Dissertations / Theses: 'Transient and stable expression'

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Godfrey, Charlotte. "Investigation of translational reprogramming during transient and stable expression of monoclonal antibodies in Chinese hamster ovary cells." Thesis, University of Kent, 2017. https://kar.kent.ac.uk/65774/.

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Translational reprogramming and mRNA translation e ciency greatly in uence global protein synthesis, cell proliferation and growth; important parameters in de ning recombinant protein expression yields. Polysome pro ling is a widely-used technique to analyse mRNA transla- tion and its e ciency that provides a snapshot of ribosomes loaded on mRNA transcripts at any particular time. A higher number of polysomes present on a given mRNA suggests that the mRNA is being more heavily translated than those mRNAs with few ribosomes. Fur- ther, a large pool of sub-polysomes (40S, 60S and 80S) compared to polysomes in a sample suggests low translational activity. Here, polysome pro ling has been applied to investigate translational reprogramming in multiple recombinant monoclonal antibody (mAb)-producing Chinese hamster ovary (CHO) cell lines, and to determine how reprogramming re ects the ability of such cells to proliferate and make recombinant proteins in stable and transient mAb expression systems, in batch and fed-batch culture mode. The impact of culture temperature on the polysome pro le and hence on reprogramming was also investigated in transient studies. Polysome pro ling revealed reprogramming di ered between recombinant cell lines. Those with the highest global translational e ciency generally had the fastest cell speci c growth rates, although total ribosome capacity did not directly relate to those with the fastest growth rates or mAb productivities. This suggests it is the ability to utilise available machinery that determines protein synthetic capacity. Recombinant cell lines with higher cell speci c produc- tivities generally maintained a higher polysome to monosome (P:M) ratio during stationary phase and had elevated recombinant mRNA copy numbers localised to translationally active heavy polysomes. In transient systems, the P:M ratio was maintained longer at reduced tem- perature cultivation and related to higher mAb yields being obtained. A number of endogenous transcripts were found to be more or less abundant on polysomes at di erent times of culture, indicative of changes in the cellular requirements of the encoded proteins. Such transcripts could be potential cell engineering targets to help tune the needs of the cell to the demands of a culture process or recombinant protein, or alternatively their untranslated regions harnessed to help preferentially load target mRNAs onto ribosomes. When upstream open reading frames (uORFs) or alternative translation start sites were engineered into recombinant transcripts a range of mAb expressions were observed allowing the tuning of mAb expression, including improvement over a standard untranslated region used industrially as a control. The ndings described in this thesis therefore reveal insights into the mechanisms involved in translational regulation and reprogramming in CHO cells during bioprocessing. These can be utilised for further improvement via targeted cell engineering strategies, cell line screen- ing approaches or modi cation of recombinant transcripts for enhanced industrial host and recombinant cell lines.

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Schmitz, Christian. "Transient and stable expression of the human papilloma virus type 16 (HPV-16) early protein 2 (E2) in human keratinocytes." Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326421.

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Ferrari, Ilse Fernanda. "Caracterização de promotores de expressão especifica de cana-de-açucar (Saccharum ssp.) em sistema modelo Micro-Tom (Solanum lycopersicum L)." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/64/64133/tde-18012013-145236/.

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O emprego de novas tecnologias, como a transgenia, associadas ao melhoramento convencional de cana-de-açúcar apresenta grande potencial no combate a pragas e desenvolvimento de novas variedades. Entretanto, a falta de especificidade na expressão temporal e/ou local dos genes introduzidos tem se mostrado um fator limitante para o sucesso de produtos derivados da transgenia. Os promotores das metalotioneínas, por exemplo, podem representar uma alternativa ao uso de promotores constitutivos, particularmente, aqueles de metalotioneínas do tipo 1 (MT1) por apresentarem níveis elevados de expressão, em diferentes tecidos/órgãos e serem responsivos a estresses bióticos e abióticos. O uso de promotores sintéticos, contendo apenas elementos-cis, como GCG-like, W boxes e JERE, tem sido relevante por induzirem a expressão gênica local em resposta ao ataque de agentes bióticos. Este trabalho teve por objetivo a caracterização funcional de promotores de genes de metalotioneínas de cana-de-açúcar e o uso de elementos regulatórios sintéticos e, em paralelo, avaliou-se o uso de promotores sintéticos no controle da expressão gênica em cana-de-açúcar. Após as análises de expressão transiente, verificou-se que o promotor SoMT1b apresentou atividade GUS e GFP em epitélios de cebola e os promotores sintéticos, 4X Wbox, 4X GCC-like, 4X JERE e 4xW 4xS-box, e o promotor SoMT1b foram capazes de dirigir a expressão do gene repórter uidA (GUS) em calos embriogênicos de cana-de-açúcar. Em análise de expressão estável, o promotor SoMT1b foi capaz de dirigir a expressão do gene GUS para os frutos e sementes de tomate \'Micro-Tom\', mas não foi responsivo a estresse por herbivoria, cádmio e cobre. Também foi realizada a transformação de plantas de cana-de-açúcar, as quais ainda estão sendo analisadas. Os resultados obtidos demonstram a funcionalidade do promotor SoMT1b
New technologies, like genetic transformation, associated with conventional breeding of sugarcane have a large potential in developing new varieties tolerant to biotic and abiotic stress. However, the lack of specificity in the spatial and/or temporal expression of the introduced genes has been a limited factor for the success of products derived from transgeny. Metallothionein promoters, for instance, can represent an alternative to the use of constitutive promoters, particularly those from metallothionein type 1 (MT1) because they present high expression levels in different tissues / organs and are responsive to biotic and abiotic stress. Another alternative is the use of synthetic promoters which contain only cis elements, as GCG-like, W-box and JERE, which induces local gene expression in response to pathogen attack. In this work, we aimed to make the functional characterization of sugarcane metallothionein promoters and synthetic regulatory elements, in parallel, we evaluated the use of synthetic promoters in the control of gene expression in sugarcane. After transient expression analyzis, it was found that SoMT1b promoter was able to control the expression of the reporter genes to GUS and GFP in onion epithelium and GUS in sugarcane embryogenic calli. Additionally, synthetic promoters 4X Wbox, 4X GCC-like, 4X JERE and 4xW 4xS-box were able to direct the expression of the gene uidA (GUS) in embryogenic calli of sugarcane. In stable transformation analysis, SoMT1b promoter was capable of directing uidA expression in fruits and seeds of tomato cv. \'Micro-Tom\', but it was not responsive to herbivory, cadmium and copper stress. It was also carried out the transformation of sugarcane plants with the construction containing the SoMT1b promoter, but these are still being analyzed. The results demonstrate the functionality of the SoMT1b promoter in sugarcane and tomato

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Kinauer, Markus. "Transient and Stable Terminal Imido Complexes of Iridium." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0003-C14D-D.

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Addison, Charlene Janet. "Microtubule-associated protein 2 (MAP2) expression in transiently and stably transfected P19 embryonal carcinoma cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq26296.pdf.

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Reed, James. "Transient expression for engineering triterpenoid diversity in plants." Thesis, University of East Anglia, 2016. https://ueaeprints.uea.ac.uk/67059/.

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The triterpenes are one of the largest and most structurally diverse classes of plant specialised metabolites, with important commercial, agronomical and medical potential. However the structural complexity and low abundance of these compounds in nature presents significant challenges for exploiting this diversity. In recent years, advances in our understanding of triterpene biosynthesis in various plant species have greatly facilitated the ability to produce these compounds in other hosts. Plant-based host systems hold great promise because they may provide substrates, cofactors and subcellular compartmentalisation mechanisms facilitating engineering production of complex triterpenoids. Agroinfiltration is a rapid and scalable process that enables transient expression of biosynthetic genes in amenable plants such as Nicotiana benthamiana. This technique holds great promise for selective production and modification of triterpenes, but studies in this area have been limited. In this PhD thesis, transient expression is used to produce and oxidise triterpenes in N. benthamiana. Triterpene yields are improved through expression of rate-limiting genes in the mevalonate pathway (Chapter 3). An enzymatic ‘toolkit’ is established from a collection of previously characterised triterpene biosynthetic genes, allowing production of milligram quantities of a diverse array of simple and oxidised triterpenes in N. benthamiana. Furthermore, combinatorial biosynthesis is utilised to engineer production of novel oxidised triterpenes in planta (Chapter 4). A set of oxidised derivatives of β-amyrin are purified and evaluated for antiproliferative and anti-inflammatory activity in human cell lines, yielding insights into structural features underlying these activities in humans (Chapter 5). Finally, N. benthamiana is used for functional screening of cytochrome P450s implicated in the biosynthesis of agronomically important antifungal triterpene glycosides (avenacins) in oat, leading to identification of new P450s required for disease resistance (Chapter 6). This work highlights the utility of N. benthamiana as triterpene engineering platform and provides a basis for future studies exploring triterpene structure-activity relationships.

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Rahman, Md Mahbubar, Jay Shockey, and Aruna Kilaru. "Characterization of Select Avocado Acyltransferases by Transient Expression." Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/etsu-works/4814.

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Carter, Emma. "EPR investigation of stable and transient oxygen centred radicals over polycristalline titanium dioxide." Thesis, Cardiff University, 2007. http://orca.cf.ac.uk/56178/.

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Electron Paramagnetic Resonance Spectroscopy (EPR) has been used to identify and characterise the nature of several oxygen centred radicals over the surface of noncrystalline TiC2. The observed radicals exhibit varying stabilities over the different polymorphs of TiO2 samples studied, namely mixed-phase P25, pure phase anatase and a pure rutile material. Paramagnetic Ti centres were formed by thermal treatment of Ti02 under vacuum. Following the addition of oxygen, the superoxide anion (O2") was formed and exhibited a distribution of sites on the surface. In particular, O2" preferentially stabilises at oxygen vacancies on the surface. However, the degree of site occupancy was found to be temperature dependent. Pronounced activity for vacancy stabilised anions under the influence of thermal, photochemical and chemical treatment was identified compared to non vacancy sites. Co-adsorption of a series of aliphatic and aromatic ketones and oxygen over P25, followed by low temperature (77K) UV irradiation led to the formation of a series of unstable (transient) alkylperoxy and peroxyacyl radicals. The sequential reaction of acetone with surface adsorbed superoxide anions was also found to result in the formation of an jacetone-02 (a) surface complex which was unstable at temperatures above 250K. Thermally produced Ti centres reacted with acetone to form an unstable organic intermediate. This species subsequently dissociated and underwent further reaction with oxygen to generate peroxy and peroxacyl radicals. The identities of these oxygen centred radicals were confirmed using isotopically enriched O2. Finally, results are presented on the work involved in the development of an Ultra-High Vacuum EPR spectrometer for investigation of paramagnetic species on single crystal oxide surfaces. Samples of Cu(acac)2 supported on quartz and TiC2 (l 10) were examined by EPR and XPS. The two techniques were combined to identify paramagnetic centres on the single crystals and to provide proof of concept in the operation of the spectrometer.

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Lakshmi, Priya Saikumar. "Stable expression of tuberculosis vaccine antigen in lettuce chloroplasts." Master's thesis, University of Central Florida, 2011. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4780.

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Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the leading reasons of death by an infectious bacterial pathogen. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, a potential candidate antigen, ESAT-6 (6 kDa early secretory antigenic target) was fused with cholera toxin B subunit (CTB). Transplastomic lettuce plants were generated expressing these fusion proteins. Site-specific transgene integration into the chloroplast genome was confirmed by polymerase chain reaction and Southern blot analysis. In transplastomic leaves, expression levels of fusion protein (CTB-ESAT6) varied depending upon the developmental stage and time of leaf harvest with highest-level of accumulation in mature leaves harvested at 6PM. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Lyophilization increased CTB-ESAT6 protein content per gram of leaf material by 22 fold. Western blot analysis of lyophilized lettuce leaves showed that the CTB-ESAT6 fusion protein was stable and can be stored for prolonged period at RT. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of ESAT-6 antigen. GM-1 binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to interact with GM1 ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens fused to CTB in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB vaccine with potential for long term storage at room temperature.
ID: 031001453; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Title from PDF title page (viewed July 3, 2013).; Thesis (M.S.)--University of Central Florida, 2011.; Includes bibliographical references (p. 42-46).
M.S.
Masters
Molecular Biology and Micro
Medicine
Biotechnology

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Powers, Sara Lawrence. "Expression and characterization of an extremely stable tetrameric hyperthermophilic protein." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file 3.68 Mb., 189 p, 2006. http://wwwlib.umi.com/dissertations/fullcit/3220724.

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Bokori-Brown, Monika. "Stable allotopic expression of subunit a of human ATP synthase." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615163.

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Doostdar, Hamed. "Stable expression of eukaryotic p450 cDNA in mammalian cell lines." Thesis, University of Aberdeen, 1992. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU602073.

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The P450 profile of HepG2 cells was studied using specific enzyme assays and well characterised monoclonal and polyclonal antibodies. These cells were shown to endogenously express cytochrome P450 1A1, P450 1A2 and P450 3A5 when the cells were treated with benz(a)anthracene or rifampicin respectively. HepG2 cells were adapted to grow in Williams' E medium supplemented with only 5% (v/v) fetal calf serum. These cells showed a significantly higher concentration of P450 1A2 protein when treated with benz(a)anthracene and could be detected by P450 1A2 specific antibodies on Western blots. Stable cell lines of HepG2 and Cos-7 cells capable of continuous expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) were constructed. P450 2A6 cDNA was successfully expressed in the EBNA-1 transformed Cos-7 cell line for over 6 weeks. The rate of expression of this cDNA and the enzyme activity of the resulting protein was very low. No stable HepG2 EBNA-1 transformed cells capable of P450 2A6 expression was obtained.

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Spofforth, David J. A. "Palaeoclimatology of the late Palaeocene to middle Eocene : geochemical records of stable and transient climate states." Thesis, University of Southampton, 2011. https://eprints.soton.ac.uk/195029/.

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The late Palaeocene to late Eocene period of Earth's history is characterised by remarkable change. Temperate ice free poles at the beginning of this period gradually cooled until permanent ice formed on Antarctica around 33.5 million years before present (Ma) and sea ice formed in the Arctic. The intervening time was not stable and data, despite relatively low resolution, appear to show that the Eocene climate was dynamic. This period was the most recent time when atmospheric pCO2 concentrations were as high as predicted by models simulating the effects of anthropogenic fossil fuel burning on Earths' climate. The ability to understand the mechanisms of climate change in the Eocene will help to understand potential climate impacts in the future. This thesis examines 3 contrasting periods of climate change. Geochemical data indicate that a 3.5 million year period of high biogenic silica deposition during the Eocene was climatically relatively stable in the Arctic basin with only infrequent communication to the world's oceans outside. This period is correlated with high organic burial in the basin and global siliceous rich deposits which acted to gradually draw down pCO2. This period of `quiet' climate compares to two periods of warming where significant carbon isotope perturbations may indicate the forcing of the Earth's climate into an alternative quasi-stable state. The Palaeocene { Eocene Thermal Maximum (PETM) represents a significant input of exogenic carbon into the atmosphere over the course of several thousand years and significant warming of the Earth. Records of bulk carbonate isotopes from a section in NE Italy show several other Delta13C perturbations both before and after the PETM event, albeit a quarter to a half of the magnitude of the PETM, and having durations of only 40 { 60 thousand years (kyr). These events are thought to be the result of a re-arrangement of the internal carbon cycle of the Earth - atmosphere and may represent orbitally forced changes in deep water ocean ventilation similar to controls seen on modern day glacial { interglacial cycles. These rapid changes in the carbon cycle are shown to be inverse at the middle Eocene Climatic Optimum (MECO), where gradual warming over 400 kyr is ended abruptly by significant cooling. From the first marginal marine section of this event rapid organic carbon burial occurs over 50 { 100 kyr and is associated with previously unrecorded low oxygen bottom water conditions and high organic burial. We hypothesize that if this burial was extended over significant shelf areas then this could rapidly have returned the middle Eocene to the general cooling trend of the Eocene.

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Galbraith, Douglas. "Development of a mammalian cell system for high-level transient expression /." [St. Lucia, Qld.], 2005. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe19335.pdf.

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Williams, Darren William. "Gene transfer and transient expression of transgenes in zebrafish Danio Refrio." Thesis, University of Southampton, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242103.

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Rahman, Md Mahbubur, Jay Shockey, and Aruna Kilaru. "Transient expression of avocado DGAT1 and PDAT1 in N. benthamiana leaves." Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/asrf/2018/schedule/8.

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The avocado mesocarp contains up to 60-70% oil by dry weight where triacylglycerol (TAG) is the major constituent. This neutral lipid, TAG is utilized by plants for the carbon and energy source when stores in seed tissue. There is significant human nutritional demand for vegetable oil, but its use in the production of renewable biomaterials and fuels has intensified the need to increase oil production. In plants, the final and committed step in TAG biosynthesis is catalyzed by diacylglycerol acyltransferases (DGAT) and/or a phospholipid: diacylglycerol acyltransferases (PDAT). However, the regulation of TAG biosynthesis is not well-studied in nonseed tissues such as mesocarp of avocado. Based on the transcriptome data of Persea americana it is hypothesized that both DGAT and PDAT are likely to catalyze the conversion of diacylglycerol to TAG. In this study, putative DGAT1 and PDAT1 were identified and comprehensive in silico analyses were conducted to determine the respective start codons, full-length coding sequences, transmembrane domains, predicted protein structures and phylogenetic relationships with other known DGAT1s and PDAT1s. These data reveal that the putative DGAT1 and PDAT1 of a basal angiosperm species retain features that are conserved not only among angiosperms but also other eukaryotes. For transient expression, DGAT1 and PDAT1 were transformed into N. benthamiana leaves by agrobacterium-mediated transformation. Lipid droplet was visualized by Nile Red staining and lipid content and compositions were analyzed by TLC and GC-MS. It was found that avocado DGAT1 and PDAT1 increase lipid content significantly when expressed in tobacco leaves. These results suggest that avocado DGAT1 and PDAT1 are functional and synthesize TAG when expressed in planta.

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Steel, Elspeth Jane. "Agrobacterium tumefaciens-mediated transient expression as a tool for crinivirus antibody production." Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435893.

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Hudson, Kevin. "Development of vectors allowing efficient heterologous-gene expression in stable myeloma-cell transfectants." Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/35313.

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This thesis describes the development of expression-plasmid vectors for use in the mouse, myeloma cell-line, J558L. Following introduction of the plasmids into J558L using spheroplast fusion, the vectors allow selection for stably-transfected cells. Selective survival depends on expression from a gpt gene in the plasmid, which encodes the enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT). Expression of XGPRT confers, on J558L, a dominant selectable-resistance to mycophenolic acid in the presence of xanthine and hypoxanthine. The resultant stable gpt+-transfectants can then be screened for expression of a protein encoded by a non-selected gene in the expression plasmid. Initially, a systematic study was carried out to compare the effect on XGPRT expression of different expression elements placed upstream of the gpt coding sequence, in single-gene plasmids. Comparative expression levels were estimated by comparing the stable gpt+-transfection frequencies of J558L obtained with each plasmid. This study demonstrated the importance of the IgH-gene enhancer for obtaining high-level expression from a transfected gene. A combination of an IgH enhancer and a promoter element, upstream of the gpt coding-sequence, resulted in the highest levels of expression, but the type of promoter used was of only secondary importance. Identified combinations of effective upstream elements were then incorporated into plasmid vectors, which were constructed for expression from heterologous genes encoding proteins of interest. Stable gpt+- transfectants containing a non-selected gene were only obtained following transfection with plasmids which also contained the gpt gene. This was in contrast to situations in which a non-selected gene was cotransfected with a gpt gene which resided on a different plasmid; here, none of the stable gpt+-transfectants screened had cointegrated the non-selected gene with the gpt gene. Different classes of the two-gene expression plasmids were constructed, which differed in the relative orientation and position of the transcription units. The efficiency of the various plasmids was estimated by measuring the expression levels from a model cDNA, encoding chicken lysozyme. Transfectants were isolated which express and secrete biologically-active chicken lysozyme, at levels greater, in molar terms, than the typical level of secretion of endogenous Ig from myeloma cells. In some cases, the stable gpt+-transfection frequencies obtained with two-gene plasmids were low, compared with the transfection frequencies obtained with plasmids containing only the gpt gene. It was proposed that this was due to transcriptional interference between the two genes on the same plasmid. As this phenomenon might also restrict the expression from the non-selected gene, overcoming this effect was considered important in optimising expression. However, attempts to identify and overcome the phenomenon were inconclusive and, consequently, a rational strategy for overcoming this potential limitation on expression levels was not obtained.

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Kinauer, Markus [Verfasser], Sven [Akademischer Betreuer] Schneider, Sven [Gutachter] Schneider, and Franc [Gutachter] Meyer. "Transient and Stable Terminal Imido Complexes of Iridium / Markus Kinauer ; Gutachter: Sven Schneider, Franc Meyer ; Betreuer: Sven Schneider." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://d-nb.info/1189904543/34.

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Shedden, John Douglas. "Transient expression of ß-glucuronidase in embryogenic Brassica napus microspores following particle bombardment." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq20698.pdf.

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Buyel, Johannes Felix [Verfasser]. "Manufacturing biopharmaceutical proteins by transient expression in Nicotiana tabacum (L.) / Johannes Felix Buyel." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2013. http://d-nb.info/1043518819/34.

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Marsian, Johanna. "Transient expression of poliovirus-like particles in plants : developing a synthetic polio vaccine." Thesis, University of East Anglia, 2016. https://ueaeprints.uea.ac.uk/62929/.

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Plants, or cell suspension cultures derived from them, are a promising platform for the production of biologics and pharmaceuticals. In this work transient expression utilising the pEAQ vector system was deployed for the expression of virus-like particles (VLPs) in Nicotiana benthamiana or N. tabacum BY-2 cell suspension cultures. The results presented in this thesis demonstrate the potential of plant systems for the production of VLP-based vaccines. VLPs of the fish virus, Nervous necrosis virus (NNV), were successfully produced in plants by transient expression of the coat protein. The protein self-assembled into T = 3 particles, which appeared to be morphologically identical to the wild-type NNV when analysed by high resolution microscopy but were devoid of nucleic acid. In addition, transgenic BY-2 cell suspension lines were generated expressing correctly assembled NNV VLPs. Poliovirus (PV)-like particles from all three PV serotypes, containing either the wt coat protein or coat proteins with stabilising mutations, were successfully expressed in plants. These were generated by co-expression of the structural polyprotein P1 and the proteinase 3CD. Sufficient quantities of purified particles could be obtained for structural and immunological analysis. Mice carrying the gene for the human PV receptor were protected from wild-type PV when immunised with the plant-made stabilised PV VLPs. Structural analysis of the stabilised mutant of PV3 at 3.6 Å resolution revealed a structure almost indistinguishable from wild-type PV3, with the stabilising mutations having no effective on the antigenic surface of the particle. To make the product more attractive to the vaccine industry, tobacco BY-2 cells have been successfully tested for the transient expression of the above-mentioned PV mutant VLPs using the cell-pack method.

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Liedeman, Kerwin. "Transient transgene expression of human Coronavirus nl63 orf3 protein in a baculovirus system." University of the Western Cape, 2020. http://hdl.handle.net/11394/8042.

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>Magister Scientiae - MSc
Insect-derived baculoviruses have been used extensively as a safe and versatile research model for transgenic protein expression. Preclinical studies have revealed the promising potential of Baculoviruses as a delivery vector for a variety of therapeutic applications, including vaccination, tissue engineering and cancer treatments. Coronaviruses are enveloped viruses containing linear, non-segmented ribonucleic acid. Human coronavirus NL63 was first discovered in the Netherlands in January 2004, where a 7-month-old girl presented with an acute respiratory tract infection that was later established to predominantly infect infants, the elderly and immunocompromised individuals. In addition to the known non-structural and structural proteins of coronaviruses, an accessory protein known as open reading frame 3 which is conserved in the Coronaviridae family has not been extensively researched. Open reading frame 3 encodes a putative membrane-bound protein. This study cloned the open reading frame 3 viral gene of 741 base pairs into the baculovirus expression construct via competent bacterial cell lines. Open reading frame 3-Baculovirus particles were generated in Spodoptera frugiperda insect cells. Recombinant cells containing the viral protein gene were used to infect healthy Spodoptera frugiperda 9 cells at varying ratios of multiplicity of infection over a fixed time-course. The open reading frame 3 viral protein was not detected by quantification methods at a molecular weight of 26 kilo Dalton, due to polyclonal antibody degradation.

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Penmetsa, Ramachandra V. "Factors influencing transient gene expression in electroporated tall fescue (Festuca arundinacea Schreb.) protoplasts." Thesis, This resource online, 1992. http://scholar.lib.vt.edu/theses/available/etd-09052009-040406/.

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Norkunas, Karlah-Jade. "Development of a transient, high-level expression platform for protein production in plants." Thesis, Queensland University of Technology, 2014. https://eprints.qut.edu.au/78980/2/Karlah-Jade_Norkunas_Thesis.pdf.

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Plants are an attractive alternative to conventional expression systems for the production of recombinant proteins and useful biologics, however, the economic viability of plant made proteins is strongly yield dependent. This study aimed to improve transgene expression levels in the plant host Nicotiana benthamiana using the Agroinfiltration transient expression platform. Independent investigation of the physical, chemical and genetic features associated with Agroinfiltration identified factors that improved transformation frequencies, elevated transgene expression levels and ultimately improved protein yield. The major outcome of this research was a novel hyper-expression system for biofarming recombinant proteins in plants.

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Kunaparaju, Raj Kumar Biotechnology &amp Biomolecular Sciences Faculty of Science UNSW. "Epi-CHO, an episomal expression system for recombinant protein production in CHO cells." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2008. http://handle.unsw.edu.au/1959.4/41499.

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

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Gaji, Rajshekhar Y. "IDENTIFICATION OF CIS-ACTING ELEMENTS CONTROLLING GENE EXPRESSION IN S. neurona." UKnowledge, 2006. http://uknowledge.uky.edu/gradschool_diss/480.

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Sarcocystis neurona is an apicomplexan parasite that is a major cause of equine protozoal myeloencephalitis (EPM). During intracellular development of S. neurona, many genes are temporally regulated. To better understand gene regulation, it is important to identify and characterize regulatory elements controlling gene expression in S. neurona. To perform this study, it was essential to establish transfection system for this parasite. Hence, the 5 flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules -galactosidase (-gal) and yellow fluorescent protein (YFP) were expressed by electroporated S. neurona, thereby confirming the feasibility of performing molecular genetic experiments in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of T. gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express -gal and YFP. These transgenic clones were shown to be useful for analyzing growth rate of parasites in-vitro and for assessing drug sensitivities. To uncover possible sequence elements involved in promoter activity, the 5 flanking regions of five S. neurona genes were subjected to comparative analysis. This revealed the presence of a 7-base conserved motif GCGTCTC. Using a dual luciferase assay system, the SnSAG1 promoter was subjected to functional analysis. The motif GAGACGC located between -136 and -129 upstream of the transcription start site was found to be essential for SnSAG1 expression. This motif functions in an orientation dependent manner and was shown to play a role in binding nuclear proteins of S. neurona.

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Joh, Lawrence Day. "High-level transient expression, extraction, and purification of recombinant [beta]-glucuronidase from agroinfiltrated lettuce /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2005. http://uclibs.org/PID/11984.

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Thesis (Ph. D.)--University of California, Davis, 2005.
Degree granted in Biological Systems Engineering. On t.p. "[beta]" appears as Greek letter. Also available via the World Wide Web. (Restricted to UC campuses)

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29

McGahan, Lynda. "Expression of immediate-early gene proteins in the rat hippocampus following transient global ischemia." Thesis, University of Ottawa (Canada), 1996. http://hdl.handle.net/10393/10454.

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The temporospatial expression patterns of the immediate-early gene (IEG) proteins Fos, FosB, $\Delta$FosB, Jun, JunB, JunD, and NGFI-A were investigated in rat hippocampus by immunonohistochemistry 2 h, 12 h, 24 h, and 48 h after forebrain ischemia. Transient global ischemia (20 min), produced by four vessel occlusion (4-VO), elicited different patterns of IEG expression in vulnerable CA1 and more resilient CA3 neurons. Cell counts revealed that initially, ischemia elevated immunoreactivity in both CA1 and CA3 hippocampal subfields for all IEGs examined, with the exception of JunD and NGFI-A. However, distinct patterns of IEG expression became evident in these regions at later time points following recirculation of blood flow. The pivotal difference was the persistence of ischemia-induced elevations of FosB and Jun expression in the CA1 region of the hippocampus. Unlike CA3 neurons where IEG immunoreactivity had subsided to basal levels by 24 h-48 h after reperfusion, CA1 neurons continued to display increased FosB- and Jun-like immunoreactivity 48 h post-ischemia. In contrast to FosB and Jun, JunB expression declined significantly below basal levels in CA1 neurons at 48 h, while JunB-like immunoreactivity remained unaltered in CA3 neurons. Given that JunB has been shown to inhibit the transactivating properties of Jun, decreased JunB levels may contribute to the apoptotic death of CA1 neurons by enhancing the transcriptional regulating activity of Jun. Also notable at 48 h was the complete loss of constitutive NGFI-A expression from CA1 neurons of ischemic animals. In summary, these findings suggest that persistent elevations in FosB and Jun expression coupled with reductions in JunB and NGFI-A levels may play a role in the apoptotic death of CA1 neurons following transient global ischemia. (Abstract shortened by UMI.)

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30

Ayeleso, Taiwo Betty. "Protoplast isolation and plant regeneration in Bambara groundnut : a platform for transient gene expression." Thesis, Cape Peninisula University of Technology, 2016. http://hdl.handle.net/20.500.11838/2003.

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Thesis (MTech (Agriculture))--Cape Peninsula University Of Technology, 2016.
Bambara groundnut (Vigna subterranea), a dicotyledonous plant is a legume which has a potential to contribute to food security and nutrition. Protoplasts are naked plant cells lacking cell walls. Viable protoplasts are potentially totipotent. Therefore, when given the correct stimuli, each protoplast is capable, theoretically, of regenerating a new wall and undergoing repeated mitotic division to produce daughter cells from which fertile plants may be regenerated through the tissue culture process. Protoplast systems are valuable and versatile cell based systems that are useful in observing cellular processes and activities. In this study, the isolation of protoplast from the leaves of Bambara groundnut plant was extensively optimised. The factors affecting protoplast isolation considered in this study were ages of plant material, mannitol concentration, combinations and concentrations of enzymes and duration of incubation. Effects of ages of Bambara groundnut plant (4, 6, 8, 10 weeks), molarities of mannitol (0.4 M, 0.5 M. 0.6 M and 0.7 M), concentration and combination of enzymes (1%, 2% and 4% cellulase, 0.5% and 1% macerozyme and, 0.5% and 1% pectinase) at different incubation duration (4, 18, 24, 42 hours) were investigated. Overall, it can be deduced from this study that the optimal protoplast yield (4.6 ± 0.14×105ml-1/gFW) and viability (86.5 ± 2.12%) were achieved by digesting the leaves of four week old Bambara groundnut plant with 2% cellulase and 0.5 % macerozyme with 0.5M mannitol for 18 hours. Freshly isolated protoplasts were then cultured at different densities of 1 × 104 - 2 ×106 protoplasts/ml using MS in three different culture (Liquid, agar and agarose bead) methods. First cell division was observed only in liquid medium. With several attempts, no division was achieved in the agar and agarose bead methods, division also did not progress in the liquid medium and hence, plant regeneration from Bambara groundnut protoplasts could not be achieved in this study. Consequently, a further study is underway to compare the proteomic profiles of freshly isolated protoplasts and cultured protoplasts in order to gain insights into the expression of proteins that could perhaps be contributing to the difficulty in regenerating Bambara groundnut plant through protoplast technology. The present study is novel because it is the first study to optimise the various factors that could affect protoplast isolation from the leaves of Bambara groundnut and thus developed an efficient protocol for protoplasts isolation from leaves of Bambara groundnut for cell manipulation studies.

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Овчинникова, И., and К. Марини. "Устойчивые выражения и обороты в научной речи (английский язык)." Thesis, Сумский государственный университет, 2015. http://essuir.sumdu.edu.ua/handle/123456789/39137.

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32

Sun, Lijuan. "Tau protein expression and the development of stable microtubules during neuronal differentiation of D310 cells." Thesis, University of Ottawa (Canada), 1994. http://hdl.handle.net/10393/9748.

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The D310 EC cell line is multipotent and differentiation can be directed along the neuronal pathway by addition of retinoic acid (RA). The correlation between the expression of tau protein and the development of stable microtubule arrays during neuronal differentiation was studied by indirect immunofluorescence, ELISA immunoblotting and Immunogold labelling techniques. By immunofluorescence staining tau protein is not detected in undifferentiated EC cells, but it starts to be expressed at day 3 of RA induction and shows a beaded morphological appearance. The expression of tau increases steadily during neuronal differentiation. Double immunofluorescence labelling demonstrates that the expression of tau protein correlates well with the development of colchicine-resistant microtubules. The levels of total cellular tubulin, total class III $\beta$-tubulin and total tau protein were measured by ELISA. The amount of class III $\beta$-tubulin and tau protein were expressed as a ratio of the relative amount of class III $\beta$-tubulin or tau protein/mg total tubulin. ELISA results support the observations of immunofluorescence staining. The relative amount of tau protein expressed after RA induction increases steadily and parallels the increased expression of class III $\beta$-tubulin, an indicator of neuronal differentiation. Immunoblotting shows that both juvenile and mature isoforms of tau protein are present in RA-induced EC cells. Two isoforms are detected in day 3 RA-induced cells and four isoforms are detected in day 6 RA cells. All four isoforms associate with colchicine-stable microtubules. A beaded morphological distribution of tau along neurite processes seen by immunofluorescence staining suggests that tau protein associates with some, but not all microtubules in neurite processes. The prediction that tau associates with colchicine-resistant microtubules was tested by immunogold labelling at the light and electron microscopic levels.

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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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St, Germain Carly. "Expression and transient nuclear translocation of protein convertase 1 (PC1) during mouse preimplantation embryonic development." Thesis, University of Ottawa (Canada), 2004. http://hdl.handle.net/10393/26778.

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Preimplantation embryos express a number of hormones, neuropeptides and membrane receptors known to derive from proteolytic activation of their precursors by proprotein convertases (PCs). The goal of this study was to determine the pattern of expression of the neuroendocrine protein convertase 1 (PC1) in mouse preimplantation embryos. Previous data have shown that PC1 transcripts are detectable by reverse transcription-polymerase chain reaction in unfertilized and fertilized eggs as well as at all stages of preimplantation embryonic development (Croissandeau and Mbikay, unpublished data). In this study, we show by immunoblotting that the zymogen and mature forms of PC1 are present at these stages. Using immunofluorescence laser confocal microscopy, we have examined its subcellular location: PC1-specific staining was observed throughout the cytoplasm of unfertilized eggs. After fertilization, surprisingly, the staining was transiently concentrated in pronuclei. It relocated to the cytoplasm at post-zygotic stages and was particularly strong at junctions between blastomeres. The nuclear translocation of PC1 in fertilized eggs is probably mediated by its prodomain. Indeed, when transfected in human colon carcinoma LoVo cells, a mutant proPC1 incapable of cleaving off its prodomain accumulated in the nucleus. Furthermore, when N-terminally fused to a green fluorescent protein, this domain was able to direct the reporter protein to the nucleus of these cells. Collectively, these data suggest that PC1 is a potential convertase for precursor proteins in preimplantation embryos. They also raise the possibility of a nuclear function for this enzyme during zygote formation.

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35

Shaifta, Yasin Mohammad. "Transient receptor potential channels (TRPC) in human cells : characterisation using over expression and knock-down." Thesis, King's College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429098.

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36

Leth, Ingrid. "Agrobacterium tumefaciens Production to Enable the Large-Scale Transient Expression of Recombinant Proteins in Plants." Thesis, University of California, Davis, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10036242.

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Production of proteins through in planta transient expression offers an alternative to conventional microbial and mammalian cell culture systems. This platform is particularly appealing because of its rapid and relatively low-cost implementation and its ease of scale-up. Transient expression occurs when a functional gene construct is inserted into a plant cell, where it is expressed over a short period of time without being stably integrated into the plant genome. Large-scale transient expression of recombinant proteins in plants is a relatively new area, and studies are underway to optimize the stages of the process in order to make it economically competitive. One area that has not been examined is the fermentation of A. tumefaciens for agroinfiltration at a large-scale. This research investigated the effects of growth conditions including temperature, pH, and media composition on Agrobacterium growth kinetics and gene transfer capability, with the goal of identifying optimal process conditions for growing Agrobacterium at large scale for use in transient agroinfiltration. Growth temperature was found to affect bacterial growth rate but not gene transfer capability, and a growth temperature of 28°C was selected as optimal. Growth in Lysogeny Broth (LB) and Yeast Extract Peptone (YEP) media was examined and subsequent transient gene expression was measured. A defined media was developed and optimized for growing Agrobacterium and growth and gene transfer capability with this media was found to be comparable to LB and YEP media. Growth of Agrobacterium strain C58C1 pTFS40 in LB, YEP, and defined media resulted in maximum specific growth rates of 0.36 ± 0.01, 0.37 ± 0.03, and 0.33 ±0.01 h⁻¹ and maximum biomass concentrations of 1.9, 3.6, and 3.9 grams dry cell weight per liter after 12, 16, and 20 hours, respectively. It was demonstrated that direct infiltration with Agrobacterium in diluted growth media was an effective method of inducing transient expression. Batch fermentation of Agrobacterium was scaled up to benchtop (5 L) scale with the three types of media. Finally, production was scaled up to a 100 L working volume reactor.

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Pereira, João Nuno dos Santos. "Establishing a high titer transient gene expression process in conditioned media for CHO-DG44 cells." Master's thesis, Faculdade de Ciências e Tecnologia, 2011. http://hdl.handle.net/10362/6149.

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Dissertação para obtenção do Grau de Mestre em Biotecnologia
Transient gene expression (TGE) allows for fast protein production in mammalian cells and has become a very important technology in the product development pipeline of biopharmaceuticals. Polyethylenimine (PEI) mediated, high-density transfections have allowed for transient processes exceeding ~300mg/L in CHO-DG44 cells. As such, the bottleneck of TGE is no more in the titers, but in the scale-up to volumes higher than 1L, because of the need for a medium exchange before transfection. It is known that if the transfection is done in a running culture, without a medium exchange (i.e in conditioned medium), the yields obtained are very low (~5 mg/L). In CHO-DG44 cells, this problem was explored from the point of view of transfection efficiency, gene delivery and transcription. A new insight is presented in this work: The low productivities are not due to a deficient gene delivery, but instead, to lower mRNA levels that we hypothesize to be related to a lower gene accessibility of the transfected plasmid. Further, the yields were improved from ~5mg/L to ~90mg/L (18-fold) by optimizing the conditions for transfecting in conditioned medium and utilizing sodium butyrate as a transcription enhancer. These results are expected to open paths for the successful scale-up of TGE.

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Egawa, Gyohei. "Transient expression of ephrin B2 in perinatal skin is required for maintenance of keratinocyte homeostasis." Kyoto University, 2009. http://hdl.handle.net/2433/126460.

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39

Tait, A. S. "PEI-mediated transient gene expression in cell culture for the rapid production of therapeutic proteins." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1445865/.

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The aim of this study was to investigate the feasibility of using transient gene expression in mammalian cell culture for the production of recombinant therapeutic proteins. This would allow rapid production of candidate molecules for pre-clinical trial testing and characterisation. For unsuccessful candidates, this would remove the time consuming and costly requirement of producing a stable cell line. For effective candidates, the removal of the immediate requirement of producing a stable cell line could reduce development times by up to 6 months. This thesis describes the development of a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells (CHO-S) for the large-scale production of recombinant therapeutic proteins. Optimisation was done in a serum free environment, to make the process industrially applicable. However, removal of serum was found to dramatically affect the cell specific growth rate and transient transfection expression rate. This problem was negated by the addition of bovine serum albumin, and a concentration of 6 mg L"1 was used in subsequent optimisations. The PEI mediated transfection process was optimised with respect to PEI nitrogen to DNA phosphate ratio and the plasmid DNA mass to cell ratio, using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI nitrogen to DNA phosphate molar ratio of 10:1 and 5 jug DNA per 106 cells. It was found that PEI is cytotoxic to CHO-S cells, and the final concentration of PEI in the growth medium appears to be the critical factor. At high PEI concentrations, cell growth is reduced, which is coupled with a reduction in the transgene expression. Therefore at ratios greater than 10:1, or DNA concentrations higher than 5 ng DNA per 106 cells, cell growth rate, and therefore transgene expression was lower. This suggested that transient gene expression is linked to cell growth, and therefore cell division. Cell cycle blocking experiments demonstrated that transition into mitosis is a pre-requisite for efficient transient gene expression.

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Sutter, Nathaniel Barrett. "Suppression of stable and variegating position effects by the 5'HS2 and inducible 3MRE enhancers /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/5038.

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Weld, Richard John. "Transient gene expression following DNA transfer to plant cells: The phenomenon; its causes and some applications." Thesis, University of Canterbury. Botany, 2000. http://hdl.handle.net/10092/4798.

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A preliminary investigation into the practicality of using Ds elements as vectors for onion transformation was undertaken. Transient transposase expression was used to mediate Ds excision following co-bombardment of a transposase expression vector and a Ds element into onion callus tissue. Ds transposition in onion was demonstrated. Further development of this transformation system was not undertaken because, at the time, the low frequency of stable transformation made further investigation impractical. Transient transposase expression as a means to mobilise Ds e1ements for gene tagging and to study transient expression was also investigated. A T-DNA construct carrying the Aetivator (Ae) transposase gene was transferred to leaf discs taken from an Hieracium aurantiaeum plant containing a chromosomal Ds element. Shoots were regenerated under selection for antibiotic resistance resulting from Ds excision. Molecular analysis suggested that regenerants carried unique transposition events. Of 84 regenerated plants, 21 (25%) did not express the T-DNA marker gene and 7 (8.3%) also lacked transposase DNA sequences. These results are consistent with the theory that expression is lost due to loss ofT-DNA sequences. Potential advantages of this gene-tagging method over conventional methods are: rapid recovery of individual transposition events; regenerated plants are isogenic; gene-tagging in clonal or apomictic tissue; and the transi'ent nature ofiransposase expression should facilitate the stabilisation of the transposed element. Different factors involved in the transient nature ofT-DNA expression shortly after co-cultivation were also studied by using the green fluorescent protein reporter gene m-gfp5-ER in Nieotiana plumbaginifolia suspension cell transformation experiments. It was confirmed that transiently expressed T-DNAs can be lost during growth of somatic cells. However, cell death (64% of transient expressers died) and gene silencing (21 % of transient expressers retained T-DNA sequences) were more important barriers to the recovery of "stably" expressing transformants than lack ofT-DNA integration (15% of transient expressers lost all T-DNA sequences). Loss of transgene expression significantly limited the efficiency of plant transformation. Understanding the causes ofloss of trans gene expression should lead to improved transformation strategies.

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Marshall, Elaine. "Analysis of transient gene expression in ovine cells : a role for the PrP gene 3'UTR." Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/15276.

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In a previous thesis, in vitro expression in mouse N2a cells using reporter gene constructs with the chloramphenicol acetyl transferase (CAT) gene linked to PrP sequences had suggested that there might be an inhibitor of translation in the PrP gene 3'UTR. Transient transfection methods were developed to generate levels for in vitro expression of the CAT/PrP-3'UTR constructs in immortalised Cheviot fetal brain-derived cell lines from both scrapie resistant and susceptible genotypes and primary cell lines derived from cerebellum and liver tissues of the Icelandic sheep breed, Ovis brachyura borealis pall. Expression of a series of CAT/PrP-3'UTR vectors in cell lines derived from different PrP genotypes found that sequence between nucleotides 2000-2700 on the PrP 3'UTR may show a tendency to reduce protein expression in the cells derived from brain tissue of scrapie-resistant genotype genotype and in a tissue specific manner. Additional vectors were produced to express ovine PrP mRNAs, similar to the endogenous 4.6kb and 2.1 mRNAs transcripts, but altered to display a monoclonal epitope site (3F4) enable transiently expressed PrP protein within ovine cell cultures to be detected. Expression of the PrP constructs in mouse neuroblastoma (N2a) cells has shown that both constructs were viable. However, the immunodetection methods employed in this thesis could not distinguish between transiently expressed 3F4-labelled ovine PrP and endogenous PrP within sheep cell lines. Results presented here confirm that the 3' UTR found in the 2.1 kb mRNA is capable of supporting gene expression. A role for a specific sequence in the ovine PrP gene 3'UTR in controlling protein expression in brain-derived cell lines has been proposed. Also, the function of the regulatory sequence may be dependent on tissue origin. PrP^cis vital for the replication of the TSE agent. Controlling the amount of available PrP^cin vivo may influence susceptibility and development of TSE disease.

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Benzle, Kyle Arthur. "Isolation of Novel Agrobacterium and Transient Expression Assays in Soybean (Glycine max) and Sunflower (Helianthus annuus)." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1405555898.

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44

Davy, Robert Carlos Barton. "Development of a transient expression system for the α7 neuronal nicotinic acetylcholine receptor in mammalian cells." Thesis, University of Bath, 1995. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295445.

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45

CLERGUE, ALINE. "Transfert de genes chimeriques chez nicotiana tabacum. Expression transitoire et expression stable du gene codant pour la gus sous le controle de differents promoteurs." Paris 11, 1992. http://www.theses.fr/1992PA112359.

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L'objectif de ce travail est l'etude en expression transitoire et stable de la partie codante du gene uida sous le controle de differents promoteurs: le promoteur de l'orf14 precede ou non des sequences enhancer du promoteur 35s. Le promoteur 35s auquel a ete integre les l. R. E. De la ssu (lre35s). Le promoteur 35s precede de tandems de 2,4 ou 5 enhancers du promoteur 35s. Le promoteur 35s est pris en reference. Cette etude a necessite: la mise en place d'une methodologie efficace de transfert pour la mesure de l'expression transitoire du gene uida apres electroporation de protoplastes. Differents parametres ont ete definis. L'optimisation a ete realisee par planification experimentale. Cette approche s'es revelee une technique efficace, et a ete appliquee, avec succes, aux protoplastes de colza. L'obtention de plantes transgeniques: elle a ete realisee par la culture de disques foliaires de tabac avec a. Tumefaciens. Les plantes transformees sont selectionnees pour leur resistance a la kanamycine. Le reperage des plantes exprimant le gene uida est realise par revelation histochimique puis dosage fluorimetrique. L'integration du gene uida a ete etablie par analyse moleculaire pour certaines plantes et la transmission a la descendance du gene nptii etudiee. La planification experimentale a permis d'augmenter d'un facteur 3 a 5 le niveau d'expression de la gus. En expression transitoire le promoteur de l'orf14 et le promoteur lre-35s conferent respectivement une activite gus tres inferieure ou peu differente de celle conferee par le promoteur 35s. Des resultats comparables sont retrouvees en expression stable

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Yunusov, Dinar. "Characterization of HIPSTR highlights the heterogeneous expression pattern of lncRNAs in human embryos and stable cell lines." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-22082016-083421/.

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There is a growing appreciation that eukaryotic genomes are transcribed into numerous, previously undetected - and thus uncharacterized regulatory long non-coding RNAs (lncRNAs). Recent studies are primarily focused on lncRNAs transcribed from intergenic regions and enhancers, leaving antisense lncRNAs the least studied group of lncRNAs. At the same time, antisense transcription occurs in up to 74 % of human gene loci, frequently - from the opposite strand of genes encoding proteins involved in regulation of transcription. Here, we identified HIPSTAR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel conserved lncRNA that is transcribed antisense to the TFAP2A gene. Unlike previously reported antisense lncRNAs, HIPSTR expression does not correlate with the expression of its antisense counterpart. Although HIPSTAR and TFAP2A are co-expressed in in vitro derived neural crest and trophoblast cells, only HIPSTAR and not TFAP2A is specifically expressed in a subset of cells within 8-cell- and morula-stage human embryos. We show that, similar to HIPSTAR, in the individual cells of developing human embryos or of stable cell lines the expression of lncRNAs is more highly heterogeneous than the expression of mRNAs. Finally, we demonstrate that HIPSTAR depletion in HEK293 and H1BP, a human embryonic stem cell line, predominantly affects the expression levels of genes involved in early organismal development and cell differentiation. Together, we show that expression of HIPSTAR and hundreds other lncRNAs is highly heterogeneous in human embryos and cell lines. We use HIPSTAR to exemplify the functional relevance of lncRNAs with heterogeneous and developmental stage-specific expression patterns.
Tem sido cada vez mais reconhecido que a transcrição dos genomas eucarióticos produz múltiplos transcritos novos, anteriormente não detectados e ainda não caracterizados, sendo que a maioria é constituida de RNAs não-codificantes longos (lncRNAs) regulatórios. Estudos recentes estão focados principalmente nos lncRNAs transcritos de regiões intergênicas e enhancers; assim, o grupo dos lncRNAs antisenso permanece o menos estudado de todos. Ao mesmo tempo, a transcrição antisenso ocorre em até 74% dos loci de genes humanos, frequentemente - a partir da fita oposta de genes que codificam proteínas envolvidas na regulação da transcrição. No presente trabalho, nós identificamos HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), um lncRNA novo conservado que é transcrito a partir da fita antisenso do gene TFAP2A. Ao contrário do anteriormente relatado para os lncRNAs antisenso, a expressão de HIPSTR não está correlacionada com a expressão do gene da fita oposta. HIPSTR e TFAP2A são co-expressos em células da crista neural e em trofoblastos derivadas in vitro, mas somente HIPSTR e não TFAP2A está especificamente expresso num subconjunto de células de embriões humanos nos estágios de 8-células e mórula. Mostramos que, semelhante a HIPSTR, a expressão de lncRNAs é mais altamente heterogênea que a expressão de mRNAs em células individuais de embriões humanos em desenvolvimento ou em linhagens estáveis de células. Finalmente, nós demonstramos que a depleção de HIPSTAR em células HEK293 e H1BP, uma linhagem de células tronco embrionárias humanas, afeta predominantemente os níveis de genes envolvidos no início do desenvolvimento do organismo e na diferenciação de células. No conjunto, nós mostramos que a expressão de HIPSTR e de centenas de outros lncRNAs é altamente heterogênea em embriões humanos e linhagens celulares. Usamos HIPSTR para exemplificar a relevância funcional de lncRNAs com padrões de expressão heterogêneos e estágio-de-desenvolvimento específicos.

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Nehmé, Rony. "Expression et purification du récepteur humain de la voie Hedgehog, Smoothened, dans une conformation native et stable." Nice, 2009. http://www.theses.fr/2009NICE4031.

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La voie de signalisation Hedgehog constitue l’une des plus importantes voies dans le développement embryonnaire et la prolifération des cellules souches chez l’adulte. Cette voie implique deux protéines membranaires, Patched et Smoothened, dont les dysfonctionnements sont associés à de très nombreux cancers. Durant ma thèse, j’ai mis au point l’expression hétérologue du récepteur humain Smoothened (hSmo) et sa purification pour une caractérisation structurale et fonctionnelle. L’expression de hSmo a été réalisée dans la levure Saccharomyces cerevisiae. Utilisant la technique SPR, j’ai démontré que hSmo exprimé à la membrane plasmique de la levure est dans sa conformation native capable de fixer son antagoniste. J’ai ensuite mis au point la purification de hSmo et testé de nouvelles classes de surfactants. J’ai ainsi trouvé les meilleures conditions qui stabilisent le récepteur hSmo en solution après purification. La caractérisation d’une mutation ponctuelle au niveau de la 3ème boucle intracellulaire combinée à l’utilisation des nouveaux surfactants ont permis d’améliorer la stabilité de hSmo en solution. Les conditions que j’ai mises au point permettront l’étude structurale de hSmo et les essais de cristallisation. D’autre part, les stratégies développées en SPR permettront la recherche des partenaires protéiques cytoplasmiques de ce récepteur, encore inconnus à ce jour, afin de mieux comprendre la signalisation en aval de Smoothened. Les données structurales ainsi que la découverte des partenaires protéiques cytoplasmiques de hSmo permettront l’élaboration de nouvelles thérapies anticancéreuses
The Hedgehog pathway is one of the most important pathways in embryogenesis and in proliferation of adult stem cells. This pathway involves two transmembrane receptors, Patched and Smoothened whose dysfunctions have been linked to many human diseases including cancers. This study reports expression and purification of the human GPCR Smoothened, for structure-function relationship characterization. Therefore I developed the heterologous expression of Human Smoothened (hSmo) in the yeast S. Cerevisiae. Using SPR technology, I showed that hSmo, expressed at the plasma membrane of yeast, is in its native conformation able to bind its antagonist, cyclopamine (CPN). Then, I developed the purification of hSmo by affinity chromatography and tested new surfactants. Results show that the new surfactants stabilize hSmo in solution after purification and are preserve antagonist-binding ability of Smo suggesting that purified hSmo maintains its native conformation in solution. In addition, characterization of a single mutation of Smoothened (hSmoG435R) combined to one of the surfactants, revealed an enhanced stability of the receptor. These established conditions will be useful for crystallization assays. SPR strategies developed in this study will also be used for the research of hSmo’s cytoplasmic partners. Together, structural and functional data will contribute to the better understanding of Smo signaling and to the development of new cancer therapies

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48

Schürig, Margitta Verfasser], and Wulf [Akademischer Betreuer] [Blankenfeldt. "Efficient expression of viral surface proteins by transient gene expression in Hi5 insect cells for functional and structural analysis / Margitta Schürig ; Betreuer: Wulf Blankenfeldt." Braunschweig : Technische Universität Braunschweig, 2019. http://d-nb.info/1176701274/34.

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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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Gudmundsson, Kjartan. "Alternative methods for analysing moisture transport in buildings : Utilisation of tracer gas and natural stable isotopes." Doctoral thesis, KTH, Byggvetenskap, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3509.

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New methods, based on tracer gas measurements and isotopicanalysis can be used to evaluate the moisture properties ofbuilding materials and provide the means for forensic analysisof the origins and history of excessive water in buildings, theimmediate practical consequences of which will be the abilityto improve the moisture performance of constructions. It is shown, in theory and through measurements how thewater vapour permeability of porous building materials can witha good degree of accuracy be estimated with tracer gasmeasurements that provide an efficient alternative to the cupmethod. Complementary measurements may be carried out in orderto evaluate the contribution of surface diffusion and theeventual enhancing effects of moisture content on the diffusioncoefficient. The Random Hopping Model is used to illustrate howthe surface diffusion coefficient depends on the amountadsorbed and the activation energy of migration that can beevaluated from the sorption isotherms. It is explained how the abundance ratios of two of the mostordinary isotopes of hydrogen and oxygen in water can be usedto determine its history. These isotopes are stable and givethe water a distinct signature that can be used to reveal itssource as shown in a case study. In a contrary manner themeasured isotopic separation can be used to determine therelevance of different transport processes and reactions. It isof central importance that not only does the magnitude ofisotopic separation for the reactions vary for deuterium andoxygen-18 but even the ratio thereof. One of the challenges hasbeen to construct an experimental method for retrieving samplesof water for comparison. Furthermore this thesis includes an evaluation of a new typeof a light weight construction with loose-fill cellulose fibre,in which the conventional polyethylene vapour barrier has beenreplaced with polypropylene fabric. With a verified model ithas been investigated how the construction would perform fordifferent internal moisture loads and reference climate fromthe literature. The results suggest that this type ofconstruction is not to be recommended. KEYWORDS:tracer gas, water vapour permeability,diffusion, surface diffusion, isotopic analysis, deuterium,oxygen-18, fractionation, vapour barrier, transient numericalmodelling of diffusion.
QC 20100611

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