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Genetic Modification of Glutamine Metabolism in Hybridomas: Effect On Cell Physiology.

Bell, Sarah Loraine. (1990) Genetic Modification of Glutamine Metabolism in Hybridomas: Effect On Cell Physiology. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

The physiology of a murine hybridoma has been investigated during stirred batch culture. Use of cubic spline interpolation and differentiation of the experimental data has revealed a three-phase culture physiology which characterises growth and antibody synthesis. During this triphasic sequence, antibody synthesis was found to be nongrowth associated, and data was obtained indicating that the energy charge of the cell is involved in the regulation of antibody synthesis. Firm evidence of ammonia assimilation was obtained, indicating that it was utilised by incorporation into alanine by glutamate dehydrogenase and subsequent transamination. Glutamine was identified as a key metabolite required for proliferation by the hybridoma, and the cell-line found to be incapable of adaptation to growth in the absence of glutamine. Levels of glutamine synthetase (GS)-mRNA and the accompanying enzyme activity were found to vary throughout the duration of batch culture in a manner consistent with the three phases. Levels of GS expressed by the hybridoma, however, were insufficient to enable the synthesis of adequate amounts of endogenous glutamine for its metabolic requirements, even after amplification of the GS gene copy number. The hybridoma was transformed to glutamine independence with a gene coding for glutamine synthetase. Southern hybridization studies showed that the gene was incorporated into the genomic DNA. Levels of GS-mRNA and the associated production of active enzyme were found to be elevated. The effect of modifying glutamine metabolism on cellular physiology was assessed, and compared to that of the untransformed hybridoma. In the GS transformed cell-line it was observed that; cell yields were increased, the specific growth rate had decreased, there was a greater overall percentage of glucose utilised, and a lower ratio of lactate-produced to glucose-consumed occurred. Neither glutamine or ammonia were detected in the growth medium. However, lower antibody titres were obtained than in the parent hybridoma.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Bell, Sarah Loraine.
Date : 1990
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1990.
Depositing User : EPrints Services
Date Deposited : 24 Apr 2020 15:27
Last Modified : 24 Apr 2020 15:27
URI: http://epubs.surrey.ac.uk/id/eprint/854869

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