A comparison of the efficiency of DNA delivery into cancer cells using electroporation and K2 transfection system

Gensko zdravljenje je naprednejši način zdravljenja v medicini. Vnos genskega materiala v tarčne celice označujemo z izrazom transfekcija. Med nevirusnimi metodami se uveljavlja elektroporacija kot fizikalna metoda, pri kateri s pomočjo zunanjega električnega polja povečamo prepustnost celične membrane in s tem omogočimo vnos molekul v celice ter kemijski načini, med katerimi je najbolj pogost vnos z lipidi. Uspešnost vnosa DNA v sesalske celice je odvisna od številnih parametrov. Poleg učinkovitosti transfekcije in stopnje izražanja reporterskega proteina, je pri vseh metodah pomembno tudi preživetje celic in aktivacija gostiteljskega imunskega sistema. V magistrski nalogi smo želeli ugotoviti uspešnost genskega elektroprenosa z uporabo elektroporacije in transfekcijskega sistema K2, kot transfekcije posredovane s kationskimi lipidi. Dokazali smo, da transfekcijski sistem K2 predstavlja boljšo izbiro za in vitro vnos plazmidne DNA v celice mišjega melanoma. V primerjavi z elektroporacijo je bil delež transfeciranih celic po transfekciji s transfekcijskim sistemom K2 večji na celicah z nižjim metastatskim potencialom B16F1, intenziteta fluorescence pa je bila statistično značilno višja na celicah z višjim metastatskim potencialom B16F10. Transfekcijski sistem K2 se je izkazal za povsem netoksično metodo. Poleg tega ni aktiviral citosolnega senzorja DDX60, s katerim celice zaznavajo prisotnost tuje DNA, kar lahko vodi v aktivacijo signalnih poti in izzove apoptozo celic. Po genskem elektroprenosu smo zaznali povečano izražanje citosolnega senzorja DDX60, majhen delež preživelih celic in razlike v učinkovitosti transfekcije glede na izbrano podvrsto celic. Transfekcijski sistem K2 je bil uspešnejši tudi od kontrolne metode lipofekcije.Gene therapy is an advanced treatment approach in medicine. The process of introducing foreign genetic material into target cells is called transfection. Among non-viral methods, electroporation is a physical method in which the permeability of the cell membrane is increased by means of an applied external electric field, thus enabling delivery of molecules into the cells. Many chemical methods, such as cationic lipids are also used as in vitro carriers of nucleic acids. For transfection to be successful many different parameters need to be considered. In addition to the transfection efficiency and the expression level of reporter protein, cell survival and activation of the host immune system are also important. The aim of our study was to determine the transfection efficiency after gene electrotransfer by using electroporation and K2 transfection system as a transfection method mediated by cationic lipids. We have shown that the K2 transfection system represents a better choice for in vitro delivery of plasmid DNA into murine melanoma cells than electroporation. The number of transfected cells after transfection with K2 transfection system was statistically significantly higher for low metastatic potential B16F1 melanoma, while the fluorescence intensity was statistically significantly higher for high metastatic potential B16F10 melanoma compared to gene electrotransfer. In addition, K2 transfection system was completely nontoxic and did not activate the cytosolic sensor DDX60, which can lead to activation of signalling pathways and induce apoptosis. After gene electrotransfer we observed an increased expression of a cytosolic sensor DDX60, a decrease in cell survival, and differences in transfection efficiency depended on the cell line. K2 transfection system was also more successful than the control method lipofection