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With the aim of better understanding and optimizing gene transfection by electroporation (GET) we tested the effect of different biological and electrical parameters on GET efficiency in vitro. Due to possibility that microenvironment in applications of in vivo is slightly acidic, we studied the effects of slightly acidic electroporation and recovery medium on cell membrane permeabilization, cell survival and GET. Slightly acidic (pH 6.5) electroporation medium did not affect permeabilization threshold, but the survival of cells was better if slightly acidic electroporation and recovery medium were used. However, slightly acidic electroporation and recovery medium decreased GET. Further, to avoid or to mitigate pain and muscle contractions associated with the application of high-voltage electrical pulses in vivo we showed that with short bipolar pulses with a repetition rate on the order of tens or hundreds of kHz delivered in bursts of low frequency (HF-BP) and nanosecond pulses we can achieve comparable GET in vitro as with longer monopolar pulses, but only with high concentrations of plasmid DNA (pDNA). Although we were able to achieve similar percent of transfected cells, the number of transgenes produced was higher with longer monopolar pulses. We showed that GET efficiency depends on pulse parameters, pDNA concentration and differs between cell lines. Time dynamics of transgene expression were comparable between different pulse protocols but differed greatly between cell lines. Finally, a review, of current understanding of the mechanisms by which electric field can aid pDNA in overcoming the barriers encountered on the way to the cell nucleus was performed with the hope that gathered knowledge will enable further and faster development of GET in vitro as well as in in vivo across various cell lines, tissues, and species. V doktorskem delu smo raziskali vpliv parametrov elektroporacije na učinkovitost genske elektrotransfekcije (GET) in vitro, z namenom boljšega razumevanja in izboljšanja učinkovitosti GET. Ker je mikrookolje pri aplikacijah in vivo lahko rahlo kislo, smo preučili tudi učinke rahlo kislega medija (pH 6,5) na permeabilizacijo celične membrane, celično preživetje in GET. Po elektroporaciji in okrevanju celic v rahlo kislem mediju nismo opazili razlik v permeabilizaciji celične membrane, preživetje celic pa je bilo boljše, kot pri celicah, ki so bile elektroporirane in so okrevale v rastnem mediju. Nasprotno sta elektroporacija in okrevanje v rahlo kislem mediju zmanjšala učinkovitost GET. Da bi se izognili ali ublažili bolečine in mišične kontrakcije, ki spremljajo dovajanje visokonapetostnih električnih pulzov za GET in vivo smo pokazali, da lahko z nizkofrekvenčnimi vlaki kratkih bipolarnih pulzov, ki si sledijo s frekvencami nekaj deset ali sto kHz (HF-BP), in nanosekundnimi pulzi dosežemo GET in vitro primerljivo z GET z daljšimi monopolarnimi pulzi, vendar le z višanjem koncentracije plazmidne DNK (pDNK). Kljub temu, da smo dosegli primerljiv odstotek transfeciranih celic, je bila količina nastalega transgena večja z daljšimi monopolarnimi pulzi. Učinkovitost GET je odvisna od parametrov pulzov, koncentracije pDNK in se razlikuje pri različnih celičnih linijah. Časovna dinamika izražanja transgena je primerljiva med milisekundami, mikrosekundami, HF-BP in nanosekundnimi pulzi, se pa močno razlikuje med celičnimi linijami. Naredili smo tudi pregled trenutnega razumevanja mehanizmov GET, z namenom, da bo zbrano znanje o mehanizmih in identifikacija obstoječih vrzeli v znanju, omogočilo hitrejše izboljšanje učinkovitosti GET in vitro kot tudi in vivo v različnih celicah, tkivih in bioloških vrstah. |
