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Author: Ricardo Valdés-Bango Curell
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Improvements in the production capabilities of Chinese Hamster Ovary (CHO) cells have relied on traditional genetic engineering strategies, such as gene overexpression and gene knockouts. However, new strategies are likely to require more sophisticated rational approaches and novel molecular tools need to be developed to facilitate more refined control of gene expression. In this thesis, the use of microRNAs to harness control of transgene expression in CHO cells has been investigated. The first part of this thesis aimed to identify miRNA expression profiles that could be used to actuate transgene expression in the context of biopharmaceutical production. miRNA expression data from cell lines with different glutamine requirements was investigated in an attempt to identify glutamine responsive miRNAs. In addition, the analysis of a novel CHO miRNA expression dataset from a fed batch process resulted in the identification of interesting miRNA clusters exhibiting expression profiles that could be matched to particular growth phases. The second part of the work involved the investigation of temperature-induced miRNA expression changes in order to build a temperature dependent transgene expression control system using miRNA sponges. Temperature responsive miRNAs were identified and validated. While providing evidence that miRNA sponges can be used as molecular sensors and modulate gene expression, our results indicate that temperature-driven changes of miRNA expression are unlikely to be used as a robust gene control system. Finally, transgene expression control by combining UTR secondary structure and miRNAs was investigated. We showed that miRNA-toehold switches are able to repress transgene expression repression in a sequence specific manner although a robust ON/OFF function could not be achieved. Using a small library of synthetic 5'-UTR, the effect of several structural and sequence features in the miRNA-toehold was also investigated. Sequence determinants such as upstream ORFs, AU-rich regions and kozak environment showed greater effects on transgene expression than the 5'-UTR local secondary structure features. In summary, the work described in this thesis represents the first attempt to implement miRNA-based strategies to directly control transgene expression in CHO cells while highlighting the difficulties that need to be overcome for this strategy to be effective.
Author: Ricardo Valdés-Bango Curell
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Improvements in the production capabilities of Chinese Hamster Ovary (CHO) cells have relied on traditional genetic engineering strategies, such as gene overexpression and gene knockouts. However, new strategies are likely to require more sophisticated rational approaches and novel molecular tools need to be developed to facilitate more refined control of gene expression. In this thesis, the use of microRNAs to harness control of transgene expression in CHO cells has been investigated. The first part of this thesis aimed to identify miRNA expression profiles that could be used to actuate transgene expression in the context of biopharmaceutical production. miRNA expression data from cell lines with different glutamine requirements was investigated in an attempt to identify glutamine responsive miRNAs. In addition, the analysis of a novel CHO miRNA expression dataset from a fed batch process resulted in the identification of interesting miRNA clusters exhibiting expression profiles that could be matched to particular growth phases. The second part of the work involved the investigation of temperature-induced miRNA expression changes in order to build a temperature dependent transgene expression control system using miRNA sponges. Temperature responsive miRNAs were identified and validated. While providing evidence that miRNA sponges can be used as molecular sensors and modulate gene expression, our results indicate that temperature-driven changes of miRNA expression are unlikely to be used as a robust gene control system. Finally, transgene expression control by combining UTR secondary structure and miRNAs was investigated. We showed that miRNA-toehold switches are able to repress transgene expression repression in a sequence specific manner although a robust ON/OFF function could not be achieved. Using a small library of synthetic 5'-UTR, the effect of several structural and sequence features in the miRNA-toehold was also investigated. Sequence determinants such as upstream ORFs, AU-rich regions and kozak environment showed greater effects on transgene expression than the 5'-UTR local secondary structure features. In summary, the work described in this thesis represents the first attempt to implement miRNA-based strategies to directly control transgene expression in CHO cells while highlighting the difficulties that need to be overcome for this strategy to be effective.
Author: Niall Barron
Publisher: Springer Science & Business Media
ISBN: 9400751281
Category : Medical
Languages : en
Pages : 126
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Book Description
Focused manuscript on the potential use/role of miRNAs in bioprocessing, specifically the production of complex proteins in mammalian cells. With that in mind I propose a draft list of topics/chapters along the following lines: Intro on CHO/bioprocessing/engineering challenges to set scene, Genomic organization, biogenesis and mode of action, Identifying miRNA targets: Computational prediction, transcriptomics, proteomices, UTR analysis, etc., miRNA expression in Chinese Hamster Ovary cells, miRNAs as engineering targets: pathway manipulation to impact bioprocess phenotypes, miRNAs as biomarkers, Detection methods: Northern, PCR, hybridization arrays, Next Gen Seq, Manipulation of expression in cultured cells: Transient/stable disregulation, Knockout.
Author: Ankur Solanki
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Chinese Hamster Ovary (CHO) cells are the biopharmaceutical industry's "mini biofactories" for the production of complex, post-translationally modified therapeutic proteins. In order to address the ever-growing market need for these recombinant proteins, various genetic engineering tools have been employed. Here, we describe genetic approaches to improve CHO cell culture longevity, with a view to increasing overall process yield via the manipulation of two miRNAs: Let-7a and miR-7. Previous miRNA profiling studies in our laboratory and in the published literature helped in the identification of these miRNAs, which have shown to be disregulated in various tumor types and are key regulators of the cell cycle. Therefore, this stimulated the interest of our research group to manipulate these miRNAs in CHO cells with a view to positively impact bioprocess-relevant CHO cell phenotypes. In the first approach, we used a Let-7 sponge decoy vector to deplete endogenous Let-7 levels with a view to increasing culture longevity and productivity of CHO-K1 SEAP expressing cells. Despite let-7 having a recognised role in deregulated cell growth no improvement was observed in stable, sponge-transfected clones. Out of a panel of 40 clones, we observed only two with improved cellular viability in 24 well plate format, however, the results were not reproduced in a 5 mL scale-up batch study. In the second approach, we targeted a previously verified miRNA for improved CHO cell growth and productivity i.e. miR-7, using a bacterial genome-editing tool, CRISPR-Cas9. A considerable amount of optimisation work was performed to establish the CRISPR system for use in the lab, initially using eGFP as model target gene in CHO cells. Finally we designed single guide RNAs to target Cas9 to the miR-7a-5p genomic locus to disrupt miR-7 in order to enhance growth of a CHO-K1 cell line producing an IgG-1. We estimated ~ 40% targeting efficiency of miRNAs using this approach. After an extensive screen, one stable clone was identified with what appeared to be a heterozygous deletion of one miR-7a copy. We demonstrate that CRISPR-Cas9 can be successfully used to target miRNA loci in the CHO genome but that functional knockout may be more difficult compared to protein coding genes.
Author: Ralph A. Bradshaw
Publisher: Academic Press
ISBN: 0080920918
Category : Science
Languages : en
Pages : 3188
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Book Description
Handbook of Cell Signaling, Three-Volume Set, 2e, is a comprehensive work covering all aspects of intracellular signal processing, including extra/intracellular membrane receptors, signal transduction, gene expression/translation, and cellular/organotypic signal responses. The second edition is an up-to-date, expanded reference with each section edited by a recognized expert in the field. Tabular and well illustrated, the Handbook will serve as an in-depth reference for this complex and evolving field. Handbook of Cell Signaling, 2/e will appeal to a broad, cross-disciplinary audience interested in the structure, biochemistry, molecular biology and pathology of cellular effectors. Contains over 350 chapters of comprehensive coverage on cell signaling Includes discussion on topics from ligand/receptor interactions to organ/organism responses Provides user-friendly, well-illustrated, reputable content by experts in the field
Author: Ralph A. Bradshaw
Publisher: Academic Press
ISBN: 0123822149
Category : Science
Languages : en
Pages : 552
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Book Description
This must-have cell signaling title will appeal to researchers across molecular biology, biochemistry, cell biology and genetics. The articles are written and edited by experts in the field and emphasize signaling to and from intracellular compartments including transcriptional responses to cytoplasmic and nuclear signaling events, chromatin remodeling and stress responses, the regulation of endoplasmic reticulum function, control of cell cycle progression and apoptosis and the modulation of the activities of mitochondria and other organelles. Articles written and edited by experts in the field Thematic volume covering regulation of endoplasmic reticulum function, regulation of cell cycle progression, and quality control and assurance in mitochondrion events Up-to-date research on events in membrane proteins and proteins of intracellular matrix
Author: Hansjörg Hauser
Publisher: Walter de Gruyter GmbH & Co KG
ISBN: 3110278960
Category : Science
Languages : en
Pages : 718
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Book Description
This book introduces fundamental principles and practical application of techniques used in the scalable production of biopharmaceuticals with animal cell cultures. A broad spectrum of subjects relevant to biologics production and manufacturing are reviewed, including the generation of robust cell lines, a survey of functional genomics for a better understanding of cell lines and processes, as well as advances in regulatory compliant upstream and downstream development. The book is an essential reference for all those interested in translational animal cell-based pharmaceutical biotechnology.
Author: Vijai Singh
Publisher: Academic Press
ISBN: 0323859860
Category : Science
Languages : en
Pages : 487
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Book Description
New Frontiers and Applications of Synthetic Biology presents a collection of chapters from eminent synthetic biologists across the globe who have established experience and expertise working with synthetic biology. This book offers several important areas of synthetic biology which allow us to read and understand easily. It covers the introduction of synthetic biology and design of promoter, new DNA synthesis and sequencing technology, genome assembly, minimal cells, small synthetic RNA, directed evolution, protein engineering, computational tools, de novo synthesis, phage engineering, a sensor for microorganisms, next-generation diagnostic tools, CRISPR-Cas systems, and more. This book is a good source for not only researchers in designing synthetic biology, but also for researchers, students, synthetic biologists, metabolic engineers, genome engineers, clinicians, industrialists, stakeholders and policymakers interested in harnessing the potential of synthetic biology in many areas. Offers basic understanding and knowledge in several aspects of synthetic biology Covers state-of-the-art tools and technologies of synthetic biology, including promoter design, DNA synthesis, DNA sequencing, genome design, directed evolution, protein engineering, computational tools, phage design, CRISPR-Cas systems, and more Discusses the applications of synthetic biology for smart drugs, vaccines, therapeutics, drug discovery, self-assembled materials, cell free systems, microfluidics, and more
Author: Wilfried Weber
Publisher: Humana Press
ISBN: 9781617794117
Category : Science
Languages : en
Pages : 393
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Book Description
The rapid expansion of synthetic biology is due to the design and construction of synthetic gene networks that have opened many new avenues in fundamental and applied research. Synthetic Gene Networks: Methods and Protocols provides the necessary information to design and construct synthetic gene networks in different host backgrounds. Divided into four convenient sections, this volume focuses on design concepts to devise synthetic gene networks and how mathematical models can be applied to the predictable engineering of desired network features. The volume continues by highlighting the construction and validation of biologic tools, describing strategies to optimize and streamline the host cell for optimized network performance, and covering how optimally designed gene networks can be implemented in a large variety of host cells ranging from bacteria over yeast and insect cells to plant and mammalian cell culture. Written in the successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Synthetic Gene Networks: Methods and Protocols serves as an invaluable resource for established biologists, engineers, and computer scientists or novices just entering into the rapidly growing field of synthetic biology
Author: Anne Le
Publisher: Springer
ISBN: 331977736X
Category : Medical
Languages : en
Pages : 186
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Book Description
Genetic alterations in cancer, in addition to being the fundamental drivers of tumorigenesis, can give rise to a variety of metabolic adaptations that allow cancer cells to survive and proliferate in diverse tumor microenvironments. This metabolic flexibility is different from normal cellular metabolic processes and leads to heterogeneity in cancer metabolism within the same cancer type or even within the same tumor. In this book, we delve into the complexity and diversity of cancer metabolism, and highlight how understanding the heterogeneity of cancer metabolism is fundamental to the development of effective metabolism-based therapeutic strategies. Deciphering how cancer cells utilize various nutrient resources will enable clinicians and researchers to pair specific chemotherapeutic agents with patients who are most likely to respond with positive outcomes, allowing for more cost-effective and personalized cancer therapeutic strategies.
Author: Mohamed Al-Rubeai
Publisher: Springer Science & Business Media
ISBN: 9048122457
Category : Medical
Languages : en
Pages : 259
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Book Description
Mammalian cell lines command an effective monopoly for the production of therapeutic proteins that require post-translational modifications. This unique advantage outweighs the costs associated with mammalian cell culture, which are far grater in terms of development time and manufacturing when compared to microbial culture. The development of cell lines has undergone several advances over the years, essentially to meet the requirement to cut the time and costs associated with using such a complex hosts as production platforms. This book provides a comprehensive guide to the methodology involved in the development of cell lines and the cell engineering approach that can be employed to enhance productivity, improve cell function, glycosylation and secretion and control apoptosis. It presents an overall picture of the current topics central to expression engineering including such topics as epigenetics and the use of technologies to overcome positional dependent inactivation, the use of promoter and enhancer sequences for expression of various transgenes, site directed engineering of defined chromosomal sites, and examination of the role of eukaryotic nucleus as the controller of expression of genes that are introduced for production of a desired product. It includes a review of selection methods for high producers and an application developed by a major biopharmaceutical industry to expedite the cell line development process. The potential of cell engineering approch to enhance cell lines through the manipulation of single genes that play important roles in key metabolic and regulatory pathways is also explored throughout.
