Micrornas As Potential Tools For Miraculous Cho Cell Phenotypes In Bioprocessing Systems
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Author | : Ankur Solanki |
Publisher | : |
Total Pages | : 0 |
Release | : 2016 |
Genre | : |
ISBN | : |
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 |
Total Pages | : 126 |
Release | : 2012-11-15 |
Genre | : Medical |
ISBN | : 9400751281 |
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 | : |
Total Pages | : |
Release | : 2013 |
Genre | : |
ISBN | : |
Author | : Debmalya Barh |
Publisher | : Academic Press |
Total Pages | : 645 |
Release | : 2017-12-01 |
Genre | : Technology & Engineering |
ISBN | : 0128047496 |
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 | : Edward Muntean |
Publisher | : Walter de Gruyter GmbH & Co KG |
Total Pages | : 186 |
Release | : 2022-10-03 |
Genre | : Technology & Engineering |
ISBN | : 3110644401 |
Among liquid chromatography methods, ion chromatography (IC) can be considered one of the most valuable analytical tools. This book covers the various applications of ion chromatography in food science, such as food quality control, food authentication and analysis of residues in food products. In addition, state-of-the-art instrumentation such as combustion IC, online eluent generation systems and capillary IC is also described.
Author | : Vipin Chandra Kalia |
Publisher | : Springer |
Total Pages | : 335 |
Release | : 2017-04-04 |
Genre | : Science |
ISBN | : 3319526669 |
This contributed volume sheds new light on waste management and the production of biofuels. The authors share insights into microbial applications to meet the challenges of environmental pollution and the ever- growing need for renewable energy. They also explain how healthy and balanced ecosystems can be created and maintained using strategies ranging from oil biodegration and detoxification of azo dyes to biofouling. In addition, the book illustrates how the metabolic abilities of microorganisms can be used in microbial fuel-cell technologies or for the production of biohydrogen. It inspires young researchers and experienced scientists in the field of microbiology to explore the application of green biotechnology for bioremediation and the production of energy, which will be one of the central topics for future generations.
Author | : Debmalya Barh |
Publisher | : CRC Press |
Total Pages | : 609 |
Release | : 2016-04-19 |
Genre | : Medical |
ISBN | : 1439850097 |
A reflection of the explosion of research and development in this field, OMICS: Biomedical Perspectives and Applications explores applications of omics in bioinformatics, cancer research and therapy, diabetes research, plant science, molecular biology, and neurosciences. A select editorial panel of experts discusses their cutting edge omics researc
Author | : Roberto Mazzoli |
Publisher | : Frontiers Media SA |
Total Pages | : 144 |
Release | : 2017-06-02 |
Genre | : |
ISBN | : 2889451852 |
Microorganisms have had a long and surprising history. They were “invisible” until invention of microscope in the 17th century. Until that date, although they were extensively (but inconsciously) employed in food preservation, beer and wine fermentation, cheese, vinegar, yogurt and bread making, as well as being the causative agents of infectious diseases, they were considered as “not-existing”. The work of Pasteur in the middle of the 19th century revealed several biological activities performed by microorganisms including fermentations and pathogenicity. Due to the urgent issue to treat infectious diseases (the main cause of death at those times) the “positive potential” of the microbial world has been neglected for about one century. Once the fight against the “evil” strains was fulfilled also thanks to the antibiotics, industry began to appreciate bacteria’s beneficial characteristics and exploit selected strains as starters for both food fermentations and aroma, enzyme and texturing agent production. However, it was only at the end of the 20th century that the probiotic potential of some bacteria such as lactic acid bacteria and bifidobacteria was fully recognized. Very recently, apart from the probiotic activity of in toto bacteria, attention has begun to be directed to the chemical mediators of the probiotic effect. Thanks also to the improvement of techniques such as transcriptomics, proteomics and metabolomics, several bioactive compounds are continuously being discovered. Bioactive molecules produced by bacteria, yeasts and virus-infected cells proved to be important for improving or impairing human health. The most important result of last years’ research concerns the discovery that a very complex network of signals allows communication between organisms (from intra-species interactions to inter-kingdom signaling). Based on these findings a completely new approach has arisen: the system biology standpoind. Actually, the different organisms colonizing a certain environmental niche are not merely interacting with each other as individuals but should be considered as a whole complex ecosystem continuously exchanging information at the molecular level. In this context, this topic issue explores both antagonistic compounds (i.e. antibiotics) and “multiple function” cooperative molecules improving the physiological status of both stimulators and targets of this network. From the applicative viewpoint, these molecules could be hopefully exploited to develop new pharmaceuticals and/or nutraceuticals for improving human health.
Author | : Muhammad Sarwar Khan |
Publisher | : CRC Press |
Total Pages | : 608 |
Release | : 2016-04-21 |
Genre | : Medical |
ISBN | : 1040056709 |
Applied Molecular Biotechnology: The Next Generation of Genetic Engineering explains state-of-the-art advances in the rapidly developing area of molecular biotechnology, the technology of the new millennium. Comprised of chapters authored by leading experts in their respective fields, this authoritative reference text:Highlights the latest omics-ba
Author | : John Flex Jackson |
Publisher | : Springer Science & Business Media |
Total Pages | : 238 |
Release | : 2003-07-11 |
Genre | : Medical |
ISBN | : 9783540002925 |
Whilst genetic transformation of plants is commonly viewed as a means of bringing about plant improvement, it has not so readily been recognised as a tool for analysing the function of plant genes. This book is unusual in that it focuses on the genetic transformation of a range of plants using a number of different methods. Many plants have been found to be quite difficult to transform, and so various techniques were developed. These techniques include: Agrobacterium suspension drops, electroporation, PEG, "whiskers", and various biolistic methods. A chapter on intellectual and property rights is included.