DNA, RNA and Protein Delivery: Breaching the Cellular Walls

The membrane surrounding a cell can be a formidable barrier to introducing large molecules— especially if the goal is to keep the cell happy and healthy. And although there is no magic bullet that works equally well to efficiently transfect DNA, small and large RNAs and proteins into all cell types for every application, researchers have a host of tailored tools to help them breach that barrier. Here we review some of the latest options available to researchers interested in modifying the expression and regulation of their cells.

Payload 

Xavier de Mollerat du Jeu, director of R&D for the Life Science Group at Thermo Fisher Scientific, always starts the decision process with two questions: What are you trying to deliver? And to what type of cells?

As to the first question, “there is a misconception that RNA vs. DNA doesn’t matter,” he explains. But it’s more about where in the cell the macromolecule is to be delivered: DNA acts in the nucleus, RNA acts in the cytoplasm, and “reagents are developed to either transport into the nucleus or not.”

At least this holds for nucleic acids. Because proteins are so diverse—some are 200 kD, while others are 5 kD; some are positively charged, while others are negatively charged; and so forth—it’s very difficult to create a general protein delivery reagent. “You can have reagents that are developed for a specific protein—for example, there are a lot of reagents out there for Cas9 protein delivery,” de Mollerat du Jeu says.

Mechanical transfection methods such as electroporation “are a lot less dependent on the type of payload … you just create a temporary pore and drive through the charged molecule,” he adds. “Electroporation can drive it into the nucleus to some level, but it also goes into the cytoplasm—you can have a little of both.”

Viral methods, on the other hand, are limited to delivering either RNA or DNA.

Each payload has its distinct advantages and disadvantages over the others, even beyond delivery considerations. 

DNA is very stable, easy to generate and relatively inexpensive, for example, but it needs to be transcribed (and usually translated) before its effects can be seen, and it can be toxic to some cells. RNA can be translated directly, so there is a time advantage, but it is very labile. Proteins have an even more direct effect, because the enzyme or receptor is present as soon as it is transfected; that might be an advantage, if you desire a very short-term effect—such as with Cas9 nuclease, for example—and want to avoid the side effects of its sustained presence, points out Andrea Toell, senior product manager at Lonza.

Cell type

The growth properties of a cell influence researchers’ selection of a particular molecule type to be introduced and the transfection method. Most commonly used are adherent cells, although scientists working with larger numbers of cells may choose suspension cultures. The use of immune and primary cells is becoming a more common practice in cell-biology labs.

For adherent cell lines, de Mollerat du Jeu recommends a chemical (lipid- or polymer-based) reagent as the first pass. “If that doesn’t work, then you want to move to electroporation. And if that doesn’t work, you have viral as a last resort.”

“Suspension cells in general are resistant to any reagent-based transfection—they’re very difficult,” he says. “We recommend to go to electroporation directly. It works really well.”

With primary cells, on the other hand, “DNA is always a problem.” he observes. That’s because, as with most nondividing cells, the DNA needs to get past the nuclear membrane. “If you want to do siRNA or mRNA, it might work well. But if you want to do DNA, you may want to think about in situ electroporation (which is not optimal and is expensive) or viruses, typically.”

Are all reagents created equal?

Generally speaking, if one chemical transfection reagent works for a particular cell and application, all the chemical transfection reagents will work, and vice versa. “But there are different degrees of working,” says de Mollerat du Jeu. “You’re not going to get the same efficiency.”

Most reagents are to some extent toxic—GeneCopoeia recommends removing them after an eight-hour or overnight incubation—but they’re generally much less so than they used to be, and are far less so than electroporation.

“Toxicity varies across all the different reagents out there, and efficiency and efficacy are also variables,” says Michael Silverman, associate professor of cellular neuroscience at Simon Fraser University, who currently uses GeneCopoeia’s EndoFectin Max to transfect DNA into primary neurons. “We pretty much tested almost every lipid-based transfection reagent that has been on the market for the past 15 years. We’ve shifted as we find ones that are better than, say, Lipofectamine 2000, which we used for many years.” Even with the best reagents, only a very small percent of the neurons is transfected—but that’s actually a benefit for imaging studies, because “if we have too many transfectants, all the axons and dendrites are running across each other, and we can’t keep track of which way they’re pointing from the cell body.”

Electroporation would be more efficacious, but it would require the cells to be in suspension. “And that means you’ve got to electroporate the neurons at the time of plating,” which doesn’t work well for studying more mature cells, Silverman says. Lonza offers a system for electroporation of cells in a 24-well plate as an option for working with adherent cells.

It's difficult, if not impossible, to determine the exact formulation of many transfection reagents. For example, EZ Biosystems, which specializes in cell-type-specific transfection reagents, makes several hundred different formulations—often mixtures of lipids, polymers and other components. The company screens the formulations to determine which works best to transfect DNA into which cell type, and then the product is given that name (such as Human Artery Endothelial Cell Avalanche™ Transfection Reagent), says technical applications scientist Gordon Ma.

Other vendors, like Thermo Fisher, take a different tack, optimizing reagents for applications—such as Cas9 or RNA delivery—rather than for cell type, notes de Mollerat du Jeu.

Which, when and why?

Much of the decision as to which method to use comes down to what you’re trying to accomplish, your lab’s capabilities, the amount of effort you’re looking to invest and frankly, history. 

For Steven Fiering, professor of microbiology and immunology and professor of genetics at the Geisel School of Medicine at Dartmouth, “it’s all about what is the most convenient way to get the job done.” The core facility that he directs uses mostly electroporation protocols—mainly developed by others—to transfect mouse embryonic stem cells. His team can get transient expression in about 60% of the cells, with about 1/1000 of these surviving drug selection. But, he notes, it’s not hard to get 10⁷ embryonic stem cells to start with, so “we’re getting more than we can screen—it’s not a choke point—there’s no reason to optimize.”

For other work, Fiering’s Dartmouth Mouse Modeling Shared Resource uses retrovirus or lentivirus that integrates into the host genome. “These work great, but are a bit of a pain to make,” he notes.

How researchers ”measure” how well their technique works can vary, as well. If only, say, half the cells survive electroporation, is efficiency considered the percentage of total starting cells, or of ”viable” cells? At what time point is that determined—after 24 hours? A week? Following drug selection? And of the successfully transfected cells, how many copies are active per cell? For protein production, that latter number is of utmost importance (although for research studies, it may be enough just to know the gene is there), points out GeneCopoeia senior scientist Xueming Xu. Several vendors offer reagents optimized for generating high copy numbers in densely packed suspension cells, such as CHO and HEK293.

Chemical reagents are relatively cheap and easy when they work, but not all cells are amenable to chemical transfection. Electroporation can transfect most payloads into most cell types, making it well-suited for complex transfections, like those needed for reprogramming, CRISPR/Cas9 editing or generating CAR-T cells, points out Toell. However, specialized equipment is required, and the process can be stressful to the cells. Viruses offer a very high level of infection but have a limited payload and require a separately constructed virus for each construct to be delivered—and some facilities are not happy about receiving virus-infected cells, points out Silverman.

As with many techniques, it pays to do a little research: Assess your needs, equipment, budget and competencies; see what your colleagues are using; talk to vendors; and most importantly, see what the data and results are telling you. Try a few methods, and when you find something that works, don’t be afraid to optimize … if it matters.

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