Angelo DePalma Ph.D. Writer GEN

Because it is versatile—it works with any cell, any organism—electroporation is uniquely advantageous.

Electroporation uses an electrical pulse to introduce new species, usually polar molecules, into cells. The technique exploits the weak interactions between phospholipid bilayers that maintain the integrity of cell membranes. In a typical cell membrane, phospholipids are arrayed with their polar head groups pointing outward and their hydrophobic tail groups pointing inward, an arrangement that impedes the passage of polar molecules. Without some type of assistance, polar molecules cannot enter.

When cells experience a controlled electric pulse, the phospholipid layer opens, creating temporary physical channels that allow molecules to enter. Under the right conditions, the channels rapidly close, returning the cell to its original state—except that the cell now contains foreign molecules.

In addition to direct introduction of genes, electroporation facilitates the direct transfer of plasmids between cells or species—for example, from bacteria to yeast.

Extensive experimentation continues on the use of electroporation for delivering medicines and vaccines directly to the cells of living organisms. This article focuses on nonmedical applications.

Electroporation is most commonly used to transfect cells transiently, although stable transfection is possible as well. In the biopharmaceutical industry, transient transfection enables production of up to a few grams of protein for characterization and preclinical studies. In this application electroporation utilizing plasmids has proved to be reliable and predictable. Electroporation similarly produces stably transfected cells provided that DNA is introduced in linearized form by first treating it with a restriction enzyme.

One Technique among Many

Electroporation is firmly established in the armamentarium of transfection techniques that include viral vectors, chemical or reagent-based methods, and mechanical gene delivery. Viral vectors are the most common method for generating stably transfected cells for the manufacture of therapeutic proteins. Viral vectors provide very high transfection efficacy but are limited in terms of the length of inserted DNA. Viral vectors also face issues related to biosafety and mutagenesis.

Other mechanical techniques such as microprecipitation, microinjection, liposomes, particle bombardment, sonoporation, laser-induced poration, and bead transfection are all employed experimentally. These mechanical techniques have a common thread. They disrupt cell membranes and thereby allow DNA to enter the cell. Some approaches—the “gene gun,” for example—involve the projection of genes directly through the membrane into the cytoplasm. From here, genes may migrate to the nucleus.

Additionally, there are hybrid techniques that exploit the capabilities of mechanical and chemical transfection methods. For example, numerous articles have appeared over the last decade on magnetofection, a transfection methodology that combines chemical transfection with mechanical methods. For example, cationic lipids may be deployed in combination with gene guns or electroporators. Most of the magnetofection literature involves delivery of genes and therapeutic molecules to living organisms.

Electroporation has several advantages: versatility (works with any cell type), efficiency, very low DNA requirements, and the ability to operate in living organisms. Disadvantages include potential cell damage and the nonspecific transport of molecules into and out of the cell.

But among chemical, mechanical, and viral transfection approaches, electroporation alone provides a reasonable certainty of success regardless of the target cell or organism.

For example, chemical transformation and electroporation are two leading methods for introducing DNA into Escherichia coli. For the latter, bacteria must first be rendered “competent” by removing buffer salts to assure that current reaches the cells, followed by application of the electrical pulse at 0°C to reduce damage to the microorganisms. Chemical transformation involves suspension in CaCl2, which creates pores, followed by a heat shock that sweeps DNA into the cells.

Another approach uses cationic lipids to pry open cell membranes. Electroporation is less cumbersome and more efficient, works on more varied cell types, and lends itself more easily to standard methods than chemical transfection. Some researchers prefer chemical transformation, however, because it does not require purchasing an instrument.

Enabling Innovation

Although first described in 1965, electroporation continues to open avenues toward innovative science in terms of instrumentation, protocols, and experiment. At least a dozen university groups have developed electroporation devices based on microelectromechanical systems (MEMS). One benefit of microchanneled devices is they may be designed to apply no more voltage than that sufficient to achieve reasonable macromolecule incorporation. That advantage is also a drawback. Unlike commercial electroporation systems, the chips do not work with all cells.

A group at the Department of Biomedical Engineering, Louisiana Tech University led by Shengnian Wang, Ph.D., has found that gold nanoparticles enhance the performance of commercial electroporation equipment.1 Wang believes that the particles, which are highly conductive, reduce the cell medium’s conductivity while acting as “virtual microelectrodes” to help open phospholipid membranes. He claims enhanced performance (improved DNA delivery efficiency) and higher cell viability by virtue of lower poration voltages.

Researchers at the Charité Universitätsmedizin Berlin2 have developed a combined square pulse electroporation strategy for the reproducible transfection of cells. Britta Siegmund, M.D., and co-workers suspend cells in buffer and subject them to an initial high-voltage pulse followed by a low-voltage pulse of differing electrical and temporal value. Dr. Siegmund claims that viability is comparable to standard electroporation and that transfection efficiencies are up to 95%. She concludes that the technique may be “easily adapted for cells considered difficult to transfect.”

In addition to common DNA transfer, transfection has been employed for introducing interfering RNA into various cell types. The technique allows the controlled, small-scale study of dosing and delivery efficiency. Problems with dosing and delivery have plagued practical RNA interference applications in therapy. But at least one study has questioned whether RNA interference genes are most effectively incorporated through transfection reagents or electroporation in primary cells.3

Kirsty Jensen, Ph.D., and colleagues at the University of Edinburgh compared the efficacy of 11 transient transfection reagent kits and electroporation for the silencing of the immunomodulatory Mediterranean fever gene (MEFV) in bovine monocyte-derived macrophages. The group tested methodologies for small interfering RNA uptake, target gene knockdown, cell toxicity, and induction of the type I interferon response.

Electroporation was approximately as effective in knocking down MEFV as transfection reagents. Unlike the reagents, electroporation induced no interferon response, but cell viability was lower. Issues of viability and transfection efficiency for electroporation are generally taken as a face-value assessment of the technique.

Dr. Jensen concluded that “the use of transfection reagents is more suitable than electroporation for our work investigating the role of host macrophage genes in the response to infection,” but that “the choice of transfecting or electroporating small interfering RNA into cells depends on the individual experiments.”

In at least some instances where electroporation results are suboptimal, investigators have neglected to optimize conditions other than electric pulse strength. As recently noted by Hu and co-workers,4 the effectiveness of electroporation is influenced by nonelectrical factors such as cell or tissue type and DNA formulation.

Electroporation has become an indispensable method for both in vitro and in vivo developmental biology. A good deal of this work occurs in single cells, contributing to a model of great interest in therapeutics, diagnostics, drug delivery, and cell biology. Direct nanoporation of single cells is difficult due to the uncertainty inherent in post-poration viability.

Researchers at Northwestern University have developed a single-cell technique that provides high viability and efficiency.5 Their approach uses a microfabricated cantilever device, the nanofountain probe (NFP). It delivers molecules to cells more gently than bulk microinjection or nanoporation. Investigators have demonstrated NFP-mediated electroporation of single HeLa cells with a transfection efficiency of better than 95%, a viability of 92%, and qualitative dosage control.

NFPs represent an improvement over older transfection technologies employing atomic force microscope probes. Techniques based on atomic force microscopy often cause cells to lose attachment or rupture. NFP inflicts less damage to cells.

Resources:
1 Zu Y, Huang, S, Liao, W-C, Lu Y, Wang S. Gold nanoparticles enhanced electroporation for mammalian cell transfection. J. Biomed. Nanotechnol. Jun 2014; 10(6): 982–92.
2 Stroh T, Erben U, Kühl AA, Zeitz M, Siegmund B. Combined pulse electroporation—a  novel strategy for highly efficient transfection of human and mouse cells. PLoS ONE 2010; 5(3): e9488.
3 Jensen K., Anderson JA, Glass EJ. Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation. Vet. Immunol. Immunopathol. Apr 15, 2014; 158(3–4): 224–32.
4 Hu J, Cutrera J, Li S. The impact of non-electrical factors on electrical gene transfer. Methods Mol. Biol. 2014;1121:47–54.
5 Kang W, Yavari F, Minary-Jolandan M, et al. Nanofountain probe electroporation (NFP-E) of single cells. Nano Lett. 2013; 13(6): 2448–57.

Previous articleEbola Vaccine Trial Launched in Mali
Next articleJobWatch: BD Swings Axe, Idling 60 in California