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microRNAs as potential tools for ‘miR’aculous CHO cell phenotypes in Bioprocessing systems

Solanki, Ankur (2016) microRNAs as potential tools for ‘miR’aculous CHO cell phenotypes in Bioprocessing systems. Master of Science thesis, Dublin City University.

Abstract
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.
Metadata
Item Type:Thesis (Master of Science)
Date of Award:November 2016
Refereed:No
Supervisor(s):Barron, Niall and Clynes, Martin
Uncontrolled Keywords:Chinese hamster ovary; CHO; miRNA; CRISPR-Cas9
Subjects:Biological Sciences > Biotechnology
Biological Sciences > Cell biology
Biological Sciences > Molecular biology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > National Institute for Cellular Biotechnology (NICB)
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:21328
Deposited On:17 Nov 2016 11:48 by Niall Barron . Last Modified 19 Jul 2018 15:08
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