2009 International Symposium on Micro-NanoMechatronics and Human Science (MHS). IEEE, 2009. http://dx.doi.org/10.1109/mhs.2009.5351980.

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Masui, Toshiyuki. "EpisoDAS." In AVI '18: 2018 International Conference on Advanced Visual Interfaces. New York, NY, USA: ACM, 2018. http://dx.doi.org/10.1145/3206505.3206593.

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Kishida, Tsunao, Noriaki Nakai, Masahiro Matsui, Koichiro Yoshimoto, Hiroshi Nakano, Masaharu Shin-Ya, Taketoshi Shimada, et al. "Gain-of-function and loss-of-function analyses in vivo of transcriptional factor and cytokine genes using Epstein-Barr virus-based episomal vectors, and their implication to novel strategies of gene therapy and regenerative medicine." In 2010 International Symposium on Micro-NanoMechatronics and Human Science (MHS). IEEE, 2010. http://dx.doi.org/10.1109/mhs.2010.5669564.

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Bingxue Wang and Yuanzhong Chen. "Fuzzy logic for mining episodal association rules in time series." In 2009 IEEE International Conference on Intelligent Computing and Intelligent Systems (ICIS 2009). IEEE, 2009. http://dx.doi.org/10.1109/icicisys.2009.5358037.

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Abrams, Austin, Kylia Miskell, and Robert Pless. "The Episolar Constraint: Monocular Shape from Shadow Correspondence." In 2013 IEEE Conference on Computer Vision and Pattern Recognition (CVPR). IEEE, 2013. http://dx.doi.org/10.1109/cvpr.2013.185.

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Hensel, K., F. Cantner, S. Wirth, and J. Postberg. "Virus-Wirt-Interaktion – Hepatitis B Virus, HBx und 3D-Zellkernarchitektur: Chromosome conformation capture und next-generation sequencing zur Detektion episomaler HBV DNA in aktiv transkribierten Chromatinregionen." In Viszeralmedizin 2017. Georg Thieme Verlag KG, 2017. http://dx.doi.org/10.1055/s-0037-1605021.

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Reports on the topic "Episomal"

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Jean-Michael H. Vos. Final report. Human artificial episomal chromosome (HAEC) for building large genomic libraries. Office of Scientific and Technical Information (OSTI), December 1999. http://dx.doi.org/10.2172/764441.

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Ilan, Judith. Episome-Based Gene Therapy Strategy for Treatment of Human Breast Cancer. Fort Belvoir, VA: Defense Technical Information Center, January 1999. http://dx.doi.org/10.21236/ada373940.

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Barnes, Teresa. Collaborative Research and Development by EpiSolar and NREL of Processes and Materials for Flexible CdS/CdTe Superstrate Devices: Cooperative Research and Development Final Report, CRADA Number CRD-14-550. Office of Scientific and Technical Information (OSTI), May 2016. http://dx.doi.org/10.2172/1255203.

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