Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation

Abstract

Detailed kinetic data suggest that the direct transfer of plasmid DNA (YEp 351, 5.6 kbp, supercoiled, Mr approximately 3.5 x 10(6)) by membrane electroporation of yeast cells (Saccharomyces cerevisiae, strain AH 215) is mainly due to electrodiffusive processes. The rate-limiting step for the cell transformation, however, is a bimolecular DNA-binding interaction in the cell interior. Both the adsorption of DNA, directly measured with [32P]dCTP DNA, and the number of transformants are collinearly enhanced with increasing total concentrations [Dt] and [Cat] of DNA and of calcium, respectively. At [Cat] = 1 mM, the half-saturation or equilibrium constant is KD = 15 +/- 1 nM at 293 K (20 degrees C). The optimal transformation frequency is TFopt = 4.1 +/- 0.4 X 10(-5) if a single exponential pulse of initial field strength E0 = 4 kV cm-1 and decay time constant tauE = 45 ms is applied at [Dt] = 2.7 nM and 10(8) cells in 0.1 ml. The dependence of TF on [Cat] yields the equilibrium constants KCazero = 1.8 +/- 0.2 mM (in the absence of DNA) and K'Ca (at 2.7 nM DNA), comparable with and derived from electrophoresis data. In yeast cells, too, the appearance of a DNA molecule in its whole length in the cell interior is clearly an after-field event. At Eo = 4.0 kV cm-1 and T = 293 K, the flow coefficient of DNA through the porous membrane patches is Kto = 7.0 +/- 0.7 x 10(3)S-1 and the electrodiffusion of DNA is approximately 10 times more effective than simple diffusion: D/D0 approximately 10.3. The mean radius of these pores is rp = 0.39 +/- 0.05 nm, and the mean number of pores per cell (of size ø approximately 5.5 microns) is Np = 2.2 +/- 0.2 x 10(4). The maximal membrane area that is involved in the electrodiffusive penetration of adsorbed DNA into the outer surface of the electroporated cell membrane patches is only 0.023% of the total cell surface. The surface penetration is followed either by additional electrodiffusive or by passive (after-field) diffusive translocation of the inserted DNA into the cell interior. For practical purposes of optimal transformation efficiency, 1 mM calcium is necessary for sufficient DNA binding and the relatively long pulse duration of 20-40 ms is required to achieve efficient electrodiffusive transport across the cell wall and into the outer surface of electroporated cell membrane patches.

Factors influencing electroporation-mediated gene transfer to Stylosanthes guianensis (Aubl.) Sw. protoplasts

“…Transient membrane pores are caused by an increase in the dipole moment of hydrophilic phospholipid heads, which move in the same direction as the applied electric field and provoke highly localized dielectric breakages in membrane structure (Kinosita and Tsong, 1977;Neumann et al, 1982;Neumann et al, 1996). Irreversible pores which lead to cellular death are caused by longer pulses and stronger electric fields (Langridge et al, 1987;Shillito et al, 1985), and we found that this occurred with energy inputs of more than 5000 mJ ( Figure 3A) or electric fields stronger than 300 V.cm -1 ( Figure 3B).…”

“…In contrast to the data reported by Senaratna et al (1991), we found that non-electroporated protoplasts incubated with plasmid DNA under the same conditions for longer periods of time never showed transient expression of the reporter gene ( Figure 6C). In exponential wave electric circuits, the voltage decays exponentially in the electroporation buffer between the electrodes and the induced energy is dependent on the initially applied voltage and the charge accumulation capacity of the capacitor (Neumann et al, 1982(Neumann et al, , 1996. In this kind of circuit, the time constant (τ) is a function of the electrical resistance (R) and the capacitance (C) of the circuit according to the equation τ = RC, so that for a given capacitor (C = constant) electroporation buffers with reduced ionic force impose less resistance to the movement of charge through the circuit and pulse decay time is longer.…”

Optimization of an electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses

“…EDTA, a chelator of Ca 2+ and Mg 2+ (necessary for the enzymatic activity of DNases) decreased electroporation efficiency and resulted in lower cell viability. Neumann et al (1996) suggested that Ca 2+ and Mg 2+ are important for electroporation due to their interaction with the plasmid DNA, decreasing its negative charge and facilitating its interaction with the cell membrane. The results of post-pulse treatment in the present work, similar to those of pre-pulse treatment, suggest however that in electroporation these ions are more important for intra-cellular processes such as the transport of the plasmid through the nuclear pores.…”

Optimization of cutaneous electrically mediated plasmid DNA delivery using novel electrode

“…[22][23][24][25] Previous studies have evaluated this approach for in vivo plasmid DNA delivery to muscle. [26][27] In skin delivery, the electroporative-electrophoretic pulse combination significantly increased gene expression levels (Po0.05) when compared to each component pulse (Figure 3).…”