MicroRNAs as Potential Tools for 'miR'aculous CHO Cell Phenotypes in Bioprocessing Systems PDF Download
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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: 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: 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: Noelia Sanchez
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Author: Ulf Schmitz
Publisher: Springer Nature
ISBN: 3031083563
Category : Medical
Languages : en
Pages : 321
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Book Description
This book provides an update on the latest development in the field of microRNAs in cancer research with an emphasis on translational research. Since the early 2000s, microRNAs have been recognized as important and ubiquitous regulators of gene expression. Soon it became evident that their deregulation can cause human diseases including cancer. This book focuses on the emerging opportunities for the application of microRNA research in clinical practice. In this context, computer models are presented that can help to identify novel biomarkers, e.g. in circulating microRNAs, and tools that can help to design microRNA-based therapeutic interventions. Other chapters evaluate the role of microRNAs in immunotherapy, immune responses and drug resistance. Covering key topics on microRNAs in cancer research this book is a valuable resource for both emerging and established microRNA researchers who want to explore the potential of microRNAs as therapeutic targets or co-adjuvants in cancer therapies.
Author: Ricardo Valdés-Bango Curell
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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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: Debmalya Barh
Publisher: Academic Press
ISBN: 0128047496
Category : Technology & Engineering
Languages : en
Pages : 645
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Book Description
Omics Technologies and Bio-Engineering: Towards Improving Quality of Life, Volume 1 is a unique reference that brings together multiple perspectives on omics research, providing in-depth analysis and insights from an international team of authors. The book delivers pivotal information that will inform and improve medical and biological research by helping readers gain more direct access to analytic data, an increased understanding on data evaluation, and a comprehensive picture on how to use omics data in molecular biology, biotechnology and human health care. Covers various aspects of biotechnology and bio-engineering using omics technologies Focuses on the latest developments in the field, including biofuel technologies Provides key insights into omics approaches in personalized and precision medicine Provides a complete picture on how one can utilize omics data in molecular biology, biotechnology and human health care
Author: W.T. Godbey
Publisher: Elsevier
ISBN: 1908818484
Category : Science
Languages : en
Pages : 435
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Book Description
An Introduction to Biotechnology is a biotechnology textbook aimed at undergraduates. It covers the basics of cell biology, biochemistry and molecular biology, and introduces laboratory techniques specific to the technologies addressed in the book; it addresses specific biotechnologies at both the theoretical and application levels.Biotechnology is a field that encompasses both basic science and engineering. There are currently few, if any, biotechnology textbooks that adequately address both areas. Engineering books are equation-heavy and are written in a manner that is very difficult for the non-engineer to understand. Numerous other attempts to present biotechnology are written in a flowery manner with little substance. The author holds one of the first PhDs granted in both biosciences and bioengineering. He is more than an author enamoured with the wow-factor associated with biotechnology; he is a practicing researcher in gene therapy, cell/tissue engineering, and other areas and has been involved with emerging technologies for over a decade. Having made the assertion that there is no acceptable text for teaching a course to introduce biotechnology to both scientists and engineers, the author committed himself to resolving the issue by writing his own. The book is of interest to a wide audience because it includes the necessary background for understanding how a technology works. Engineering principles are addressed, but in such a way that an instructor can skip the sections without hurting course content The author has been involved with many biotechnologies through his own direct research experiences. The text is more than a compendium of information - it is an integrated work written by an author who has experienced first-hand the nuances associated with many of the major biotechnologies of general interest today.
Author: Mallappa Kumara Swamy
Publisher: Springer Nature
ISBN: 9811372055
Category : Science
Languages : en
Pages : 492
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Book Description
Nature has consistently provided human beings with bioactive compounds that can be used directly as drugs or indirectly as drug leads. Some of the major classes of natural bioactive compounds include phenolics, alkaloids, tannins, saponins, lignin, glycosides, terpenoids, and many more. They possess a broad range of biological activities and are primarily useful in the treatment of various health issues. At the same time, the search for new and novel drugs is never-ending and, despite major advances in synthetic chemistry, nature remains an essential resource for drug discovery. Therefore, more and more researchers are interested in understanding the chemistry, clinical pharmacology, and beneficial effects of bioactive compounds in connection with solving human health problems. This book presents a wealth of information on natural metabolites that have been or are currently being used as drugs or leads for the discovery of new drugs. In addition, it highlights the importance of natural products against various human diseases, and their applications in the drug, nutraceuticals, cosmetics and herbal industries. Accordingly, the book offers a valuable resource for all students, educators, and healthcare experts involved in natural product research, phytochemistry, and pharmacological research.
Author: Jayanta Kumar Patra
Publisher: Springer
ISBN: 981106847X
Category : Medical
Languages : en
Pages : 489
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Book Description
This edited book, is a collection of 20 articles describing the recent advancements in the application of microbial technology for sustainable development of agriculture and environment. This book covers many aspects like agricultural nanotechnology, promising applications of biofuels production by algae, advancements and application of microbial keratinase, biocontrol agents, plant growth promoting rhizobacteria, bacterial siderophore, use of microbes in detoxifying organophosphate pesticides, bio-surfactants, biofilms, bioremediation degradation of phenol and phenolic compounds and bioprospecting of endophytes. This book intends to bring the latest research advancements and technologies in the area of microbial technology in one platform, providing the readers an up-to-date view on the area. This book would serve as an excellent reference book for researchers and students in the agricultural, environmental and microbiology fields.
Author: Malay K. Das
Publisher: Springer Nature
ISBN: 9811562555
Category : Science
Languages : en
Pages : 634
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Book Description
This book reviews the application of Nanobiotechnology in the development of Nanomedicine, while also discussing the latest trends and challenges in the clinical translation of Nanomedicine. Nanomedicine refers to the application of Nanotechnology to medicine and holds tremendous potential for achieving improved efficiency, bioavailability, dose response, personalized medicine and enhanced safety as compared to conventional medicines. The book first introduces readers to the basic concepts of Nanomedicine, and to technological advances in and applications of Nanotechnology in treatment, diagnosis, monitoring, and drug delivery. In turn, it reviews the current status of multi-functionalization strategies for using Nanoparticles in the targeted delivery of therapeutic agents. The book’s third and final section focuses on the regulatory and safety challenges posed by Nanomedicine, including industry and regulatory agencies’ efforts to address them.
