Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Some cells, like HeLa cells, are easy to transfect with lipid reagents. Others, like stem cells, primary neurons and unstimulated T and B cells, not so much. For these, electroporation has become the method of choice, with researchers resorting to more involved methods, such as viral transduction, as a backup.
Historically, electroporators delivered a single, large pulse of electricity designed to blast open cells and allow foreign material to diffuse in by Brownian motion, explains Mike Mortillaro, owner of Bulldog Bio, the North American distributor of the NEPA21 system. The process was harsh, often leaving far more dead cells than transfected ones. Many of the more sophisticated systems available today can vary the parameters of the shock to offer a more nuanced delivery of charged nucleic acids into a cell, with data demonstrating high efficiencies and little (if any) damage from the electroporation itself.
However, sometimes even following published protocols or manufacturer’s recommendations doesn’t yield the hoped-for results. But don’t start cloning your gene of interest into those adeno- and lentiviruses just yet: There may be simple ways to optimize and tweak conditions enough, from the way the cells and DNA are prepared, through the electroporation itself, to how the cells are treated afterward, so as to avoid the hassles and pitfalls of using viruses to transfect difficult-to-transfect cells.
Culture conditions
The cells should be healthy (and mycoplasma-free), to start. The problem could be that the cells are being stressed or damaged by the preparation procedure, which adding an electrical pulse only exacerbates. Something as simple as lowering the centrifugation speed may help improve results, says Andrea Toell, senior product manager at Lonza, manufacturer of the Amaxa Nucleofector™ system.
Teresa Rubio, R&D manager of the Cell Biology Unit at Bio-Rad Laboratories, says the use of enzymes like trypsin to suspend the cells can sometimes negatively impact electroporation. It’s also “important to remove antibiotics when you’re doing any kind of transfection, and then resume them afterward,” she says.
When dealing with cultured cells, it’s best that they are in log-phase growth, having been split (or at least fed) the day before transfection, points out Jim Brady, director of technical applications at MaxCyte. DNA is much more likely to find its way into the nucleus of an actively dividing cell, in which the nucleus is disassembling and reassembling.
This is one reason that primary, nondividing cells are notoriously more difficult to transfect. Yet even these nondividing cells can benefit from good, consistent handling. Kristin Wiederholt, senior manager for R&D at Life Technologies, says that researchers often blame variable transfection efficiencies on the electroporation itself, when factors such as how the cells were pulled out and disrupted, how they were cared for and how long after plating they were electroporated were not consistent. She recommends optimizing those procedures “and locking down those individual parameters, being very consistent from isolation to isolation to isolation.”
Along similar lines, Toell says that if primary human T-cell transfection, for example, doesn’t go well it could be because of the cells’ donor. “So therefore it may make sense just to give it a second try with another donor, and see if this gets better.” Cell lines also can change their properties after many passages, she adds, and it may be worth looking into acquiring a fresh stock from ATCC when transfection efficiencies don’t meet expectations.
Shocking
Electroporation has the advantage over lipid-based transfection in that pulses are designed not only to open up pores in the outer membrane but also to create gaps in the nuclear membrane.
Modern electroporators have the ability to control the duration, amplitude (voltage), number and perhaps even the polarity or waveform of the pulses it delivers—choices that can literally make the difference between success and failure, life and death.
Some, such as Life Technologies’ Neon®, Bio-Rad’s Gene Pulser and the NEPA21 systems are so-called open systems that enable the user to control each parameter individually. The manufacturer typically has a database with a recommended setting for a given cell type, which can then be optimized and tweaked.
Others, including the MaxCyte and Amaxa Nucleofector, are considered black boxes. The user is supplied with a code based on the company’s optimization databases for that cell type. The individual parameters it encodes, and the makeup of the buffer, remain proprietary. “How you deliver the electricity is very critical,” explains Brady. “Using the wrong parameter can lead to cell death. So unless you’re an expert in biophysics, you’re not really going to know how to play around with all those different variables.” If a researcher’s results aren’t meeting expectations, a call to the company’s technical-support team will provide troubleshooting help and perhaps a different code (or buffer) to try.
Oh, and be sure to use controls. When calling tech support, the first question may be: Did the vendor-supplied control plasmid yield good results? If not, the problem could be with the DNA. Several vendors recommend using Qiagen EndoFree or similar endonuclease-free DNA-preparation kits. Toell says that internal ribosome entry site (IRES) sequences and long repeats in the plasmid backbone can have a negative impact, as well.
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The DNA is the only thing about the electroporation itself that customers need to optimize, Brady says: “High DNA will give high transfection efficiency, but it will lower your viability, because putting in a lot of DNA is toxic to the cells.” Similarly, large plasmids containing multiple genes (like those used for iPS cell reprogramming) show diminished efficiency, says Wiederholt, who recommends instead using multiple, smaller plasmids (but not more total DNA).
After the fact
How the cells are treated after electroporation—like what media they’re put into, the density at which they’re plated and even the temperature at which they’re incubated—can have an impact. Mortillaro suggests also that the sooner the cells are (back) into culture “the healthier they typically are and the better their viability.”
Viability and efficiency typically are measured about 24 hours after electroporation, after cells have had a chance to recover (or die) and proteins express.
If the number of healthy cells expressing the protein of interest still isn’t satisfactory after all the optimizing and tweaking, try RNA instead. It doesn’t need to enter the nucleus to be expressed, so mRNA or siRNA (for knockdown experiments) delivery sometimes succeeds where DNA transfection fails.
Image: High transfection efficiency of Jurkat cells with Life Technologies' Neon® Tranfection System. Intracellular uptake of reporter vector encoded with EGFP at 24 hr following transfection of Jurkat cells with Neon Transfection System. B is the corresponding fluorescence image of A.