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1
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 & 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/.
Full textAbstract:
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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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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Schnödt, Maria Angelika [Verfasser], Dagmar [Gutachter] Mörsdorf, Mirka [Gutachter] Uhlirova, and Hildegard [Gutachter] Büning. "Improving safety and establishing episomal maintenance of Adeno-associated viral vectors / Maria Angelika Schnödt ; Gutachter: Dagmar Mörsdorf, Mirka Uhlirova, Hildegard Büning." Köln : Universitäts- und Stadtbibliothek Köln, 2016. http://d-nb.info/1122713827/34.
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Liu, Jing. "Reprogramming peripheral blood mononuclear cells using an efficient feeder-free, non-integration method to generate iPS cells and the effect of immunophenotype and epigenetic state on HSPC fate." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/10031.
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Background and objectives: In 2006 Shinya Yamanaka successfully reprogrammed mouse fibroblasts back to an embryonic stem cell-like state (called induced pluripotent cells, iPS cells) using retrovirus to introduce four genes that encode critical transcription factor proteins (Oct4, Sox2, KLF4, and c-Myc). This ability to reprogram has promising future applications in clinical and biomedical research for study of diseases, development of candidate drugs and to support therapeutic treatments in regenerative medicine. However, the clinical applications have to meet GMP requirements without the risk of insertional mutagenesis associated with retrovirus. Chromatin modifying agents are widely used in many protocols to generate iPS cells and culture of blood CD34+ cells with chromatin-modifying agents can lead to an increase in marrow repopulating cells and in the case of valproic acid increased erythroid cell colony formation. We undertook research to help understand what effects these reagents have on mobilised peripheral blood (mPB) CD34+ cells and optimised the expansion medium protocol to facilitate reprogramming work. This project aims to utilize peripheral blood mononuclear cells (MNC), one of the most easily accessible tissues to generate iPS cells using an efficient non-viral, feeder cell free methodology, with the ultimate goal of moving this methodology towards clinical use. Materials and Methods: G-CSF mobilised peripheral blood, buffy coat, cord blood and fetal liver were obtained from patients and donors under informed consent and ethics committee approval. Haematopoietic stem/progenitor cells CD34+ or CD133+) isolated by magnetic separation were flow cytometry sorted into CD34+/CD133+, CD34+/CD133-, and CD34-/CD133+ sub-populations and their lineage potential were assessed in colony forming unit assays. The effect of epigenetic modifiers valproic acid and 5-aza-2-deoxycytidine used singly or in combination with each other and with IL3 on phenotype and lineage potential of cultured CD34+ cells from mobilised peripheral blood were assessed by flow cytometry and colony-forming unit assays. Prior to reprogramming mononuclear cells from peripheral blood or CD34+ cells from blood were expanded in culture medium supplemented with stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L) and Interleukin- 3 (IL-3) for several days. Actively proliferating cells were reprogrammed by electroporation using episomal vectors with an oriP/EBNA-1 backbone to deliver five reprogramming genes, Oct4, Sox2, Lin28, L-Myc, and Klf4. Electroporated cells were seeded onto matrigel coated plates immediately after transfection or were reseeded after three days’ culture. Subsequently, cells were cultured in specific medium on different days. When iPS colonies appeared, they were picked and cultured as for ES cells. Once established, iPS cell lines were immunophenotyped using flow cytometry and immunofluorescence and their potential to differentiate into the three germ layers was assessed in vitro. Results and Conclusion: The largest subpopulation of CD34+ cells was CD34+/CD133+ population which was essentially committed to myeloid colony production, while much smaller CD34+/CD133- subpopulation had a greater capacity to generate erythroid colonies. Optimised cytokine cocktail for expansion of CD34+ cells included IL-3, important in improving expansion and maintaining functionality of CD34+ cells. The optimised cytokine cocktail comprised 100 ng/ml SCF, 10 ng/ml Flt3L, and 20 ng/ml IL-3, which maintained CD34+ cells and MNC in an active proliferating state. In addition, valproic acid and IL3 were found to act synergistically, to increase the numbers of CD34+/CD36+ positive cells. However, we found that an apparent increase in red cell colony formation actually resulted from a decrease in white cell colonies, so no overall increase in red cell colonies was seen when equivalent numbers of CD34+ cells were plated. Proliferating MNC maintained in optimised cytokine cocktail were amenable to electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) within a backbone of oriP/EBNA-1. We successfully developed an efficient and simple method for reprogramming MNC from fresh or frozen samples to generate induced pluripotent cells using episomal vectors in a feeder-free system without any requirement for small molecules and the highest reprogramming efficiency is 0.033% (65 colonies from 2 ◊ 105 seeding MNC). The cytokine cocktail and reprogramming methods work better in CD34+ cells from cord blood or fetal liver, and we obtained 148 iPS colonies from 105 seeding cells (0.148%) at most. In addition, fibroblasts from adult and fetal liver can be successfully reprogrammed using the same reprogramming method. The use of episomal vectors with an oriP/EBNA-1 backbone to deliver reprogramming genes, and efficient electroporation were the most important factors in efficiency of the reprogramming process. In addition, it is pivotal to initiate transfection when cells are actively proliferating. The iPS cell lines we generated maintained the successful expression of ES markers including Oct4, Nanog, SSEA3. SSEA4, TRA-1-60 and TRA-1-81, and had the capacity to successfully differentiate into cell types of ectoderm, mesoderm and endoderm layers in vitro.
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Gallaher, Sean David. "In vivo delivery and persistence in mouse of an episomal expression cassette by a helper dependent adenovirus - Epstein-Barr viru hybrid gene therapy vector." Diss., Restricted to subscribing institutions, 2007. http://proquest.umi.com/pqdweb?did=1472159431&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Pirlo, Steven Dominic. "Identification of viral-based replicating vectors suitable for the development of a sugarcane bioreactor." Thesis, Queensland University of Technology, 2007. https://eprints.qut.edu.au/16548/1/Steven_Dominic_Pirlo_Thesis.pdf.
Full textAbstract:
The circular, single-stranded (ss) DNA genomes of plant viruses in the families Geminiviridae and Nanoviridae are replicated within the nucleus of a host cell by a mechanism called rolling circle replication (RCR). Although this process relies almost exclusively on the replication machinery of the host cell, initiation occurs via the interaction of the viral replication initiation protein (Rep) with regulatory DNA sequences within the viral genome. The use of a virus-based episomal amplification technology as a plant bioreactor platform exploits the process of Rep-mediated RCR for the high-level amplification of virus-based episomes in plants and subsequent expression of heterologous proteins; such an approach offers advantages over existing gene expression technologies. This PhD thesis describes research towards the development of a virus-based episomal amplification system for use in sugarcane. Such a crop is ideally suited for a plant bioreactor system due to the efficient high-level production of plant biomass and the existence of established production, harvesting and processing infrastructure.
In order to rapidly assess the potential of a virus-based episomal amplification system in sugarcane, a transient assay system was established. Sugarcane callus was identified as the most suitable cell preparation; providing rapid cell regeneration, uniform experimental samples and upon isolation, total DNA suitable for Southern analysis. This assay system once established, proved effective in rapidly identifying virus-based episomes capable of undergoing RCR within sugarcane host cells.
This transient assay system was then used to test the functionality of a virus-based episomal amplification system based on the ssDNA virus, Banana bunchy top virus (BBTV) in sugarcane. BBTV-based episomal amplification vectors were constructed with a reporter gene expression cassette flanked by two copies of the BBTV regulatory DNA sequences. The episomal amplification vectors were bombarded into sugarcane and banana host cells in various combinations and evidence of RCR was assessed through Southern blot analysis. RCR products were identified in banana host cells bombarded with the BBTV-based episomal amplification vectors in combination with vectors encoding BBTV Master-Rep (M Rep). RCR products were not identified within sugarcane cells bombarded with the same construct combinations.
Integrated InPAct (In Plant Activation) episomal vectors based on BBTV were then employed to confirm the transient results, in addition, the functionality of an InPAct vector based on an alternate virus, Tobacco yellow dwarf virus (TYDV) was also assessed. InPAct vectors based on BBTV were constructed with an untranslatable expression cassette for integration within the sugarcane genome. Transient experiments were performed to assess the ability of BBTV M-Rep and TYDV Rep to initiate RCR of their respective InPAct vectors. Visual observation of GFP expression indicated that BBTV M-Rep was capable of initiating RCR of the BBTVbased InPAct vectors within banana host cells but no evidence was observed in sugarcane host cells. TYDV Rep was capable of initiating RCR of the TYDV-based InPAct vector within sugarcane host cells with a 100-fold increase in the number of fluorescent foci compared to cells bombarded with the TYDV InPAct vector alone. The BBTV-based InPAct vector was stably integrated within the sugarcane genome and the ability for BBTV M-Rep to initiate episome formation and RCR was assessed by Southern blot analysis. Evidence of BBTV M-Rep mediated RCR was not detected within the transgenic sugarcane bombarded with BBTV M-Rep. Transgenic sugarcane containing the TYDV-based InPAct vectors was assessed for the ability to be activated by TYDV Rep and undergo RCR. Southern blot analysis demonstrated that TYDV Rep was capable of recognising the integrated TYDVbased InPAct vector and RCR was detected within the transgenic sugarcane.
The observation that episomal vectors based on TYDV were functional within sugarcane host cells and BBTV-based vectors were not, was unexpected. It had been hypothesised that an episomal vector based on a monocot-infecting virus would replicate in an alternate monocot host, while an episomal vector based on a dicot infecting virus would not. Virus replication is thought to be host-specific however most host range studies have been conducted with full length infectious clones and not deconstructed virus-based episomes. The implication that viral Reps may be functional in plant cells of non-host species was then investigated. The ability for viral Reps to recognise their cognate IR and initiate RCR of virus-based episomes in different host cells was assessed through cross-replication experiments. Four ssDNA plant viruses; BBTV, TYDV, Chloris striate mosaic virus (CSMV) and Tomato leaf curl virus - Australia (ToLCV-Au) were assessed via Southern blot analysis for their ability to initiate both autonomous replication of infectious clones and episomal amplification within three different plant hosts; tobacco, sugarcane and banana.
Results from cross replication studies indicated a complex interaction between viral and host replication components. BBTV infectious clones and episomal vectors were restricted to replication within banana host cells providing a clear indication that episomal amplification vectors based on BBTV are restricted to Musa spp.
BBTV M-Rep was unable to recognise the viral regulatory DNA sequences of the other three ssDNA viruses. TYDV infectious clones and episomal vectors were capable of replicating within all three host cells tested, indicating that TYDV is capable of undergoing RCR within a broad range of plant hosts. TYDV Rep was also capable of recognising the viral regulatory DNA sequences of both CSMV and BBTV given favourable conditions within specific plant hosts. Replication of the CSMV infectious clone was not detected in any of the three host cells, although fidelity of this clone requires further confirmation. CSMV episomal vectors were functional within banana host cells only, indicating that although closely related to TYDV, episomal amplification vectors based on CSMV have a restricted host range.
CSMV Rep could not initiate RCR of episomal amplification vectors containing the viral regulatory DNA regions of the other three viruses in any of the plant host cells. ToLCV-Au infectious clones were capable of replicating within banana and tobacco host cells. Episomal amplification vectors based on ToLCV-Au extended the host range to sugarcane. ToLCV-Au Rep was unable to recognise the viral regulatory
DNA sequences of the other three viruses in any of the plant host cells. The ability for a viral Rep to recognise its own cognate regulatory DNA sequences within alternate plant host cells is variable. Episomal amplification vectors based on TYDV and ToLCV-Au appear to be the most suitable for the further development of a virusbased bioreactor system in sugarcane.
This study details the initial steps taken towards the development of a virus-based episomal amplification system in sugarcane. In doing so, fundamental knowledge into the mechanisms involved in Rep recognition of viral regulatory DNA sequences has been gathered. These research findings will provide a solid foundation for the further development of a sugarcane-based bioreactor.
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Pirlo, Steven Dominic. "Identification of viral-based replicating vectors suitable for the development of a sugarcane bioreactor." Queensland University of Technology, 2007. http://eprints.qut.edu.au/16548/.
Full textAbstract:
The circular, single-stranded (ss) DNA genomes of plant viruses in the families Geminiviridae and Nanoviridae are replicated within the nucleus of a host cell by a mechanism called rolling circle replication (RCR). Although this process relies almost exclusively on the replication machinery of the host cell, initiation occurs via the interaction of the viral replication initiation protein (Rep) with regulatory DNA sequences within the viral genome. The use of a virus-based episomal amplification technology as a plant bioreactor platform exploits the process of Rep-mediated RCR for the high-level amplification of virus-based episomes in plants and subsequent expression of heterologous proteins; such an approach offers advantages over existing gene expression technologies. This PhD thesis describes research towards the development of a virus-based episomal amplification system for use in sugarcane. Such a crop is ideally suited for a plant bioreactor system due to the efficient high-level production of plant biomass and the existence of established production, harvesting and processing infrastructure. In order to rapidly assess the potential of a virus-based episomal amplification system in sugarcane, a transient assay system was established. Sugarcane callus was identified as the most suitable cell preparation; providing rapid cell regeneration, uniform experimental samples and upon isolation, total DNA suitable for Southern analysis. This assay system once established, proved effective in rapidly identifying virus-based episomes capable of undergoing RCR within sugarcane host cells. This transient assay system was then used to test the functionality of a virus-based episomal amplification system based on the ssDNA virus, Banana bunchy top virus (BBTV) in sugarcane. BBTV-based episomal amplification vectors were constructed with a reporter gene expression cassette flanked by two copies of the BBTV regulatory DNA sequences. The episomal amplification vectors were bombarded into sugarcane and banana host cells in various combinations and evidence of RCR was assessed through Southern blot analysis. RCR products were identified in banana host cells bombarded with the BBTV-based episomal amplification vectors in combination with vectors encoding BBTV Master-Rep (M Rep). RCR products were not identified within sugarcane cells bombarded with the same construct combinations. Integrated InPAct (In Plant Activation) episomal vectors based on BBTV were then employed to confirm the transient results, in addition, the functionality of an InPAct vector based on an alternate virus, Tobacco yellow dwarf virus (TYDV) was also assessed. InPAct vectors based on BBTV were constructed with an untranslatable expression cassette for integration within the sugarcane genome. Transient experiments were performed to assess the ability of BBTV M-Rep and TYDV Rep to initiate RCR of their respective InPAct vectors. Visual observation of GFP expression indicated that BBTV M-Rep was capable of initiating RCR of the BBTVbased InPAct vectors within banana host cells but no evidence was observed in sugarcane host cells. TYDV Rep was capable of initiating RCR of the TYDV-based InPAct vector within sugarcane host cells with a 100-fold increase in the number of fluorescent foci compared to cells bombarded with the TYDV InPAct vector alone. The BBTV-based InPAct vector was stably integrated within the sugarcane genome and the ability for BBTV M-Rep to initiate episome formation and RCR was assessed by Southern blot analysis. Evidence of BBTV M-Rep mediated RCR was not detected within the transgenic sugarcane bombarded with BBTV M-Rep. Transgenic sugarcane containing the TYDV-based InPAct vectors was assessed for the ability to be activated by TYDV Rep and undergo RCR. Southern blot analysis demonstrated that TYDV Rep was capable of recognising the integrated TYDVbased InPAct vector and RCR was detected within the transgenic sugarcane. The observation that episomal vectors based on TYDV were functional within sugarcane host cells and BBTV-based vectors were not, was unexpected. It had been hypothesised that an episomal vector based on a monocot-infecting virus would replicate in an alternate monocot host, while an episomal vector based on a dicot infecting virus would not. Virus replication is thought to be host-specific however most host range studies have been conducted with full length infectious clones and not deconstructed virus-based episomes. The implication that viral Reps may be functional in plant cells of non-host species was then investigated. The ability for viral Reps to recognise their cognate IR and initiate RCR of virus-based episomes in different host cells was assessed through cross-replication experiments. Four ssDNA plant viruses; BBTV, TYDV, Chloris striate mosaic virus (CSMV) and Tomato leaf curl virus - Australia (ToLCV-Au) were assessed via Southern blot analysis for their ability to initiate both autonomous replication of infectious clones and episomal amplification within three different plant hosts; tobacco, sugarcane and banana. Results from cross replication studies indicated a complex interaction between viral and host replication components. BBTV infectious clones and episomal vectors were restricted to replication within banana host cells providing a clear indication that episomal amplification vectors based on BBTV are restricted to Musa spp. BBTV M-Rep was unable to recognise the viral regulatory DNA sequences of the other three ssDNA viruses. TYDV infectious clones and episomal vectors were capable of replicating within all three host cells tested, indicating that TYDV is capable of undergoing RCR within a broad range of plant hosts. TYDV Rep was also capable of recognising the viral regulatory DNA sequences of both CSMV and BBTV given favourable conditions within specific plant hosts. Replication of the CSMV infectious clone was not detected in any of the three host cells, although fidelity of this clone requires further confirmation. CSMV episomal vectors were functional within banana host cells only, indicating that although closely related to TYDV, episomal amplification vectors based on CSMV have a restricted host range. CSMV Rep could not initiate RCR of episomal amplification vectors containing the viral regulatory DNA regions of the other three viruses in any of the plant host cells. ToLCV-Au infectious clones were capable of replicating within banana and tobacco host cells. Episomal amplification vectors based on ToLCV-Au extended the host range to sugarcane. ToLCV-Au Rep was unable to recognise the viral regulatory DNA sequences of the other three viruses in any of the plant host cells. The ability for a viral Rep to recognise its own cognate regulatory DNA sequences within alternate plant host cells is variable. Episomal amplification vectors based on TYDV and ToLCV-Au appear to be the most suitable for the further development of a virusbased bioreactor system in sugarcane. This study details the initial steps taken towards the development of a virus-based episomal amplification system in sugarcane. In doing so, fundamental knowledge into the mechanisms involved in Rep recognition of viral regulatory DNA sequences has been gathered. These research findings will provide a solid foundation for the further development of a sugarcane-based bioreactor.
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Šmíd, Jiří. "Vývoj protokolu pro transientní transfekci buněčné linie HEK293 EBNA1." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2009. http://www.nusl.cz/ntk/nusl-216484.
Full textAbstract:
Recombinant proteins belong to considerable biofarmaceutics products used in biomedical research and in the treatment of human disease. Recombinant protines can be produced by stable transfection in big amount or by faster transient transfection with smaller amounts. To provide regular biological activity, it is necessary for the protein to be properly folded and post-translationally modified. As these modifications can be accurately performed only in mammalian cells, they have become the major host for complex r-protein expression. In this thesis is described transient transfection HEK 293 EBNA1 cells with linear polyethylenimines. These cells has been adapted to suspension cultivation in serum free medium. The cells were transfected with pcDNA3.1, pCI, pEBSV1, pCEP4, pEAK8 a pcDNA5/FRT/TO plasmids, everyone contained repoter gene SEAP. Concentration of SEAP in cell culture supernatants were determined in order to compare efficiencies of individual transfections. DNA:PEI ratio was another factor which was optimised and two different PEIs were compared. Highest achieved expresion was 50 mg per litre with transfection in 24 well plate when DNA:PEI ratio was 1:5. Comparison of six different plasmids give the bigest expresion pCEP4/SEAP, in well plate as well as in scaled up system.
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Nehlsen, Kristina. "Molekulare Grundlagen der episomalen Replikation Charakterisierung zirkulärer, nichtviraler Vektoren /." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971750637.
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La, Bella Tiziana. "Adeno-associated virus in the liver : natural history of the infection and consequences in tumor development." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC263.
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Le virus adéno-associé (AAV) est un virus à ADN monobrin, défectif et endémique dans la population humaine. L'infection à AAV a longtemps été considérée comme non pathogénique. Cependant, il y a quelques années, nous avons identifié pour la première fois des insertions clonales récurrentes d'AAV2 impliquées dans la pathogenèse du carcinome hépatocellulaire humain (CHC) développé sur un foie normal. Ces insertions virales clonales ciblent des oncogènes et entraînent leur surexpression. À ce jour, peu de travaux ont étudié l'infection à AAV de type sauvage dans le foie humain. Dans ce travail, nous avons étudié l'histoire naturelle de l'infection virale dans les tissus hépatiques et ses conséquences sur le développement de tumeurs dans une vaste cohorte de patients (n = 1464). La présence de l'AAV est observée chez 21% des patients, plus fréquemment dans la contrepartie non-tumorale (18%) que dans la tumeur (8%) et significativement enrichie chez les femmes, les patients jeunes et non cirrhotiques. Deux sous-types d’AAV ont été identifiés dans le foie, l’AAV2 classique et un génotype hybride AAV2-AAV3-AAV13, avec une fréquence égale dans notre cohorte. Nous avons détecté la présence de formes épisomales d'AAV dans 27% des tissus non tumoraux positifs pour l'AAV, associée de manière significative à l'expression de l'ARN viral et à la co-infection par des virus auxiliaires, suggérant une infection active en cours. Nous avons identifié le virus de l'herpès humain de type 6 (HHV6) comme étant le virus auxiliaire naturel de l'AAV dans le foie. En revanche, l'ADN de l'adénovirus n'a été détecté que chez 0,5% des patients et aucune association avec l'AAV n'a été constatée. Nous avons confirmé la sélection positive des insertions d'AAV clonales au cours du développement du CHC chez les patients sans cirrhose dans 2% des tumeurs ciblant CCNA2, CCNE1, TERT, TNFSF10, KMT2B et INHBE / GLI1. De plus, les altérations de CCNA2 et de CCNE1 dues à des insertions virales d'AAV et de VHB ou à des réarrangements structurels ont défini une nouvelle sous-classe de CHC (CCN-HCC) et un nouveau mécanisme de développement du CHC sur le foie normal, améliorant nos connaissances sur l'hépatocarcinogénèse sur le foie non cirrhotique. Les CCN-HCC présentent également des caractéristiques moléculaires particulières qui pourraient être ciblées par un traitement spécifiqueAdeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population. AAV infection has long been considered as non-pathogenic, however few years ago we reported for the first time recurrent clonal AAV2 insertion in the pathogenesis of human hepatocellular carcinoma (HCC) developed on normal liver. These clonal viral insertions target cancer driver genes leading to their overexpression. To date, little is known about wild type AAV infection in human liver. In this work we investigated the natural history of the viral infection in the liver tissues and the consequences in tumor development in a large cohort of patients (n=1464). The presence of AAV was observed in 21% of patients, more frequently in the non-tumor counterpart (18%) than in tumor (8%) and significantly enriched in young, female and non-cirrhotic patients. Two AAV subtypes were identified in the liver, the classical AAV2 and a hybrid AAV2-AAV3-AAV13 genotypes, with an equal frequency in our cohort. We detected the presence of episomal AAV forms in 27% of AAV positive non-tumor tissues significantly associated with viral RNA expression and co-infection with helper viruses suggesting an ongoing active infection. We identified human herpes virus type 6 (HHV6) as the natural AAV helper virus in the liver. In contrast, adenovirus DNA was detected in only 0.5% of patients and no association with AAV was found. We confirmed the positive selection of clonal AAV insertions during HCC development in patients without cirrhosis in 2% of tumors targeting CCNA2, CCNE1, TERT, TNFSF10, KMT2B and INHBE/GLI1. Moreover, the alterations in CCNA2 and CCNE1 due to viral insertions of AAV and HBV or structural rearrangements defined a new subclass of HCCs (CCN-HCC) and a novel mechanism of HCC development on normal liver improving our knowledge on hepatocarcinogenesis on non-cirrhotic liver. CCN-HCCs display also peculiar molecular features that could be targeted by specific treatment
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Ziegler, Manja. "Vergleichende Untersuchung der regulierten Genexpression von integrierter und episomaler HIV-1 LTR." Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-69127.
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Vitone, Francesca <1973>. "Tecniche di biologia molecolare per la determinazione quantitativa di HIV-DNA (integrato ed episomale) in soggetti HIV-1 infetti." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/76/.
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Lima, Daniel Chaves de. "Characterization of RadA/Sms from Chromobacterium violaceum and discovery of a new episome." PROGRAMA DE P?S-GRADUA??O EM BIOQU?MICA, 2016. https://repositorio.ufrn.br/jspui/handle/123456789/22058.
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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq)
Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)
Chromobacterium violaceum is a ?-proteobacteria commonly found around tropical and subtropical regions throughout the globe. It produces many metabolites with biotechnological properties such as antitumoral peptides, antibiotics and polymers that have potential to replace the oil-based ones. Although it has been extensively studied over the past 40 years, there are many aspects of C. violaceum that remains unclear until today. We have conducted a biochemical study on the homologous recombination (HR) machinery of C. violaceum, mainly in RecA and its paralog, RadA/Sms. We performed in vitro assays from initial and late steps of HR such as D-loop formation and branch migration, respectively, with their corresponding molecular actors and how RadA/Sms influenced each one. We observed cvRadA/Sms influences negatively D-loop formation promoted by cvRecA and through pull-down assay we have observed an interaction between these two proteins. We also observed the DNA-binding preference of cvRadA/Sms and cvRecA and observed that this protein binds preferentially to dsDNA instead ssDNA, unlike cvRecA. No involvement of cvRadA/Sms on branch migration reactions was detected. In this work, we also described, for the first time, the isolation, sequencing and annotation of a new plasmid from C. violaceum, which we named ChVi1 and has 44,236 base pairs, 39 predicted open reading frames (ORFs) and, possibly, two origins of replication. Most of the ORFs codes for hypothetical and structural bacteriophage proteins. By using restriction digestion and Next-generation sequencing (NGS) we also looked for the presence of a similar plasmid in other seven C. violaceum strains isolated from amazon region. Our analysis suggest the presence of a plasmid similar to ChVi1 in two of these strains. The present work describes for the first time a biochemical characterization of RadA/Sms and RecA from C. violaceum which have different roles in HR. Moreover, the discovery of ChVi1 opens a path to further explore C. violaceum?s biology.
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Brown, Hannah Frances. "Generation of an HVS-based episomally maintained gene delivery system for reprogramming adult somatic cells." Thesis, University of Leeds, 2012. http://etheses.whiterose.ac.uk/4445/.
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iPSC technology has the potential to generate patient specific pluripotent cells for use in stem cell therapies and disease modelling. However, current reprogramming methods utilise retroviral vectors, which integrate into the host cell genome disrupting normal gene function. Transient gene delivery methods have been investigated as safer alternatives but demonstrate poor reprogramming efficiency. Therefore, there is a requirement for iPSC gene-delivery vectors which are capable of providing prolonged transgene delivery without integrating into the host cell genome. Herpesvirus saimiri (HVS) is a prototype member of the gamma-2 Herpesviridae, and is capable of persisting as a non-integrated episome in dividing and differentiating cell populations, providing sustained transgene expression. Therefore, this thesis has explored the potential of HVS-based vectors for iPSC generation. This thesis focuses on the generation of three HVS-based vectors expressing the iPSC reprogramming transgenes, Oct4, Lin28 and Nanog. The potential of these HVS-iPSC vectors to reprogram both primary and cancerous cells has been investigated. Human primary Neural Stem Cells demonstrated cytopathology upon transduction with HVS-based vectors, indicating the need for further improvements to the biosafety of these vectors before they are suitable for the generation of clinical grade iPSCs. However, reprogramming attempts utilising the Ewing‟s sarcoma cell line, A673 cells, successfully generated induced pluripotent cancer stem cell (iPC)-like colonies upon transduction with all three recombinant vectors. Results from detailed analysis of these colonies suggest some form of reprogramming has taken place, albeit incomplete, as indicated by elevated expression of Oct4, Rex1 and Klf4; in addition to positive alkaline phosphatase staining and SSEA4 expression. Furthermore, these iPC-like colonies were capable of differentiation down the ectodermal lineage, as evidenced by upregulation of MSX1, MAP2, and Nestin. In conclusion, this thesis has demonstrated the potential of HVS-based reprogramming vectors through the generation of A673-iPCs.
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Nicolaides, L. "Interactions of the human papillomavirus E6 protein and their role in the persistence of viral episomes." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1333251/.
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The E6 protein from high-risk Human Papillomaviruses (HPVs) has previously been shown to be necessary for the persistence of viral episomes in cells, however, the mechanism for this remains unclear. High-risk E6 proteins have many activities including the ability to degrade p53 and the ability to bind to and degrade PDZ proteins. In this study I aimed to further elucidate the role of E6 in the persistence of viral episomes. I used two HPV16 mutant genomes with mutations in the E6 open-reading frame; one that is unable to degrade p53 (16E6p53m), and one that lacks the PDZ-binding motif (16E6PDZ). I found that both are unable to persist episomally in cells thereby implicating these two activities of E6 in HPV episomal persistence. Upon closer investigation of the two mutant genomes, I found that the 16E6p53m genome does not replicate as efficiently as the wild-type genome. This result suggests a function for p53-degradation in genome replication, and consequently in genome persistence. Furthermore, by carrying out a more detailed analysis of the relationship between E6 and the PDZ protein hScrib, I showed that the wild-type E6 protein is stabilised by virtue of the PDZ-binding motif, present on its C-terminus. On the other hand, the mutant E6 protein that lacks the PDZ-binding motif (E6PDZ) is more susceptible to proteasomal degradation. These findings provide evidence for a previously unknown outcome of the E6-PDZ protein interaction, in stabilising wild-type E6 protein. In addition to the implications of this stabilisation in the persistence of viral episomes, it is also significant when considering the activities and properties of E6 that contribute to the development of neoplasia. Finally, I have also found that wild-type HPV16 genomes cannot persist in cells that constitutively express E6 protein, suggesting that the correct regulation of E6 expression is crucial.
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Jensen, Ryan Lee. "Molecular Characterization of Adeno-Associated Virus in the Natural Host." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1211229929.
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Broll, Sandra Verena Ursula [Verfasser], and Jürgen [Akademischer Betreuer] Bode. "Selbstreplizierende nicht-virale Episomen (Minicircles): Expressionseigenschaften und Etablierung im Zellkern / Sandra Verena Ursula Broll ; Betreuer: Jürgen Bode." Braunschweig : Technische Universität Braunschweig, 2009. http://d-nb.info/1175829501/34.
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Wade-Martins, Richard. "Developing Epstein-Barr virus-based stable episomes for gene expression from large genomic inserts to complement cell phenotypes." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301648.
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Piolot, Tristan. "Analyse des interactions de la protéine EBNA1 du virus Epstein-Barr et des chromosomes cellulaires : proposition d'un nouveau modèle de persistance des épisomes viraux." Paris 7, 2001. http://www.theses.fr/2001PA077233.
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Deschamps, Thibaut. "Rôle de l'interaction entre la protéine virale EBNA1 et le facteur cellulaire RCC1 dans la persistance du génome du virus d'Epstein-Barr." Thesis, Lyon, École normale supérieure, 2015. http://www.theses.fr/2015ENSL1011/document.
Full textAbstract:
Le virus d’Epstein-Barr (EBV) est un herpesvirus dont la séroprévalence est d’environ 90 % de la population adulte mondiale. EBV est associé à de nombreuses pathologies tumorales. La primo infection conduit à l’établissement du virus sous forme latente dans les lymphocytes B mémoires. Au sein de ces cellules B, le génome viral est sous la forme d’un épisome, un ADN circulaire double brin, et une fraction restreinte de gènes viraux est exprimée. Afin de se maintenir aux cours des divisions cellulaires, le génome viral est répliqué en phase S par la machinerie cellulaire et ségrégé lors de la mitose dans chaque cellules filles. La réplication et la ségrégation du génome viral nécessitent 2 facteurs viraux que sont la protéine virale EBNA1 (Epstein-Barr Nuclear Antigen 1) et la région oriP sur le génome viral. En phase S, EBNA1 interagit directement avec l’oriP et y recrute le complexe de pré-réplication de l’ADN. En mitose, EBNA1 ancre l’épisome à la chromatine ce qui permet une ségrégation efficace. Les mécanismes d’interaction entre EBNA1 et la chromatine reste encore flou. Au cours de notre travail, nous avons identifié la protéine RCC1 comme un partenaire potentiel pour la protéine EBNA1 pouvant être impliqué dans l’ancrage d’EBNA1 à la chromatine. Nous avons validé cette interaction et caractérisé les régions d’interactions pour ces deux protéines. Par ailleurs nous avons démontré que RCC1 est recrutée sur l’oriP en présence d’EBNA1 et que ces deux protéines interagissent en mitose. À la lumière de nos résultats et des données de la littérature, nous proposons que l’interaction d’EBNA1 avec la chromatine est dynamique et implique à la fois des interactions directes (AT-Hook, interaction avec les nucléosomes) mais aussi des facteurs cellulaires (RCC1, EBP2 et HMGB2)Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with several human cancers. In proliferating latently-infected cells, the EBV genome persists as a circular plasmid that is replicated once per cell cycle and partitioned at mitosis. Both of these processes require a single viral protein, Epstein Barr nuclear antigen 1 (EBNA1), which binds to two clusters of cognate binding sites within the origin of plasmid replication (oriP). EBNA1 plays an essential role both in viral episome replication, by recruiting the cellular complex of DNA replication onto the oriP, and in the efficient segregation of the viral episomes, by tethering the viral DNA onto the mitotic chromosomes. Whereas the mechanisms of viral DNA replication have been well documented, the mechanisms involved in tethering EBNA1 to the cellular chromatin are far from being understood. Here we have identified Regulator of Chromosome Condensation 1 (RCC1) as a novel EBNA1 cellular partner. RCC1 is the only known nuclear guanine nucleotide exchange factor (RanGEF) for the small GTPase Ran enzyme. RCC1, associated with chromatin, is involved in the formation of RanGTP gradients critical for nucleo-cytoplasmic transport, mitotic spindle formation, and nuclear envelope reassembly after mitosis. We have used several approaches to demonstrate a direct interaction between these two proteins and to identify the regions. involved Moreover, by using Chromatin ImmunoPrecipitation assay (ChIP) we have shown that RCC1 is enriched in the oriP region of mini viral replicons in a manner dependent on EBNA1. Finally, by using a combination of confocal microscopy and FRET analysis to follow the dynamics of interaction between the two proteins throughout the cell cycle, we have demonstrated that EBNA1 and RCC1 closely associate on the chromosomes during metaphase. Taken together, our data strongly suggest an essential role for RCC1 in tethering EBNA1 - linked to the viral episome - to the metaphasic chromosomes. Our results and those of others lead us to the idea that the interaction between EBNA1 with the cellular chromosomes requires several factors such as direct interactions or cellular proteins and these interactions are complementary and / or redundant
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Huang, Nien-Chen, and 黃念貞. "Episomal replication of human papillomavirus type 16 from a cerv- vical carcinoma cell line CC7T." Thesis, 1994. http://ndltd.ncl.edu.tw/handle/69039474505063588037.
Full textAbstract:
碩士國立師範大學
生物學研究所
82
Observing the replication of human papillomavirus is difficult because of lacking an in vitro system to support a stable episomal viral DNA replication. In 1980, Dr.C. P. Hu established a cervical carcinoma cell line CC7T, which contains both episomal and integrated HPV 16 DNA. Compare the DNA sequences with the prototype HPV 16 DNA, there are point mutations in both E1 andE7 ORFs in epi some, and these two genes are responsible for viral DNA replication. In order to prove whether these mutations could effect the existence of episomal DNA in CC7T, we transfected the cloned episome (pCC7T-BS) and the prototype HPV 16 DNA (pHPV-BS) into re lated cervical carcinoma and larynx carcinoma cell lines, HeLa and Hep-2, respectively. Our results showed that there were neither apparent changes in morphology nor cell death in transfected cells except pCC7T-BS transfected cell line HeLa. In HeLa ， t wo days after transfection with pCC7T-BS ， the growth of the cells delayed and the cells appeared to die 。 On the other hand ， the replication patterns of both pCC7T-BS and pHPV-BS were similiar in HeLa and Hep-2. The amount of DNA were papillomavirus replication reduced gradually a Most of the informations about nd there were no apparent DNA replication in both cell lines. It indicated that the mutations occured in episome might not as important as we expected. come from researches of the bovine papillomavirus type 1.
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Loh, Yuen Yung, and 羅源榮. "The Potential of Episomal Induced Pluripotent Stem Cell Therapy on Peripheral Nerve Regeneration in Vascularized Composite Allotransplantation." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/g8974n.
Full textAbstract:
碩士長庚大學
顯微手術國際碩士學位學程
104
Since the discovery of the capability of somatic cells to be reprogrammed into pluripotent stem cells or what is termed induced pluripotent stem cells, the potentials that these pluripotent stem cells provide is endless. Pluripotent stem cells possess the ability to unrestrictedly differentiate into any somatic cell in the body. As such, they are able to regenerate a whole host of cells in the body. This thesis aims to investigate and review the potential capabilities of iPSCs in accelerating the recovery of peripheral nerves. Peripheral nerves when severed, undergo Wallerian degeneration which is a time dependent process. The longer the time taken to recovery, the poorer the functional outcome due to the wasting and degeneration of motor end plates at the end target muscle organ. The various options used in the generation of iPSCs are reviewed in this thesis and will provide an up to date insight into the current methods for cell reprogramming and their individual pros and cons. Lastly, we explore the potential of iPSCs in the regeneration of peripheral nerves in a murine model which mimics clinical scenarios experienced and the functional assessment of recovery in mice.
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Lin, Deng-Yi, and 林登藝. "A Basic Study of the Replication of Human Papillomavirus Type16 Episomal DNA from a Cervical Carcinoma Cell Line CC7T/VGH." Thesis, 1995. http://ndltd.ncl.edu.tw/handle/50895382668459957644.
Full textAbstract:
碩士國立師範大學
生物學研究所
83
HPV16 基因體常在子宮頸癌檢體中被檢出，具有游離(episome)及插入兩種 形式，但其無法在體外培養，癌細胞衍出的細胞株又多只具插入形式HPV16 DNA使其複製和寄主同步，故其複製研究不易。1980年榮總胡承波博士建立 一子宮頸癌細胞株-CC7T/VGH，同時具有游離及插入形式HPV16 DNA ，其游 離基因體之E1、E7及L1皆發生突變，為研究HPV16複製的理想材料，本實驗 即將直線狀之重組HPV16 prototype或/及由CC7T/VGH選殖之游離基因體DNA 轉染至另一子宮頸癌(HeLa)或咽喉癌(HEp-2)細胞株中觀察其複製情形，以 判別CC7T中HPV16 episome之存在是否為episome DNA 本身的突變所造成。 實驗結果發現轉染五天內，細胞皆無明顯生長遲滯情形，形態亦正常，而 不論轉染重組HPV16 prototype、游離基因體DNA或二者，HPV16 DNA在HeLa 及HEp-2細胞中的量皆隨時間的增加而逐漸減少，並無複製現象，故HPV16 游離基因體本身的變異應不是其穩定存在CC7T/VGH細胞中的主因。將 CC7T/VGH抽取出來的低分子量DNA以限制酵素BamHI切割，可觀察到7.7kb的 HPV16游離基因體DNA片段及一段約3.1kb大小的DNA片段，此3.1kb片段經不 同限制酵素切割，得知其上至少有EcoRI、KpnI及PstI的切割區。 Human papillomavirus (HPV) type16 genome is usually detected from the biopsies of patients who carry cervical intraepithelial neoplasma (CIN). Both episomal and integrated forms of HPV16 genome have been found in these cells but viruses can not be produced when cultured in vitro. Most cell lines derived from CIN are found to have only integrated form of HPV16 DNA and that makes the integrated HPV16 DNA replicating synchronously with the host cell, thereby, it becomes difficult to study the independent replication of HPV16 DNA in this regard. In 1980, Dr. C. P. Hu established a cervical carcinoma cell line CC7T/VGH, which contains both episomal and integrated HPV16 DNA. The episomal DNA in the CC7T/VGH cell contains mutations in its E1, E7 and L1 open reading frames and can be a good system for comparative study the replication of HPV16 genome. We transfected the linear recombinant HPV16 prototype or episomal (cloned from CC7T/VGH)DNA into other cervical carcinoma (HeLa) or larynx carcinoma (HEp-2) cell lines to observe the consequence of transfected HPV16 genome . Our results showed that the morphology and growth rate did not change distinctively in all transfected cells. The amount of HPV16 DNA was gradually decreased after transfection for 5 days when recombinant HPV16 prototype or episomal DNA or both were tranfected. No any HPV16 DNA replication in HeLa or HEp-2 was shown which indicated that the mutation of episome might not play an important role for existence of episomal HPV16 DNA in the CC7T/VGH cell. We also observed an unusual DNA fragment from the BamHI restriction endonuclease digestion of low molecular weight DNA of the CC7T/VGH cell. The fragment is about 3.1kb and has EcoRI, KpnI and PstI recognition sites. The significant existence of this small fragment is currently under investigation.
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Μπαβέλη, Μαρία. "Ανάπτυξη επισωματικού φορέα για τη γονιδιακή μεταφορά του φυσιολογικού γονιδίου της β-σφαιρίνης." Thesis, 2008. http://nemertes.lis.upatras.gr/jspui/handle/10889/933.
Full textAbstract:
Η γονιδιακή θεραπεία θεωρείται μια πολλά υποσχόμενη προσέγγιση για την αντιμετώπιση διαφόρων ασθενειών, καθώς για αρκετές από αυτές δεν υπάρχει ουσιαστική θεραπεία παρά μόνο αντιμετώπιση των συμπτωμάτων. Οι αιμοσφαιρινοπάθειες ως μονογονιδιακές ασθένειες έχουν θεωρηθεί εδώ και πολλά χρόνια εξαιρετικά μοντέλα ασθενειών για τη γονιδιακή θεραπεία. Επιπλέον, ο μοριακός μηχανισμός της νόσου είναι από τους καλύτερα μελετημένους. Οι στρατηγικές που χρησιμοποιούνται στις προσπάθειες γονιδιακής θεραπείας των αιμοσφαιρινοπαθειών περιλαμβάνουν:
Μεταφορά του φυσιολογικού γονιδίου β, κυρίως σε αρχέγονα αιμοποιητικά κύτταρα, και
Προσπάθεια ενεργοποίησης των γ γονιδίων, καθώς ασθενείς με β- θαλασσαιμία και κληρονομική παραμονή της εμβρυϊκής αιμοσφαιρίνης παρουσιάζουν σχεδόν φυσιολογικό φαινότυπο.
Όσον αφορά τη γονιδιακή μεταφορά, αυτή γίνεται κατά κύριο λόγο με ιϊκά συστήματα. Ωστόσο, οι φορείς αυτοί παρουσιάζουν ορισμένα μειονεκτήματα με κυριότερα την πρόκληση ανοσολογικών αντιδράσεων και την in vitro μεταλλαξιγένεση λόγω ενσωμάτωσης. Για το λόγο αυτό, τα τελευταία χρόνια οι έρευνες έχουν στραφεί στην κατασκευή επισωματικών φορέων που θεωρούνται γενικά πιο ασφαλείς καθώς δεν ενσωματώνονται στο γενετικό υλικό των κυττάρων. Σημαντικό βήμα στην ανάπτυξη επισωματικών φορέων για την γονιδιακή θερπαπεία των αιμοσφαιρινοπαθειών, επιτεύχθηκε με την κατασκευή του φορέα pEPI-eGFP ο οποίος έχει την ικανότητα να διατηρείται σε επισωματική κατάσταση χάρη στην αλληλουχία S/MAR που περιέχει και η οποία προέρχεται από το 5΄ άκρο του ανθρώπινου γονιδίου της ιντερφερόνης β. Επιπλέον, έρευνες στο παρελθόν έχουν δείξει ότι το mini LCR της β-σφαιρίνης είναι απαραίτητο για την υψηλή και ιστο-ειδική έκφραση του γενετικού τόπου της β-σφαιρίνης.
Σκοπός της παρούσας εργασίας είναι η κατασκευή ενός επισωματικού φορέα που θα βασίζεται στον φορέα pEPI-eGFP και ο οποίος αποτελεί ενδιάμεσο στάδιο για την κατασκευή ενός κοσμιδιακού φορέα που θα περιλαμβάνει το mini locus της β-σφαιρίνης.Gene therapy is considered many promising approach for the confrontation of enough various illnesses, while for from them does not exist essential treatment only that confrontation of symptoms. Haemoglobinopathies as monogenic disorders have been considered for many years exceptional models for gene therapy. Moreover, the molecular mechanism of haemoglobinopathies is very well studied. The strategies that are used in the efforts of gene therapy haemoglobinopathies include: Transport of physiologic gene b, mainly in original stem cells, and Effort of activation of γ-globin genes, since patients with b thalassaemia and hereditary presence of embrionic haemoglobin show almost physiologic phrenotype. With regard to the pertaining to genes transport, this becomes in the first place with virus systems. However, this vectors present certain disadvantages with mainly the challenge of immunological reactions and in vitro metallaxigenesi because incorporation. For this reason, in the past few year the researches have been turned in the manufacture of episomal vectors that is considered in general sure while is not incorporated in the genetic material of cells. Important step in the growth of episomal vectors for gene therapy of haemoglobinopathies, was achieved with the manufacture of pEPI-eGFP which has the faculty to be maintained in episomal situation thanks to the concatenation S/MAR that it contains and which is emanated from the 5΄ of the human gene of interferon β. Moreover, researches in the past have shown that mini LCR of b-globin is essential for high and specific expression of genetic place of b-globin. Aim of present work is the manufacture of episomal vector that will be based on the institution pEPI-eGFP and which constitutes intermediary stage for the manufacture of cosmidial vector that will include the mini locus b-globin.
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Λάζαρης, Βασίλειος. "Μελέτη της γονιδιακής μεταφοράς επισωματικών φορέων σε αρχέγονα αιμοποιητικά κύτταρα." Thesis, 2010. http://hdl.handle.net/10889/4967.
Full textAbstract:
Στη γονιδιακή θεραπεία, οι κλινικές μελέτες μέχρι τώρα χρησιμοποιούν ιϊκούς φορείς για την μεταφορά του διαγονιδίου στα κύτταρα στόχους. Το DNA των ιϊκών φορέων ενσωματώνεται στο γενετικό υλικό των κυττάρων, και αυτό εμπεριέχει τον μεγάλο κίνδυνο της παρεμβολής στο ενδογενές πρόγραμμα γονιδιακής έκφρασης (μεταλλαξιγένεση λόγω ένθεσης). Μια λύση σε αυτή την ανεπιθύμητη κατάσταση είναι η χρήση επισωματικών φορέων και ιδιαίτερα όσων φέρουν χρωμοσωμικά στοιχεία. Παλαιότερα είχε αναφερθεί ότι ο πρότυπος επισωματικός φορέας pEPI που βασίζεται στο Scaffold /Matrix Attachment Region (S/MAR), παραμένει ως σταθερό επίσωμα για πολλές γενεές σε κυτταρικές σειρές ανθρώπου και ποντικού, αλλά δεν παραμένει για πολλές γενεές σε ανθρώπινα CD34+ κύτταρα. Για να ενισχυθεί η ικανότητα του φορέα να υποστηρίξει την γονιδιακή μεταφορά και συγκράτηση του σε πρωτογενή αρχέγονα/προγονικά αιμοποιητικά κύτταρα, πρώτον ενισχύθηκε η μεταγραφή του S/MAR χρησιμοποιώντας τους ισχυρούς υποκινητές EF1/HTLV ή SFFV για το διαγονιδίο της eGFP και δεύτερον προστέθηκε μια αλληλουχία έναρξης της αντιγραφής (IR) από το γενετικό τόπο των β σφαιρινικών γονιδίων. Στην εργασία αυτή έγινε μεταφορά των νέων αυτών φορέων με την μέθοδο της πυρινικής ηλεκτροδιάτρησης σε κύτταρα CD34+ που απομονώθηκαν από κινητοποιημένο περιφερικό αίμα δοτών μυελού των οστών τα οποία διαμόλυναν επιτυχώς. Στην συνέχεια τα διαμολυσμένα κύτταρα CD34+ επιλέχθηκαν με FACS και καλλιεργήθηκαν σε θρεπτικό υλικό μεθυλοκυτταρίνης. Μετά την πάροδο 14 ημερών, ανιχνεύτηκε, με μικροσκοπία φθορισμού, έκφραση της eGFP στις τελικά διαφοροποιημένες αιμοποιητικές αποικίες που προέκυψαν.Gene therapy clinical trials are currently based on integrating viral vectors; this approach presents the major risk of insertional mutagenesis. A solution to this side effect could be the use of episomal vectors and particularly the ones carrying chromosomal elements.We previously reported that the prototype episomal vector pEPI, based on a Scaffold /Matrix Attachment Region (S/MAR), functions as a stable episome for many generations in human and murine hematopoietic cell lines, but mediates very low long term retention in human CD34+ cells. To enhance the vector’s potential for gene transfer into primary hematopoietic stem/progenitor cells, (a) was enforced transcription through the S/MAR by using the strong hybrid EF1/HTLV or SFFV promoters to drive expression of the upstream transgene (eGFP) and (b) was included the replication initiation region (IR) from the β-globin gene locus. In this thesis the new vectors where delivered by nucleofection in CD34+ cells isolated from mobilized peripheral blood of healthy donors; th cells were efficiently transfected. Moreover the the transfected CD34+ cells were separated with FACS and cultured in methylocyttarine containing medium. After 14 days, eGFP expression was readily detected by fluorescence microscopy in the differentiated hematopoietic colonies.
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Σταύρου, Ελεάνα. "Ενεργοποίηση του γονιδίου της γ-σφαιρίνης του ανθρώπου με επισωματική μεταφορά συνθετικού ενεργοποιητή." Thesis, 2010. http://nemertes.lis.upatras.gr/jspui/handle/10889/4355.
Full textAbstract:
Η αύξηση της έκφρασης του γονιδίου της γ-σφαιρίνης και κατ’ επέκταση και της εμβρυικής αιμοσφαιρίνης (HbF), μέσω ενεργοποίησης με φαρμακολογικούς παράγοντες ή μεταφοράς του γονιδίου της γ-σφαιρίνης, αποτελούν σημαντικές στρατηγικές για την θεραπεία της δρεπανοκυτταρικής και μεσογειακής αναιμίας. Καινοτομία αποτελεί η δημιουργία ενός ειδικού συνθετικού ενεργοποιητή της γ-σφαιρίνης, του Zif-VP64, με δομή δακτύλων ψευδαργύρου, ειδικά σχεδιασμένη για πρόσδεση σε αλληλουχία 18bp, περί την θέση -117HPFH του υποκινητή της γ-σφαιρίνης. Επιδίωξη της εργασίας αυτής ήταν η ανάπτυξη ενός μη ιϊκού, επισωματικού φορέα, που φέρει τον συνθετικό ενεργοποιητή του γονιδίου της γ-σφαιρίνης, ικανού να λειτουργεί με επάρκεια σε κύτταρα του αιμοποιητικού ιστού.
Η φορέας αυτός, Zif-VP64-Ep1, περιλαμβάνει τον ενεργοποιητή της γ-σφαιρίνης και την μεταγραφική κασέτα CMV-eGFP-S/MAR, για την εξασφάλιση της επισωματικής του κατάστασης μέσω του στοιχείου S/MAR. Τα αποτελέσματά μας δείχνουν ότι ο φορέας Zif-VP64-Ep1: i. διαμολύνει επιτυχώς, K562 κύτταρα σε ποσοστό 45% παραμένοντας σε επισωματική κατάσταση και υποστηρίζοντας έκφραση του διαγονιδίου για τουλάχιστον 200 γενεές, προγονικά κύτταρα μυελού τον οστών ποντικού β-YAC BMCs σε ποσοστό 23% και CD34+ κύτταρα περιφερικού αίματος κινητοποιημένου υγιούς δότη σε ποσοστό 22,5%. ii. ο φορέας Zif-VP64-Ep1 υποστηρίζει σημαντική αύξηση των επιπέδων της γ-σφαιρίνης κατά 3.3±0.2 φορές σε Κ562 και 3.0±1 φορές σε CD34+ κύτταρα, και ενεργοποίηση του ανενεργού γονιδίου γ-σφαιρίνης σε κύτταρα β-YAC BMCs. Επιτυγχάνεται έτσι, για πρώτη φορά, επισωματική γονιδιακή μεταφορά του ενεργοποιητή Zif-VP64 και trans-ενεργοποίηση του γ-γονιδίου σε επίπεδο θεραπευτικής σημασίας.The increase of HbF through activation of gamma-globin gene is a valid strategy for the treatment of hemoglobinopathies. Zif-VP64 is a selective, synthetic gamma-globin activator, containing a zinc-finger DNA protein that binds the gamma-globin promoter -117HPFH area and a transcription inducer that induces gamma globin gene in K562 cells, after viral transfer. We report the study of an episomal vector of this activator, which is based on a Scaffold/Matrix attachment region (S/MAR) that supports retention of episomes in the nucleus of the host cell. We constructed an episomal vector, Zif-VP64-Ep1, containing the activator Zif-VP64, the reporter gene cassette CMV-eGFP and the S/MAR element. Gene transfer into cells was done by electroporation or nucleofection. Expression of eGFP was documented by Florescent Microscopy and Flow Cytometry, while the fate of vector molecules in the cells was studied by Southern Blot and plasmid rescue experiments. Real time PCR, Western blotting and Intracellular Flow Cytometry were used to investigate gamma-globin mRNA, gamma-globin protein and HbF protein levels respectively. Binding specificity of the activator was determined by Chromatin Immunoprecipitation (ChIP). Gene transfer was done in K562 cells producing long term stable cell lines; murine beta-YAC cells, where the YAC contains the complete human, beta-globin gene locus; and human progenitor hemopoietic CD34+ cells from healthy, mobilized individuals, with transfection efficiencies of 65%, 25% and 23% respectively. In K562 cells, gamma-globin mRNA levels showed an increase of 250%, gamma-globin protein of 350% and HbF protein of 165%, as compared to the corresponding levels in the untransfected K562 cells, at least 200 generations post-transfection. Interestingly, vector Zif-VP64-Ep1 was able to mediate the activation of expression of the silent, human gamma-globin gene of the murine beta-YAC cells, at a level matching the (active) human beta-globin gene of the YAC, as well as the murine beta-globin gene, showing that it can efficiently activate the gamma-globin gene from within a heterochromatic region. Finally and most significantly, vector Zif-VP64-Ep1 was able to transfect the human, hemopoietic progenitor CD34+ cells and to mediate a 3.0±1 fold increase of gamma globin mRNA, compared to untrasnfected CD34+ cells, as estimated in cultures of 7-8 days after transfection. In conclusion, activation of human gamma-globin by episomal gene transfer of a synthetic activator, in three different hemopoietic cells, is documented, including the CD43+ cells, that are the target cells for gene therapy of the Hemoglobinopathies. This is the first time that an S/MAR based episomal vector is used for gene transactivation in a cell line and progenitor cells, aiming at specific gene therapy.
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Γιαννακόπουλος, Αριστείδης Π. "Ανάπτυξη και εκτίμηση του δυναμικού επισωματικών και ιϊκών φορέων για την γονιδιακή μεταφορά σε κυτταρικά συστήματα και σε κλινικές δοκιμές στην γονιδιακή θεραπεία." Thesis, 2006. http://nemertes.lis.upatras.gr/jspui/handle/10889/1385.
Full textAbstract:
Οι επισωματικοί φορείς αποτελούν την εναλλακτική επιλογή για την μεταφορά
γονιδίων σε εφαρμογές γονιδιακής θεραπείας. Διπλασιάζονται αυτόνομα χωρίς να
ενσωματώνονται στα χρωμοσώματα του κυττάρου και έτσι στερούνται της σοβαρής
παρενέργειας του φαινομένου της μεταλλαξιγένεσης μέσω ενσωμάτωσης. Το
ενδιαφέρον ως προς την ανάπτυξη επισωματικών φορέων για την γονιδιακή μεταφορά
αυξήθηκε κατακόρυφα τα τελευταία χρόνια μετά την ανάπτυξη του φορέα pEPI-1, ο
οποίος περιείχε το στοιχείο S/MAR του γονιδίου της ανθρώπινης βήτα- ιντερφερόνης. Ο
φορέας αυτός είχε την ιδιότητα να διατηρείται επισωματικά χωρίς την ανάγκη
μεταγραφής κάποιου ιϊκού γονιδίου. Τα στοιχεία S/MAR αποτελούν ετερογενείς
περιοχές DNA οι οποίες συνδέονται στην θεμέλια ουσία του πυρήνα, συμμετέχοντας
στην οργάνωση του ευκαρυωτικού γονιδιώματος, στην αντιγραφή του DNA και στην
ρύθμιση της μεταγραφής. Αποφασίσαμε στην μελετήσουμε την δράση του S/MAR
στοιχείου σε ένα διαφορετικό περιβάλλον DNA. Ξεκινώντας από τον φορέα pCEP4 που
βασίζεται στα στοιχεία OriP και EBNA-1 του ιού η EBV κατασκευάσαμε τα εξής
πλασμίδια: 1) pEBS/eGFP με το S/MAR να είναι κλωνοποιημένο μετά το γονίδιο της
eGFP όπως στο pEPI-1. 2) pESdER / eGFP προερχόμενο από το pEBS/eGFP με
διαγραφή του γονιδίου EBNA-1 και.3) pCEP4 / eGFP (πλασμίδιο μάρτυρας). Σε
αντίθεση με τις προσδοκίες μας μόνο το πλασμιδίου pCEP4 / eGFP ήταν ικανό να
εγκαταστήσει σταθερές κυτταρικές σειρές Jurkat. Ο υπολογισμός της
αποσταθεροποίησης υπό τάση των παραπάνω πλασμιδίων απέδειξε μια υψηλή ουδό για
την αποσταθεροποίηση της περιοχής του OriP, σε αντίθεση με το μητρικό φορέα pCEP4,
γεγονός που οφείλεται στον ενεργειακό ανταγωνισμό μεταξύ του OriP και της υψηλά
αποσταθεροποιημένης περιοχής του S/MAR. Η αντικατάσταση του OriP στο πλασμίδιο
pESdER / eGFP από την περιοχή έναρξης της αντιγραφής το γονίδιο της β-σφαιρίνης
(IR) (pESdER-IR/eGFP) έχει ως αποτέλεσμα την αποκατάσταση της επισωματική της
κατάστασης, προσδίδοντας επιπλέον και υψηλή μιτωτική σταθερότητα χωρίς την ανάγκη
ύπαρξης πίεσης επιλογής σε καλλιέργειες για χρονικό διάστημα τριών μηνών. Συμπεράνουμε ότι το δυναμικό πολλαπλασιασμού ενός επισωματικού συστήματος που
περιέχει ένα στοιχείο S/MAR εξαρτάται από την ύπαρξη μιας δεύτερης υψηλά
αποσταθεροποιημένης έννοιες περιοχής ικανής να ξεπεράσει την σταθεροποιητική δράση
της αλληλουχίας S/MAR. Το γεγονός αυτό προσδίδει μια νέα διάσταση στη σχεδίαση
αυτόνομων επισωματικών φορέων, που περιέχει τον υπολογισμό της αποσταθεροποίησης
της διπλής έλικας DNA υπό τάση. Η ευρεία χρήση των ρετρο-ιϊκών φορέων ως συστήματα γονιδιακής μεταφοράς
σε κλινικές δοκιμές γονιδιακής θεραπείας έφερε στην επιφάνεια την ανάγκη για μια
ακριβή εκτίμηση της ασφάλειας των δοκιμών αυτών όσον αφορά το φαινόμενο της
μεταλλαξιγένεσης μέσω ενσωμάτωσης. Στην εργασία αυτή αναφέρεται μια νέα μέθοδος
για την ανίχνευση των θέσεων ενσωμάτωσης των ρετρο-ιϊκών φορέων που έχει κλινικό
προσανατολισμό και ονομάζεται DSCP-PCR( partially Double stranded, Sterically
Hindered Primer – PCR). Η μέθοδος αυτή έχει αυξημένη ευαισθησία και ειδικότητα,
δεδομένου ότι δεν εξαρτάται από στάδια κατάτμησης με ένζυμα περιορισμού και
αντιδράσεις λιγάσης που αποτελούν μέρος των υπαρχόντων μεθόδων (όπως η LAMPCR).
Η μείζονα διαφορά ανάμεσα στην DSCP-PCR και στις προηγούμενες PCR
μεθόδους συνιστάται στο στάδιο της σύνδεσης της συμπληρωματικής αλυσίδας του DNA
όπου χρησιμοποιείται ένας εκκινητής που το ένα άκρο του είναι εκφυλισμένο ενώ το
άλλο αποτελείται από διπλή έλικα DNA. Η σχεδίαση αυτή τον καθιστά ικανό να
προσδένεται μόνο στο άκρο οποιασδήποτε μονής αλυσίδας. Η μέθοδος αυτή παράγει ένα
υψηλά πληροφοριακό σύνολο δεδομένων για τον χαρακτηρισμό των θέσεων
ενσωμάτωσης των φορέων. Επίσης μπορεί να χρησιμοποιηθεί για τη σύνθεση του προφίλ
των κλώνων που συμμετέχουν στην αιμοποίηση σε κάποια χρονική περίοδο. Η
παρακολούθηση το προφίλ αυτού μέσα στον χρόνο δίνει ενδείξεις για την κινητική της
αιμοποίησης και κατά συνέπεια της εμφάνιση νέων κλώνων.Episomal vectors have been considered valid alternatives to viral vectors for gene therapy applications, as they are replicating extrachromosomally and are devoid of the adverse effect of insertional mutagenesis. Interest in the episomal systems was boosted by the development of the pEPI-1 vector, containing the scaffold matrix attachment region (S/MAR) element of the human beta-interferon gene, which confers to it stable episomal status, without the need for transcription of any viral element. S/MARs are heterogeneous DNA regions that attach to the nuclear matrix, participating in the organization of the eukaryotic genome, initiation of DNA replication and regulation of transcription. We decided to study the performance of the S/MAR element in a completely different vector DNA context. Starting from vector pCEP4 (Invitrogen) based on OriP EBV and EBNA-1 latent retention system, we constructed plasmids: (1) pEBS/eGFP with SMAR element cloned after eGFP gene as in pEPI-1. (2) pESdER/eGFP derived from pEBS/eGFP by deletion of EBNA-1 gene and (3) pCEP4/eGFP as reporter plasmid. Contrary to expectations, only plasmid (3) was able to establish stable Jurkat cell line cultures. Calculation of stress-induced duplex destabilization of the above plasmids demonstrated a high threshold for the destabilization of the OriP region, unlike the parental vector pCEP4, caused by the energy competition of OriP with the highly destabilized S/MAR region. Substitution of OriP in plasmid (2) with the β-globin initiation region (IR) (pESdER-IR/eGFP) results in the restoration of episomal status, providing high mitotic stability without selection pressure for up to 3 months of culture. We deduce that the replication potential of an episomal system carrying an S/MAR element depends on the existence of highly destabilized vector sequences, sufficient to counteract the S/MAR effect of stabilization on vector’s DNA backbone molecule and maintain the plasmid’s accessibility to the cellular replication machinery. Thus emerges the concept of including the calculation of stress-induced duplex destabilization in the design of self-replicative extrachromosomal units. The widespread use of retroviral-based vectors as gene transfer systems in the context of gene therapy clinical trials has emerged the need for accurately assessing their safety profile in order to identify the potential risks of insertional mutagenesis. Here we report a new PCR - based method, DSHP-PCR ( partially Double stranded, Sterically Hindered Primer – PCR), a clinically orientated method for analysing the integration sites with high sensitivity and reliability, devoid of the restriction digestion and cloning bias present in the existing methods. The difference between DSHP-PCR and previous PCR-based methods (such as LAM-PCR) consists in the step of second strand synthesis where the use of a partially double stranded –degenerated primer that binds at the end of the DNA single strands bypasses the need for the restriction digestion step that inserts a bias in integration site detection and for the cassette ligation step that renders the whole procedure inefficient. This method generates a highly informative integration site library used for the sequencing of the human genomic – retroviral junctions, by creating a PCR product pool that contains a high percentage of specific fragments of similar length that can be subcloned with the same efficiency in a sequencing vector. It can also be used for the creation of the clonal composition profile of patient’s each cell lineage in time allowing the monitoring of clonal kinetics of the haematopoietic or immune system by detecting the emergence of new clones that contribute to haematopoiesis.
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Σταύρου, Ελεάνα. "Γονιδιακή μεταφορά σε αιμοποιητικά κύτταρα με επισωματικά αυτο-αναπαραγόμενα οχήματα." 2005. http://nemertes.lis.upatras.gr/jspui/handle/10889/437.
Full textAbstract:
Tο πρώτο μέρος της εργασίας αφορούσε την παρακολούθηση της ερυθρολευχαιμική σειρά ποντικού (MEL) διαμολυσμένη με το επισωματικό αυτό-αναπαραγώμενο όχημα pEPI-EGFP, τα κύτταρα της οποίας δεν παρουσίαζαν φθορισμό ενώ παρέμεναν ανθεκτικά στο αντιβιοτικό (G418). Αποδείχθηκε η επισωματική κατακράτηση του οχήματος για δύο μήνες μετά τη δημιουργία της διαμολυσμένης σειράς. Η διαπίστωση επίσης ότι το μεταφερόμενο γονίδιο μπορεί να μην εκφράζεται αλλά το όχημα να παραμένει για τουλάχιστον 60 ημέρες επισωματικό και χωρίς ενσωμάτωση μας ώθησε σε νέες προοπτικές αναζήτησης. Το δεύτερο μέρος της εργασίας έγινε παρασκευή δύο οχημάτων τα οποία διαφέρουν ως προς τον υποκινητή του γονιδίου αναφοράς ΕGFP τόσο μεταξύ τους όσο και από το όχημα pEPI-EGFP, το βασικό όχημα μελέτης έως τώρα. Η παρασκευή αυτή βασίστηκε στην αντικατάσταση του υποκινητή pCMV από: 1 τον υποκινητή pSFFV για την παρασκευή του οχήματος pEPI-pSFFV και 2 τον υποκινητή της β-σφαιρίνης (pβ-globin) για την παρασκευή του αντίστοιχου οχήματος pEPI-pβ-globin. Η παρασκευή των δύο αυτόν οχημάτων, του pEPI-pSFFV και pEPI-pβ-globin αποτελεί ένα σημαντικό βήμα στη ανάπτυξη νέων οχημάτων που αποτελέσουν οχήματα μεταφοράς γονιδίων σε αρχέγονα αιμοποιητικά κύτταρα.The first part of this work concerned the observation of the mouse erithroid cell line (MEL) transected with the Replicating Episomal Vector pEPI-EGFP. The cells even thought that they had lost their florescence a few days after the transfection with the Vector pEPI-EGFP, they still endure to the antibiotic (G418). In the fist place we have shown that the Replicating Episomal Vector pEPI-EGFP remains in episomatic condition at list two months after the transfect ion of the cell line with out any insertion eventhought the transferred gene is not translated. The second part of this work concerned the construction of two new vectors. The new vectors have the same reference gene with the pEPI-EGFP vector the EGFP gene but they have tow totally deferent new promoters for the EGFP gene. This contract was based on the replacement of the promoter pCMV 1. with the promoter pSFFV for the contraction of the pEPI-pSFFV new vector 2. with the promoter pβ-globin for the contraction of the pEPI-pβ-globin new vector These two new vectors the pEPI-pSFFV and the pEPI-pβ-globin were tested for their ability of being used as Replicating Episomal Vectors to Hematopoietic Stem Cells, HSC sach as CD34+ cells.
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Δρύλλης, Γιώργος. "Ανάπτυξη επισωματικού φορέα για τη γονιδιακή μεταφορά του τεχνητού μεταγραφικού παράγοντα ενεργοποίησης της γ-σφαιρίνης." Thesis, 2008. http://nemertes.lis.upatras.gr/jspui/handle/10889/930.
Full textAbstract:
Οι αυτοαναπαραγόμενοι επισωματικοί φορείς γονιδιακής μεταφοράς αποτελούν πολλά υποσχόμενους φορείς γονιδιακής θεραπείας. Στην παρούσα εργασία δημιουργήθηκε ο φορέας Zif-VP64-EP2 στα πλαίσια των μελετών γονιδιακής θεραπείας για τις αιμοσφαιρινοπάθειες. Πρόκειται για ένα κυκλικό πλασμίδιο, που φέρει το γονίδιο ενός τεχνητού μεταγραφικού παράγοντα της γ-σφαιρίνης του Zif-VP64 υπό την επενέργεια του ισχυρού υποκινητή pSFFV καθώς και τo γονίδιο της eGFP με το S/MAR στοιχείο από την περιοχή 5’ του γονιδίου της ανθρώπινης ιντερφερόνης β υπό την επενέργεια του υποκινητή pCMV.
Διαπιστώθηκε ότι το Zif-VP64-EP2 μεσολαβεί γονιδιακή μεταφορά σε διαμολυσμένα κύτταρα της ανθρώπινης κυτταρικής σειράς Κ562. Η μακράς διάρκειας διαμολυσμένη καλλιέργεια (3 μήνες) καταδεικνύει ότι το όχημα είναι λειτουργικό και διατηρείται ως επισωματικό σε Κ562 κύτταρα διαμολυσμένα κύτταρα με το Zif-VP64- ΕΡ2.Self-replicating episomal vectors for gene transfer are a new and very promising experimental approach to gene therapy. In this study, it was created the vector Zif-VP64-EP2, within the context of developing self-replicating episomal vectors for the gene therapy of hemoglobinopathies. Zif-VP64-EP2 is a circular plasmid which includes the gene of an artificial transcription factor for gamma globin: Zif-VP64 under the control of pSFFV promoter and the gene of eGFP with the S/MAR element from the region 5’ of the human interferon β gene under the control of pCMV promoter. It was established that Zif-VP64-EP2 was retained within the transfected Κ562 hematopoietic progenitor cell. Its episomal situation for a long time (3 months) and its normal expression in K562 human cells constitutes a proof of the utility of Zif-VP64- ΕΡ2 system in gene therapy applications.
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Παπαπέτρου, Ειρήνη. "Γονιδιακή μεταφορά με μη ιϊκά επισωματικά." 2005. http://nemertes.lis.upatras.gr/jspui/handle/10889/328.
Full textAbstract:
Τα επισωματικά αυτο-αναπαραγώμενα συστήματα αποτελούν υποσχόμενα εναλλακτικά οχήματα γονιδιακής μεταφοράς για εφαρμογές της γονιδιακής θεραπείας. Η πρόσφατη κατανόηση της ικανότητας των αλληλουχιών S/MAR να διαμεσολαβούν την επισωματική διατήρηση γενετικών στοιχείων επέτρεψε την ανάπτυξη ενός πρότυπου κυκλικού επισωματικού φορέα που λειτουργεί χωρίς να κωδικοποιεί πρωτεΐνες ιϊκής προέλευσης. Σε αυτή τη μελέτη, διερευνήθηκε για πρώτη φορά η δυνατότητα αυτού του φορέα, pEPI-eGFP, να μεσολαβεί γονιδιακή μεταφορά σε κυτταρικές σειρές προγονικών αιμοποιητικών κυττάρων καθώς και σε πρωτογενή ανθρώπινα κύτταρα και, κυρίως, σε ανθρώπινα προγονικά αιμοποιητικά κύτταρα. Δείχνουμε ότι ο φορέας pEPI-eGFP διατηρείται επισωματικά και υποστηρίζει παρατεταμένη έκφραση του γονιδίου αναφοράς eGFP, ακόμα και χωρίς πίεση επιλογής, στην ανθρώπινη κυτταρική σειρά K562, καθώς και σε πρωτογενείς ανθρώπινους ινοβλάστες. Αντίθετα, στην κυτταρική σειρά ερυθρολευχαιμίας ποντικού MEL, η έκφραση της eGFP αποσιωπάται μέσω αποακετυλίωσης ιστονών, παρά την επισωματική διατήρηση του φορέα. Προγονικά αιμοποιητικά κύτταρα με κλωνογόνο ικανότητα, προερχόμενα από αίμα ομφάλιου λώρου, διαμολύνονται αποτελεσματικά με το φορέα μέσω ηλεκτροδιάτρησης. Ημιστερεές αποικίες προερχόμενες από διαμολυσμένα CD34+ κύτταρα διατηρούν το φορέα και εκφράζουν eGFP. Μετά από 4 εβδομάδες ο φορέας διατηρείται επισωματικά σε περίπου 1% των θυγατρικών κυττάρων. Τα αποτελέσματά μας αποδεικνύουν για πρώτη φορά ότι ένα πλασμίδιο βασιζόμενο σε μια αλληλουχία S/MAR μπορεί να λειτουργεί ως σταθερό επιίσωμα σε πρωτογενή ανθρώπινα κύτταρα και, ιδιαίτερα, σε προγονικά αιμοποιητικά κύτταρα, υποστηρίζοντας παρατεταμένη έκφραση του διαγονιδίου. Η μελέτη αυτή αναδεικνύει τη χρησιμότητα του συστήματος αυτού για τους σκοπούς της γονιδιακής θεραπείας. Παράλληλα, καταδεικνύει τους στόχους στους οποίους πρέπει να επικεντρωθεί η μελλοντική έρευνα προς την κατεύθυνση της βελτίωσής του.Episomally maintained self-replicating systems present attractive alternative vehicles for gene therapy applications. Recent insights into the ability of chromosomal scaffold/matrix attachment regions (S/MARs) to mediate episomal maintenance of genetic elements cloned in cis allowed the development of a small circular episomal vector that functions independently of virally encoded proteins. In this study, we investigated the potential of this vector, pEPI-eGFP, to mediate gene transfer in hematopoietic progenitor cell lines as well as in primary human cells and, importantly, in human hematopoietic progenitor cells. pEPI-eGFP was episomally maintained and conferred sustained eGFP expression even in nonselective conditions in the human cell line, K562, as well as in primary human fibroblast-like cells. In contrast, in the murine erythroleukemia cell line, MEL, transgene expression was silenced through histone deacetylation, despite the vector’s episomal persistence. Hematopoietic semisolid cell colonies derived from transfected human cord blood retained the vector and expressed eGFP. After 4 weeks, the vector was maintained in approximately 1% of progeny cells. Our results provide the first evidence that a S/MAR-based plasmid can function as a stable episome in primary human cells, supporting long-term transgene expression. The present study constitutes a proof of principle for the utility of this system in gene therapy applications and points at targets for future improvements.
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Nehlsen, Kristina [Verfasser]. "Molekulare Grundlagen der episomalen Replikation : Charakterisierung zirkulärer, nichtviraler Vektoren / von Kristina Nehlsen." 2004. http://d-nb.info/971750637/34.
Full text
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Ziegler, Manja [Verfasser]. "Vergleichende Untersuchung der regulierten Genexpression von integrierter und episomaler HIV-1 LTR / Manja Ziegler." 2007. http://d-nb.info/984562796/34.
Full text
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Chang, Keng-Ming. "Stable propagation of the yeast 2 micron plasmid : equal segregation by hitchhiking on chromosomes." Thesis, 2014. http://hdl.handle.net/2152/24799.
Full textAbstract:
The 2 micron plasmid of Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number of 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-encoded partitioning system and an amplification system to ensure its stable, high-copy propagation. The partitioning system consists of the plasmid encoded proteins, Rep1 and Rep2 and a cis-acting partitioning locus STB. The Rep proteins, together with several host factors, assembled at STB couple plasmid segregation to chromosome segregation. A plasmid lacking an active partitioning system is subject to a ‘diffusion barrier’, which causes it to be retained in the mother cell with a strong bias (mother bias). Currently available evidence favors the hitchhiking model for plasmid segregation, in which the tethering of plasmids to chromosome provides the basis for faithful plasmid partitioning. However, direct evidence to support this hypothesis has been difficult to obtain because of the small size of the budding yeast nucleus and the poor resolution of chromosomes in live cells or in chromosome spreads. In this study, we have attempted to verify the hitchhiking model using single copy derivatives of the 2 micron plasmid as reporters. We demonstrate, using two single copy reporters present in the same nucleus, that plasmid association with chromosome spreads is authentic, and is dependent on the partitioning system. By using a strategy that forces all chromosomes to stay in either the mother or the daughter compartment, we show that plasmid segregation can be uncoupled from nuclear envelope segregation. However, plasmid segregation cannot be uncoupled from chromosome segregation under this condition. This tight coupling between plasmid and chromosome segregation is consistent with the hitchhiking model for plasmid segregation. The plasmid partitioning complex is assembled de novo at STB during each cell cycle during the G1-S window. Plasmid replication or cell cycle cues that signal cellular DNA replication appear to trigger this assembly. Furthermore, there is an apparent temporal hierarchy in the association and dissociation of protein factors at STB. When DNA replication is delayed or blocked, the dissociation of factors from STB from the previous portioning cycle and the association of factors for the new partitioning cycle are delayed or blocked, respectively. The precise role of replication in plasmid segregation has not been elucidated. We have addressed this question by blocking either plasmid replication or all cellular DNA replication. We find that replication is not required for plasmid to overcome mother bias. However, replication is critical for the equal segregation of sister plasmid copies. These results provide a refinement of the hitchhiking model by suggesting that sister plasmids tether to sister chromatids in a replication-dependent manner and hitchhike on them during chromosome segregation. Finally, we have attempted to reconstitute the 2 micron plasmid partitioning system in mammalian cells with the goal of exploiting their larger nuclear size and the considerably higher chromosome resolution they provide. In experiments completed so far, we show that Rep2 expressed in COS7 cells localizes to chromosomes, and Rep1 does so in the presence of Rep2. Furthermore, they show co-localization on sister chromatids in a symmetric fashion, implying that plasmids associated with them are likely to follow suit. These observations suggest, by extrapolation, the Rep1-Rep2 assisted association of sister plasmids with sister chromatids in yeast as well, and are consistent with the refined hitchhiking model for plasmid segregation.text
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Wucherpfennig, Frank [Verfasser]. "Transfer und Freisetzung von Episomen für die somatische Gentherapie durch einen Herpesvirus-, Adenovirus-Hybridvektor / vorgelegt von Frank Wucherpfennig." 2007. http://d-nb.info/983139660/34.
Full text
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Liu, Yen-Ting 1980. "The segregation of native and foreign extra-chromosomal genetic elements in Saccharomyces cerevisiae : stable propagation by hitchhiking on chromosomes." 2012. http://hdl.handle.net/2152/22068.
Full textAbstract:
The 2 micron plasmid of the budding yeast Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number around 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-coded partitioning system and an amplification system to exhibit nearly chromosome-like stability in its host. Plasmid behavior under conditions that missegregate chromosomes suggest that the partitioning system couples plasmid segregation to chromosome segregation. However, the mechanism of this coupling has not been elucidated. A plausible model, consistent with current evidence, is the hitchhiking model, in which plasmid-chromosome tethering provides the basis for faithful plasmid partitioning. Testing this hypothesis unequivocally has been difficult, primarily because of the technical limitations posed by the small size of the budding yeast nucleus and poor resolution of chromosomes. As a result, cell biological assays based on fluorescence microscopy have had only modest success in addressing this problem. In the present study, I devised an experimental verification of the hitchhiking model using a single copy derivative of the 2 micron plasmid as a reporter. The rationale was to establish various conditions that force sister chromatids to co-segregate during mitosis in a bias-free manner or with a bias towards the daughter. The segregation patterns of plasmid sisters were followed under these conditions. The sum of the results from this analysis is accommodated by the hitchhiking model, with sister plasmids associating with sister chromatids in a one-to-one fashion. Episomes of mammalian viruses belonging to the gamma-herpes and papilloma families utilize a hitchhiking mechanism to persist in cells during the latent phase of their infection. Two of the viral partitioning systems have been reconstituted in S. cerevisiae. We wished to exploit these systems to characterize the efficiency of non-native chromosome tethering systems in promoting equal segregation of viral plasmids in S. cerevisiae. We find that the 2 micron plasmid partitioning system is considerably superior to the viral systems. This could be due to the higher efficiency of plasmid-chromosome association and/or due to the ability of plasmid sisters to tether to sister chromatids.text
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"The stable expression of linear bovine papillomavirus type 1 episome and its association with the nuclear matrix." Tulane University, 1994.
Find full textAbstract:
The goal of this work was to study the interaction of bovine papillomavirus type 1 (BPV-1) episomal DNA with the nuclear matrix (NM). Two cell lines, BPV-1.4R and BPV-1.4H, were generated by cotransfection of C127 cells with BamHI-linearized BPV-1 and T8-pMV7, a neomycin selection vector. Both transfectants were morphologically different from each other and from parental cells. Studies were conducted to determine if NM protein composition changed with transfection of BPV-1. 2-D IEF PAGE analysis revealed that maintenance and expression of BPV-l causes changes in the expression of a small subset of nuclear matrix proteins. Southern blot analysis indicated that both transfectants carry episomal copies of linear BPV-l. Bal 31 nuclease digestion and anchored-PCR experiments further demonstrated the existence of linear episomal BPV-1 molecules. No alteration of sequences was observed by sequencing in the 5$\prime$ end and 3$\prime$ end of linear episomes. Northern blot analysis indicated that linear extrachromosomal BPV-1 molecules are transcribed. Studies of viral DNA interactions with the nucleus suggested that linear BPV-1 DNA is not free to diffuse in the nucleus and is associated with the nuclear matrix. However, weak interactions of the viral genome with the nuclear matrix were detected rather than high affinity associations usually found for cellular genesacase@tulane.edu
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Shaw, Aaron Marcus. "Advancing the Safety of Lentiviral Vector Mediated Gene Therapy." Thesis, 2015. http://hdl.handle.net/1805/7925.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)Lentiviral vector mediated gene therapy has made great strides in recent years with several successful clinical trials. However, adverse events encountered with some early trials have highlighted the necessity to improve upon its safety. Improvements can range from early steps in vector production to evaluation of insertion sites post-transduction. We have evaluated an FDA approved DNase for removal of residual plasmid DNA during vector production, developed novel non-integrating lentiviral vectors and employed modified insertion site analysis post-transduction to improve the safety of lentiviral vector mediated gene therapy. To prevent the exposure of gene therapy patients to HIV-1 DNA it is essential to remove residual plasmid DNA during vector production. We evaluated a recombinant human DNase which has been FDA approved for use in patients as an alternative to a bacterially derived DNase. Our results indicate this DNase is an effective alternative with a potentially safer profile for use in patients. The ability of lentiviral vectors to stably integrate their genome into a host cell’s DNA can have negative side-effects due to the risk of insertional mutagenesis. Non-integrating lentiviral vectors have been developed to alleviate this risk in applications where integration is not necessary. However, a low frequency of illegitimate integration persists when using these vectors. We have developed a novel non-integrating vector mutation and evaluated the efficacy of combining it with other mutations for reducing the frequency of illegitimate integration. We demonstrate that combining mutations that inhibit integration can further reduce the frequency of illegitimate integration. Several methodologies have been developed for evaluating the insertion sites of normal integrating lentiviral vectors. Illegitimate integration by non-integrating vectors demonstrates mechanisms which result in insertions and/or deletions at the vector-genome junction. Current methods lack the sensitivity to account for these variables in a high-throughput manner. We have adapted modifications to current methods to improve the capture of these variable insertion sites for analysis. The results of these studies improve the safety of lentiviral vector mediated gene therapy by improving the purity of the vector product, providing a safer vector for non-integrase mediated applications, and allowing more sensitive analysis of insertion sites post-transduction.
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Πολυβίου, Σταύρος. "Οχήματα γονιδιακής μεταφοράς για τη γονιδιακή θεραπεία των αιμοσφαιρινοπαθειών." 2005. http://nemertes.lis.upatras.gr/jspui/handle/10889/454.
Full textAbstract:
Τα επισωματικά οχήματα γονιδιακής μεταφοράς αποτελούν ελκυστική εναλλακτική πειραματική προσέγγιση της γονιδιακής θεραπείας σε σχέση με τα ιϊκά οχήματα. Στην παρούσα εργασία και στο πλαίσιο της ανάπτυξης επισωματικών οχημάτων για την γονιδιακή θεραπεία των αιμοσφαιρινοπαθειών, μελετάται το όχημα hβ-SMAR(Α), ένα κυκλικό πλασμίδιο, που φέρει το γονίδιο της ανθρώπινης β-σφαιρίνης και το μLCR και βασίζεται σε ανθρώπινα χρωμοσωμικά στοιχεία, το S/MAR στοιχείο από την περιοχή 5’ του γονιδίου της ανθρώπινης ιντερφερόνης β. Διαπιστώθηκε ότι το hβ-SMAR(Α) συγκρατήθηκε εντός της διαμολυσμένης κυτταρικής σειράς MEL για περισσότερες από 300 γενιές με διαπιστωμένη την επισωματική του κατάσταση για τουλάχιστο 180 γενιές. Επιπλέον, διατήρησε υψηλά επίπεδα έκφρασης του διαγονιδίου, τα οποία θα αντιστοιχούσαν σε θεραπευτικά επίπεδα, αν αναπαράγονταν in vivo.Episomal vehicles for gene transfer are an attractive alternative experimental approach to gene therapy in the place of viral vectors. The vehicle hβ-SMAR(Α) studied here, within the context of developing efficient episomal vectors for the gene therapy of hemoglobinopathies, is a circular plasmid bearing the human β globin gene and the μLCR and is based on human chromosomal elements, the S/MAR element from the region 5’ of the human interferon β gene. It was established that hβ-SMAR(Α) was retained within the transfected MEL cell line for more than 300 generations, with its episomal state ascertained for at least 180 generations. Furthermore, it retained high level expression of the transgene, which would be therapeutic, if reproduced in vivo.