Expression of DNA sensors and determination of cell death after gene electrotransfer of plasmid DNA in normal murine cells

Gensko zdravljenje je eden od novejših pristopov zdravljenja različnih bolezni. Z vnosom nukleinskih kislin v celico lahko popravimo napako ali pa celici dodamo novo funkcijo. Ena od metod vnosa nukleinskih kislin je genski elektroprenos, pri katerem za vnos genov uporabljamo elektroporacijo. Elektroporacija je fizikalna metoda, ki s pomočjo električnega polja začasno omogoči večjo prepustnost membrane in s tem poveča učinkovitost prenosa snovi. V magistrski nalogi smo želeli ugotoviti, ali z genskim elektroprenosom plazmidne DNA v celico povzročimo povišano izražanje citosolnih senzorjev DNA in ali ti posledično aktivirajo posebno obliko celične smrti, piroptozo. Pri tem smo uporabili dve normalni mišji celični liniji, endotelijske celice 2H11 in keratinocite Kera-308. S qPCR smo določali, kateri senzorji DNA se v celicah povišano izražajo. Pri celicah Kera-308 se je povišano izražal tudi gen za senzor Aim2, ki je pomemben v aktivaciji piroptoze. Ker nas je še posebej zanimala ta oblika celične smrti, smo nadaljnje teste izvajali le na celicah Kera-308. Označili smo izpostavljen fosfatidilserin, določili koncentracijo izplavljenega ATP in aktivnost kaspaze-1. Kljub povišanemu izražanju Aim2 smo na podlagi nadaljnjih testov ugotovili, da do piroptoze v Kera-308 ne pride.Gene therapy is a newly developed new approach for the treatment of different diseases. With the introduction of nucleic acids into cells, we can repair the cell’s deficiency or add a new function to the cell. One of the methods for the introduction of nucleic acids into the cells is electroporation, a physical method which is using an electric field to increase the cell membrane permeability and thus allowing the uptake of molecules. The aim of our master thesis was to determine the increased expression of cytosolic DNA sensors after gene electrotransfer of plasmid DNA and to find out whether this leads to pyroptosis. We used two murine cell lines, the endothelial line 2H11 and keratinocytes from line Kera-308. The increased expression of cytosolic DNA sensors and cytokines was demonstrated by qPCR. In Kera-308 cells, we also observed a significant increase in expression of mRNA for Aim2, which is involved in the activation of pyroptosis. Since we were interested in pyroptosis, we continued our research with Kera-308 cells. We stained the cells for phosphatidylserine exposure, measured the concentration of ATP in the cells’ medium and measured the caspase-1 activity. Even though we detected significantly increased expression of Aim2, further tests for pyroptosis, could not confirm that pyroptosis is present in Kera-308 cells after gene electrotransfer