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Phenotypic changes in Chinese Hamster Ovary (CHO) cells during continuous fermentation.
Štor, Jerneja (Author), Oven, Irena (Mentor) More about this mentor... This link opens in a new window, Borth, Nicole (Co-mentor)

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Abstract
CHO 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.

Language:English
Keywords:CHO, continuous bioprocess, DNA methylation, restriction enzymes, HpaII, MspI, fluorescein-12-dUTP, DAC, resveratrol, RNA interference
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2019
Publisher:[J. Štor]
UDC:602.4:576.34:575.21(043.2)
COBISS.SI-ID:18598659 This link opens in a new window
Views:498
Downloads:61
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Secondary language

Language:Slovenian
Title:Fenotipske spremembe ovarijskih celic kitajskega hrčka (CHO) med kontinuirnim bioprocesom.
Abstract:
S kontinuirnim bioprocesom smo gojili sesalski celični liniji CHO K1 8mM L-Gln in CHO K1 0mM L-Gln. S konstantim pretokom medija skozi sistem smo ustvarili okolje, v katerem se lastnosti sistema s časom ne spreminjajo. To imenujemo stabilna faza sistema. S spreminjanjem pretoka skozi sistem smo celicam ustvarili različna stabilna okolja. Odziv celic na spreminjanje okolja smo ovrednotili z določanjem viablinosti kulture, celične gostote kulture, velikosti celic in stopnjo porabe/produkcije glutaminske kisline, glukoze, glutamina, amoniaka in laktata ter z meritvami metilacije DNA. Rezultati so pokazali, da spreminjanje okolja celic s pretokom vpliva na aktivnost celic, prav tako pa smo opazili razlike med testiranima celičnima linijama. Opazili smo tudi vpliv spreminjajočega okolja na metilacijo DNA, vendar razlik med celičnima linijama ni bilo opaziti. Namen naloge je bil tudi razviti metodo za določanje nivoja metilacije DNA z uporabo restrikcijskega encima odvisnega od metilacije DNA, HpaII, in barvanjem nastalih fragmentov s fluorescentnim barvilom, fluorescin-12-dUTP. Izkazalo se je, da je metoda obetaven način za določanje metilacije DNA, vendar bo za pridobitev natančnih rezultatov potrebna optimizacija. Težave, s katerimi smo se soočali, so bile določanje koncentracije DNA v barvanih vzorcih. To je ključno za natančno določitev nivoja metilacije DNA, saj je količina vezanega barvila odvisna od količine DNA v vzorcu. Prav tako smo za natančno določitev metilacije vključili encimski par MspI, ki je neodvisen od metilacije, vendar se je izkazalo da metilacija vseeno vpliva na aktivnost le-tega.

Keywords:CHO, kontinuirni bioproces, metilacija DNA, restrikcijski encimi, HpaII, MspI, fluorescein-12-dUTP, DAC, resveratrol, RNA interferenca

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