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Genska elektrotransfekcija je biotehnološka metoda, ki za vnos genskega materiala v biološke celice izkorišča pojav elektroporacije. Celice izpostavimo kratkim visokonapetostnim električnim impulzom, ki povzročijo začasno povečano prepustnost celične membrane in na ta način olajšajo vstop genskega materiala v celice. V magistrskem delu smo se posvetili modelu, s katerim lahko izračunamo število pDNK v celici in celičnih podstrukturah ob določenem času. Število pDNK molekul, ki vstopa v celico, ni za vse celice enako. Odvisno je od lokacije celice in pDNK molekul v transfekcijskem pufru ter aktivnosti celičnih podstruktur. Model predvideva, da se za vstop molekul pDNK v celico in transport po njej izkorišča endocitoza. Kot rezultat modela dobimo število pDNK molekul v celičnih podstrukturah. [1] Eksperimenti so bili izvedeni na celični liniji, pridobljeni iz ovarija kitajskega hrčka (CHO-K1). Kot pDNK pa so uporabili pEGFP-N1, ki kodira zeleni fluorescentni protein (GFP). Iz meritev fluorescence skupkov pDNK molekul na celični membrani in meritev fluorescence v jedru celic smo izračunali delež pDNK molekul. Eksperimentalne rezultate smo primerjali z rezultati modela. Določil smo tudi vse hitrostne konstante modela tako, da se krivulja, ki predstavlja število pDNK molekul v jedru, prilega časovnemu poteku genske ekspresije. Genetic electrotransfection is a biotechnological method that exploits the phenomenon of electroporation to entering genetic material into cells. The cells are exposed to short, high voltage electrical impulses, which cause temporarily increased cell membrane permeability, making it easier for genetic material to enter in cells. In the master's thesis, we focused on a model that can calculate the number of pDNA in a cell and cell substructures at a given time. The number of pDNA molecules entering a cell is not the same for all cells. It depends on the location of the cell and the pDNA molecules in the transfection buffer and the activity of cellular substructures. The model assumes that endocytosis is used to enter and transport pDNA molecules into the cell. As a result of the model, we obtain the number of pDNA molecules in cellular substructures. [1] The experiments were carried out on a cell line derived from a Chinese hamster ovary (CHO-K1). PEGFP-N1, which encodes a green fluorescent protein (GFP), was used as a pDNA. From measurements of the fluorescence of the pDNA molecules on the cell membran and the measurement of fluorescence in nucleus, we calculated the proportion of pDNA molecules that were successfully expressed in the core. Experimental results were compared with the results of the model. We have also determined all the model rate constants so that the curve representing the number of pDNA molecules in the core fits the time course of genetic expression. |
