The overarching objective of this thesis was the development of an improved expression cell line for recombinant proteins, in which a transgene of interest can be inserted into a highly active gene locus using recombinase mediated cassette exchange. Random integration of transgenic DNA is a common route to achieve transgene expression. However, randomly integrated transgenes are susceptible to gene silencing over time, and do not show stable expression for extended periods in culture. Furthermore, every new cell clone generated requires regulatory approval. The improvement of expression strategies may significantly reduce the time required to bring novel recombinant protein products to the market. In order to identify a suitable expression locus for the integration of transgene expression cassettes, total protein samples were derived from the production cell lines HEK293 and CHO. Highly expressed proteins were isolated via 2D PAGE, and identified via peptide mass fingerprinting. Their promoter regions were then validated to express a recombinant transgene in HEK293 cells. The long term stability of these promoter regions was also assessed. Direct gene targeting of the highly active gene loci may or may not be possible in typical producer cell lines. Targeting of a murine homologue to these highly expressed CHO/HEK293 loci may be more efficient in a murine stem cell line. The transfer of a modified allele from HM1 murine embryonic stem cells, into a somatic cell line (HC11) was demonstrated in this thesis. These validated methods were then explored for the generation of viable HM1-HEK293 and HM1-CHO fusion hybrids. For these experiments, a fluorescence based fusion assay was generated, validated and used for in-situ monitoring of the cell fusion process. The random integration of transgenic DNA into mammalian genomes typically results in a highly unpredictable integration architecture. RMCE at such loci would be inefficient. However, a highly efficient RMCE reaction at (rare) single copy transgene integrations, may be possible under the correct conditions. RMCE at randomly integrated loci could therefore be more beneficial (for transgene expression) than random integration alone. This thesis explores this concept with the use of a randomly integrated RMCE platform, and subsequent selection of cell lines post RMCE attempts at these loci CRISPR/Cas9 technology was also applied to a highly expressed locus in HEK293 cells. A framework for successful direction of double strand breaks to a defined locus is demonstrated in this work. The methods used to achieve this can therefore be built upon for the homologous recombination of a transgenic cassette, into a highly expressed locus in HEK293 cells. Monoclonal antibodies have dominated the biologics market for over two decades, and mammalian expression systems are well suited to their production. The work in this thesis attempts to raise and verify antibody molecules against a potential tumour marker using hybridoma and phage display technologies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:715451 |
| Date | January 2016 |
| Creators | Knowles, Christopher |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=231905 |
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