Membrane Binding of Plasmid DNA and Endocytic Pathways Are Involved in Electrotransfection of Mammalian Cells

Abstract

Electric field mediated gene delivery or electrotransfection is a widely used method in various studies ranging from basic cell biology research to clinical gene therapy. Yet, mechanisms of electrotransfection are still controversial. To this end, we investigated the dependence of electrotransfection efficiency (eTE) on binding of plasmid DNA (pDNA) to plasma membrane and how treatment of cells with three endocytic inhibitors (chlorpromazine, genistein, dynasore) or silencing of dynamin expression with specific, small interfering RNA (siRNA) would affect the eTE. Our data demonstrated that the presence of divalent cations (Ca2+ and Mg2+) in electrotransfection buffer enhanced pDNA adsorption to cell membrane and consequently, this enhanced adsorption led to an increase in eTE, up to a certain threshold concentration for each cation. Trypsin treatment of cells at 10 min post electrotransfection stripped off membrane-bound pDNA and resulted in a significant reduction in eTE, indicating that the time period for complete cellular uptake of pDNA (between 10 and 40 min) far exceeded the lifetime of electric field-induced transient pores (∼10 msec) in the cell membrane. Furthermore, treatment of cells with the siRNA and all three pharmacological inhibitors yielded substantial and statistically significant reductions in the eTE. These findings suggest that electrotransfection depends on two mechanisms: (i) binding of pDNA to cell membrane and (ii) endocytosis of membrane-bound pDNA.

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

“…76 Recent study from our lab showed that pDNA could bind to membrane during electrotransfection and continuously be internalized by cells over a period of time that was much longer than the theoretical lifetime of transient pores induced by pulsed electric fields. 48 In addition, we demonstrated that a clathrin-mediated endocytosis mechanism could actively transport plasmids across the membrane and facilitate electrotransfection. 77 The accumulating evidence supports the notion that electrotransfection of pDNA is mediated by endocytosis.…”

“…It is known that macropinocytosis can be distinguished from other endocytic pathways, such as clathrin-mediated endocytosis (CME) and phagocytosis, because it is independent of a GTP-binding protein, dynamin 2. 82 In our previous study, 48 we have addressed the role of dynamin-2-dependent endocytosis played in electrotransfection. By using a specific inhibitor (i.e., dynasore), we demonstrated dynamin-2-dependent endocytosis could be responsible for electrotransfection in certain cells.…”

“…Cells were obtained from Duke University Cell Culture Facility (CCF) and cultured as previously described. 48,77 In brief, cells were grown as monolayers in high-glucose DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone) and Antibiotic-Antimycotic (Invitrogen) at 37 C in a humidified incubator with 5% CO 2 and passaged every 36-48 hr.…”

“…17,34,44,45 Whereas controversies remain over the biophysical nature of electrotransfection, there has been increasing evidence from various studies implicating the involvement of biological processes in the uptake and intracellular transport of electrotransfected pDNA. 46,47 Specifically, electrotransfection may involve endocytic pathways, 48 which are essential for nutrient uptake and regulation of receptor-mediated cell signaling. Based on the types of vesicles involved in molecular transport, endocytosis can be divided into several categories, and one of them is macropinocytosis, which is a mode of nonselective fluid phase endocytosis.…”

A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors

“…In particular, it has been suggested that the relatively low-pressure renal venous system would provide a feasible site for hydrodynamic injections, by facilitating exogenous transgene delivery and limiting injection-related tissue damage (38,47). Plasmids (7,16,25,30,31,36,(45)(46)(47)49), baculovirus (1,2,23,24,39), and adenovirus (21,29,35,41,45) vectors have been previously identified as useful agents for in vitro and in vivo mammalian gene delivery. These vectors are reported to generate relatively low levels of pathogenicity and toxicity, are capable of carrying large DNA inserts, and can be generated in sufficiently high concentrations to provide sustained transgene expression (22,23,29,30).…”