Effects of pulse length and pulse strength on transfection by electroporation

The relative importance of pulse field strength E and pulse length tau 1/2 (half decay time of an exponential decay pulse) on the stable transfection frequency for HeLa or HUT-78 cells was investigated. Cells were transfected with plasmids containing the promoter and drug resistant genes pRSVgpt or pRSVneo by electroporation. The stable transfection frequency was assayed using the marker rescue technique. The transfection frequency increases with increasing values of E tau 1/2. For a given pulse length, the transfection frequency is proportional to the power of the pulse (E2 tau 1/2). Pulses with half decay times of 2.2 to 4.6 ms appear to be more efficient than 0.275 to 0.31 ms for stable transfection of HeLa cells.     

In vivo electroporation using an exponentially enhanced pulse: a new waveform

In vivo electroporation is currently accomplished by one of two types of common waveforms: exponential decay or square-wave pulses. The purpose of this report is to present a new electroporation waveform, the exponentially enhanced pulse (EEP). Pulsing protocols including the EEP resulted in high levels of luciferase expression in muscle and skin, equal to or greater than expression resulting from low-voltage, millisecond square-wave pulses. This high level of expression requires fewer pulses when using an EEP protocol. Therefore, similar or greater plasmid DNA expression levels are obtained using fewer pulses with the EEP protocol than with current protocols. This is the first report of this new waveform and shows the success of using protocols employing the EEP to deliver plasmid DNA to various tissue types.     

Quantitation of transient gene expression after electroporation.

The combination of a photometric reporter-gene assay, with transfection by electroporation, is potentially a rapid and sensitive tool for the study of genetic regulatory elements in many types of cells. We have found that the sensitivity, accuracy, and reproducibility of the technique is greatly improved by the inclusion of appropriately chosen carrier DNA as the primary DNA species present during electroporation. By using high levels of carrier, the activities of constructs of differing sizes can be quantitatively compared, active constructs can be assayed with sub-microgram amounts of plasmid, and the activities of the constructs are linear over a wide concentration of DNA. In addition, the activity of miniprep DNA can be screened without purification on CsCl gradients giving activities equal to CsCl-purified DNA. This is extremely useful when doing preliminary screening of large numbers of constructs for promoter or enhancer activities. We report the results of testing various types of DNA as carrier, and the parameters for optimizing its use.     

Temperature measurement on neurological pulse generators during MR scans

According to manufacturers of both magnetic resonance imaging (MRI) machines, and implantable neurological pulse generators (IPGs), MRI is contraindicated for patients with IPGs. A major argument for this restriction is the risk to induce heat in the leads due to the electromagnetic field, which could be dangerous for the surrounding brain parenchyma. The temperature change on the surface of the case of an ITREL-III (Medtronic Inc., Minneapolis, MN) and the lead tip during MRI was determined. An anatomical realistic and a cubic phantom, filled with phantom material mimicking human tissue, and a typical lead configuration were used to imitate a patient who carries an IPG for deep brain stimulation. The measurements were performed in a 1.5 T and a 3.0 T MRI. 2.1°C temperature increases at the lead tip uncovered the lead tip as the most critical part concerning heating problems in IPGs. Temperature increases in other locations were low compared to the one at the lead tip. The measured temperature increase of 2.1°C can not be considered as harmful to the patient. Comparison with the results of other studies revealed the avoidance of loops as a practical method to reduce heating during MRI procedures.     

A quantitative study of electroporation showing a plateau in net molecular transport.

Electroporation is believed to involve a temporary structural rearrangement of lipid bilayer membranes, which results in ion and molecular transport across the membrane. The results of a quantitative study of molecular transport due to electroporation caused by a single exponential pulse are presented; transport of four molecules of different physical characteristics across erythrocyte ghost membranes is examined as a function of applied field strength. Flow cytometry is used to quantitatively measure the number of molecules transported for 10(4) to 10(5) individual ghosts for each condition. This study has four major findings: 1) Net transport first increases with field strength, but reaches a plateau at higher field strengths. Significant transport is found at or below 1 kV/cm, and transport plateaus begin at field strengths between 2 and 5 kV/cm depending on the molecule transported. 2) A single population of ghosts generally exists, but exhibits a wide distribution in the amount of molecular transport. 3) Under the conditions used, the direction of transport across the ghost membrane does not appear to affect molecular transport significantly. 4) Large numbers of ghosts may be destroyed by the electroporation procedure.     

Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 μm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).     

Survival and growth of pea protoplasts after transformation by electroporation

Protoplasts from two different pea cultivars, Belman and Filby, were stably transformed by direct gene transfer using electroporation. Transgenic calli could be obtained after selection, when hygromycin resistance was used as the selective trait introduced into the protoplasts, while no transformants were obtained when kanamycin resistance was used as selective marker in either of the two pea cultivars tested. The effect of the field strength on survival and division rates of the protoplasts was studied. Two different culture systems and osmotica were compared for induction of sustained divisions in and regeneration of transgenic callus from the protoplasts. The choice of the culture system had a considerable effect on the initial division frequency of the treated protoplasts, as well as on the later growth of the colonies. Transformation efficiency was monitored by histochemical GUS assay, and the transgenic nature of the calli selected for resistance against antibiotics was confirmed by DNA analysis.     

Calorimetric measurement of water transport and intracellular ice formation during freezing in cell suspensions

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0–0.8) at various cooling rates (0.5–250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q_IIF) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q_CW) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.     

The OPTIMIZOR: a graphic pulse analyzer to monitor electro cell fusion and electroporation experiments

BTX has developed an instrument, the OPTIMIZOR, which allows the accurate monitoring and adjusting of electrical parameters to aid in electro cell fusion and electroporation experiments. The OPTIMIZOR can be used with the family of BTX generators and chambers, as well as home-made or other commercial systems with the appropriate adaptors. The microprocessor-based design allows great flexibility in further expansion of functions and possible mating to a fully-automated laboratory system with robotic features.     

The residual protonmotive force in mitochondria after an oxygen pulse

Both from irreversible thermodynamics and from mass-action kinetics it can be derived that upon anaerobiosis in an oxygen-pulse experiment the protonmotive force across a mitochondrial membrane undergoes a sudden drop. Under representative conditions the protonmotive force after the drop (the residual protonmotive force) is shown to be less than 3 kJ . mol-1 as opposed to steady-state values for the protonmotive force of 19 kJ . mol-1. It is concluded that correction for proton leakage in pulse experiments by back extrapolation underestimate proton leakage. Consequently the observed H+/O stoichiometries must underestimate the true H+/O ratios.     

Recovery of transgenic trees after electroporation of poplar protoplasts

Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 10⁵ to 10⁴ protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 M paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 M phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated.Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.     

Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts.

The methodology has been developed to measure Cl activity and transport in cultured cells grown on a monolayer using the entrapped Cl-sensitive fluorophore 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ). The method was applied to a renal epithelial cell line, LLC-PKI, and a nonepithelial cell line, Swiss 3T3 fibroblasts. SPQ was nontoxic to cells when present for greater than h in the culture media. To load with SPQ (5 mM), cells were made transiently permeable by exposure to hypotonic buffer (150 mOsm, 4 min). Intracellular fluorescence was monitored continuously by epifluorescence microscopy using low illumination intensity at 360 +/- 5 nm excitation wavelength and photomultiplier detection at greater than 410 nm. Over 60 min at 37 degrees C, there was no photobleaching and less than 10% leakage of SPQ out of cells; intracellular SPQ fluorescence was uniform. SPQ fluorescence was calibrated against intracellular [Cl] using high K solutions containing the ionophores nigericin and tributyltin. The Stern-Volmer constant (Kq) for quenching of intracellular SPQ by Cl was 13 M-1 for fibroblasts and LLC-PKl cells. In the absence of Cl, SPQ lifetime was 26 ns in aqueous solution and 3.7 +/- 0.6 ns in cells, showing that the lower Kq in cells than in free solution (Kq = 118 M-1) was due to SPQ quenching by intracellular anions. To examine Cl transport mechanisms, the time course of intracellular [Cl] was measured in response to rapid Cl addition and removal in the presence of ion or pH gradients. In fibroblasts, three distinct Cl transporting systems were identified: a stilbeneinhibitable Cl/HCO3 exchanger, a furosemide-sensitive Na/K/2Cl cotransporter, and a Ca-regulated Cl conductance. These results establish a direct optical method to measure intracellular [Cl] continuously in cultured cells.     

Transformation of Acidiphilium by electroporation and conjugation.

Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigated. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 degrees C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 degrees C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10-15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 micrograms/mL. Transformation efficiencies in these experiments ranged up to 10(4) transformants/micrograms DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10(-5)-10(-9) per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain.     

Pore disappearance in a cell after electroporation: theoretical simulation and comparison with experiments.

The process of pore disappearance after cell electroporation is analyzed theoretically. On the basis of the kinetic model, in which the formation and annihilation of a metastable hydrophilic pore are considered as random one-step processes, a distribution function of cell resealing times, Fr(t), is derived. Two cases are studied: 1) the rate of pore resealing, k(r), is significantly greater than the rate of pore formation, k(f); and 2) the rate of pore formation, k(f), is comparable with k(r). It is determined that the shape of the distribution function depends on the initial number of pores in a cell, n(i). If in the absence of an external electric field the rate of pore formation, k(f), is significantly less than the rate of pore resealing, k(r) (case 1), pores disappear completely, whereas when k(f) approximately k(r) (case 2), the cell achieves a steady state in which the number of pores is equal to k(f)/k(r). In case 1, when n(i) = 1, the distribution function Fr(t) is exponential. The developed theory is compared with experimental data available in the literature. Increasing the time of incubation at elevated temperature increases the fraction of resealed cells. This indicates that the time necessary for the resealing varies from cell to cell. Although the shape of experimental relationships depends on the electroporation conditions they can be described by theoretical curves quite well. Thus it can be concluded that the disappearance of pores in the cell membrane after electroporation is a random process. It is shown that from the comparison of presented theory with experiments, the following parameters can be estimated: the average number of pores, n(i), that appeared in a cell during an electric pulse; the rate of pore disappearance, k(r); the ratio k(f)/k(r); and the energy barrier to pore disappearance deltaWr(0). Estimated numerical values of the parameters show that increasing the amplitude of an electric pulse increases either the apparent number of pores created during the pulse (the rate of pore resealing remains the same) or the rate of pore resealing (the average number of pores remains the same).     

Quantitative study of molecular transport due to electroporation: uptake of bovine serum albumin by erythrocyte ghosts.

Electroporation is believed to involve the creation of aqueous pathways in lipid bilayer membranes by transient elevation of the transmembrane voltage to approximately 1 V. Here, results are presented for a quantitative study of the number of bovine serum albumin (BSA) molecules transported into erythrocyte ghosts caused by electroportion. 1) Uptake of BSA was found to plateau at high field strength. However, this was not necessarily an absolute maximum in transport. Instead, it represented the maximum effect of increasing field strength for a particular pulse protocol. 2) Maximum uptake under any conditions used in this study corresponded to approximately one-fourth of apparent equilibrium with the external solution. 3) Multiple and longer pulses each increased uptake of BSA, where the total time integral of field strength correlated with uptake, independent of inter-pulse spacing. 4) Pre-pulse adsorption of BSA to ghost membranes appears to have increased transport. 5) Most transport of BSA probably occurred by electrically driven transport during pulses; post-pulse uptake occurred, but to a much lesser extent. Finally, approaches to increasing transport are discussed.     

Millisecond fading and recovery phenomena in fluorescent biological objects.

Cytofluorometric signals derived from some frequently used fluorophores were studied during illumination times in the millisecond range. These rapid signals were recorded on a storage oscilloscope. The objects studied included (1) Berberine sulphate stained mast cell heparin, (2) Acriflavine-Feulgen stained DNA, (3) Acridine orange stained mast cell heparin, (4) Acridine orange stained DNA and (5) Fluorescein isothiocyanate-conjugated IgG in an antinuclear factor test. A new rapid fading phenomenon, appearing as an initial peak upon the familiar slowly declining fluorescence signal, is reported. This fading, which had a duration of about 10 ms, also showed a very rapid recovery. The influence of this phenomenon on fluorometric measurement techniques is discussed. The millisecond fading phenomenon occurred in all the fluorophores studied except Fluorescein isothiocyanate-conjugated IgG. In the case of acridine orange the phenomenon was present when the dye was bound to nuclear DNA but absent when the dye was bound to mast cell heparin. This suggests that the millisecond fading and recovery phenomenon may be used in fluorescent microprobe studies.     

Optimization of an electroporation procedure for Kluyveromyces lactis transformation

Summary An electroporation method using a Bio-Rad Gene Pulser has been optimized for introducing heterologous DNA into Kluyveromyces lactis yeasts. The plasmid pCR1, derived from a native Kluyveromyces plasmid, was used to transform K. lactis. This plasmid produces a wheat -amylase and contains both the biosynthetic marker URAA and G418 resistance genes. Transformation was optimal at 4500 V/cm, 25 F, and with 0.2 g plasmid DNA. Transformation efficiencies in the range 10⁴–10⁵ transformants/10⁷ cells/g DNA were obtained.     

Recovery of adherent cells after in situ electroporation monitored electrically

Here we describe various experiments that address the efficiency of loading extracellular probes into the cytoplasm of adherent mammalian cells (normal rat kidney, Madin-Darby canine kidney, and African green monkey) by means of in situ electroporation. Subsequent cell recovery from the electroporation pulse was monitored electrically in real time for each condition. In this study, small, gold-film electrodes (5 x 10(-4) cm2) are used as culture substrates and at the same time as an electrode for both the application of the electroporating voltage pulse and the noninvasive electrical monitoring of cell recovery, using a technique referred to as ECIS. Electroporation has been performed by using ac sinusoidal voltage pulses of varying frequency, amplitude, and duration. Permeabilization and re-closure of the plasma membrane were evaluated by the uptake of the fluorescence probe, Lucifer Yellow, from the extracellularfluid. With the experimental setup described here, efficient electroporation was achieved with voltages less than 5 V. Using ECIS, we followed the morphological response of the cells to the electricfield-induced membrane permeabilization. For optimized electroporation conditions, cell recovery was completed in less than 1 h. The introduction of membrane-impermeable substances by electroporation and in situ monitoring of the cellular response mayfind many applications in cell biology.