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: 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:
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Author: Stefan de Folter
Publisher: Humana Press
ISBN: 9781493990412
Category : Science
Languages : en
Pages : 363
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Book Description
This detailed volume provides a collection of protocols for the study of miRNA functions in plants. Beginning with coverage of miRNA function, biogenesis, activity, and evolution in plants, the book continues by guiding readers through methods on the identification and detection of plant miRNAs, bioinformatic analyses, and strategies for functional analyses of miRNAs. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Plant MicroRNAs: Method and Protocols aims to ensure successful results in the further study of this vital area of plant science.
Author: Gunter Jagschies
Publisher: Elsevier
ISBN: 0128125527
Category : Technology & Engineering
Languages : en
Pages : 1310
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Book Description
Biopharmaceutical Processing: Development, Design, and Implementation of Manufacturing Processes covers bioprocessing from cell line development to bulk drug substances. The methods and strategies described are essential learning for every scientist, engineer or manager in the biopharmaceutical and vaccines industry. The integrity of the bioprocess ultimately determines the quality of the product in the biotherapeutics arena, and this book covers every stage including all technologies related to downstream purification and upstream processing fields. Economic considerations are included throughout, with recommendations for lowering costs and improving efficiencies. Designed for quick reference and easy accessibility of facts, calculations and guidelines, this book is an essential tool for industrial scientists and managers in the biopharmaceutical industry. Offers a comprehensive, go-to reference for daily work decisions Covers both upstream and downstream processes Includes case studies that emphasize financial outcomes Presents summaries, decision grids, graphs and overviews for quick reference
Author: Krishnarao Appasani
Publisher: Cambridge University Press
ISBN: 9780521118552
Category : Science
Languages : en
Pages : 580
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Book Description
MicroRNAs (miRNAs) are RNA molecules, conserved by evolution, that regulate gene expressions and their recent discovery is revolutionising both basic biomedical research and drug discovery. Expression levels of MiRNAs have been found to vary between tissues and with developmental stages and hence evaluation of the global expression of miRNAs potentially provides opportunities to identify regulatory points for many different biological processes. This wide-ranging reference work, written by leading experts from both academia and industry, will be an invaluable resource for all those wishing to use miRNA techniques in their own research, from graduate students, post-docs and researchers in academia to those working in R&D in biotechnology and pharmaceutical companies who need to understand this emerging technology. From the discovery of miRNAs and their functions to their detection and role in disease biology, this volume uniquely integrates the basic science with industry application towards drug validation, diagnostic and therapeutic development. Forewords by: Sidney Altman, Yale University, Winner of the Nobel Prize in Chemistry, 1989 and Victor R. Ambros, Dartmouth Medical School, Co-discoverer of MicroRNAs
Author: Ralf Pörtner
Publisher: Springer Nature
ISBN: 3030798712
Category : Medical
Languages : en
Pages : 552
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Book Description
This contributed volume is dedicated towards the progress achieved within the last years in all areas of Cell Culture Engineering and Technology. It comprises contributions of active researchers in the field of cell culture development for the production of recombinant proteins, cell line development, cell therapy and gene therapy, with consideration of media development, process scale-up, reactor design, monitoring and control and model-assisted strategies for process design. The knowledge and expertise of the authors cover disciplines like cell biology, engineering, biotechnology and biomedical sciences. This book is conceived for graduate students, postdoctoral fellows and researchers interested in the latest developments in Cell Engineering.
Author: Tristan Vaughan
Publisher: John Wiley & Sons
ISBN: 3527340866
Category : Medical
Languages : en
Pages : 762
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Book Description
Branchenführende Big-Pharma-Unternehmen und erstklassige Forscher präsentieren grundlegende Konzepte und Herausforderungen bei proteinbasierten Pharmazeutika. Beinhaltet auch eine Einführung in die aus Sicht der Arzneimittelentwicklung fünf wesentlichen Anwendungsbereiche.
Author: Cesar Lopez-Camarillo
Publisher: CRC Press
ISBN: 1466576774
Category : Medical
Languages : en
Pages : 426
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Book Description
MicroRNA (miRNA) biology is a cutting-edge topic in basic as well as biomedical research. This is a specialized book focusing on the current understanding of the role of miRNAs in the development, progression, invasion, and metastasis of diverse types of cancer. It also reviews their potential for applications in cancer diagnosis, prognosis, and th
