Phenotypic changes in Chinese Hamster Ovary (CHO) cells during continuous fermentation.Štor, Jerneja (Avtor) Oven, Irena (Mentor) Borth, Nicole (Komentor) CHOcontinuous bioprocessDNA methylationrestriction enzymesHpaIIMspIfluorescein-12-dUTPDACresveratrolRNA interferenceCHO K1 8mM L-Gln and CHO K1 0mM L-Gln cells were cultured in a continuous culture where steady state was used to study cell activity under stable environment. Moreover, by changing the dilution rate we studied how changes in environment affect cell performance. Culture viability, viable cell density, cell size and uptake/secretion of glutamic acid, glucose, glutamine, ammonium and lactate were evaluated. Our results show that changing the cell environment introduced changes in cell functionality. Also a change in cell activity between the tested cell lines was observed. An attempt was also made to develop a method to measure DNA methylation using the DNA methylation dependent restriction enzyme HpaII and fluorescein-12-dUTP. The effect of changing cell environment on DNA methylation was observed, but a difference between cell lines was not observed. The method appeared to be a promising way for determination of DNA methylation level although some steps of the method still need to be improved. We were facing a problem to determine DNA concentration in samples dyed with fluorescein-12-dUTP. This is necessary for quantification of the level of DNA methylation since the amount of bound dye depends on the amount of DNA in the sample. Moreover, for precise quantification of DNA methylation, DNA methylation non-dependent isoschizomer MspI was used, which appeared not to be completely nondependent on DNA methylation.[J. Štor]20192019-11-21 07:45:03Magistrsko delo/naloga112908UDK: 602.4:576.34:575.21(043.2)VisID: 175014COBISS_ID: 18598659sl