|Title:||Characterizing influences of specific miRNAs on phenotypic properties of mammalian cells||Language:||English||Authors:||Leberbauer, Lena Anna||Qualification level:||Diploma||Keywords:||miRNAs; mammalian cells; metabolic phenotyping; MFA; GFP; media development||Advisor:||Herwig, Christoph||Issue Date:||2017||Number of Pages:||103||Qualification level:||Diploma||Abstract:||
MicroRNAs (miRNAs) are short, non-coding, hairpin shaped RNAs, that exhibit many functions within dierent cells. Their main function is to interfere with the post-transcriptional step of protein expression, called RNA interference. Development of diseases have been linked to the malfunction of these small molecules. As the requirement of recombinant therapeutic proteins rises continuously, every direction to increase production qualities and titers of active agents is pursued. Lately, miRNAs have also been investigated for this matter, as their abundance within cells across all species is well known and this could hold the key to new engineering procedures to enhance cellular productivity. Their potential to modulate complete biochemical pathways without burdening the metabolism could mean a highly advanced approach to modify complicated biochemical system of mammalian cells. This research aimed to investigate new ways to visualize eects of miRNAs on cell characteristics, by transiently transfecting HEK 293T cells with preselected miRNAs, and analyze the outcome using ow cytometry. Evaluation on the increase of protein expression, described promising trends in several specic miRNAs, pointing out that this line of research holds a reassuring future. An investigation into the possible involvement of specic miRNAs, stably cloned into the genome of Chinese hamster ovary cells, in cell metabolism was conducted, using metabolic ux analysis (MFA). This gave an opportunity to explore their eects on a complex mammalian biochemical network. Metabolic ux analysis, media development experiments and process parameter analysis of CHO cells with altered miRNA expression patterns did show signicant dierences in central metabolism. Especially extracellular measurable uxes, like glucose and glutamine consumption, and glutamate and glutamine production decreased in anti-apoptotic cells, suggesting a more ecient handling of resources. Transient transfection of existing cell lines, parental, negative control and anti-miRNA cell line, with plasmids, carrying the sequences of complex glycoproteins, EPO and HubChe, facilitated the comparison of protein expression levels across cell clones. The production level of these proteins increased signicantly in the anti-apoptosis cell line.
|Library ID:||AC13674233||Organisation:||E166 - Institut für Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften||Publication Type:||Thesis
|Appears in Collections:||Thesis|
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checked on Feb 27, 2021
checked on Feb 27, 2021
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