Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
You can learn a lot from in vitro assays. But if you want to know what a given nucleic acid is doing, you pretty much have to put it into cells.
For the most part, that means transfecting DNA. When researchers want to determine whether, say, a given promoter sequence is active, they can test that by sticking the promoter in front of luciferase, putting the resulting construct into cells and testing for light production a few days later.
Such an assay cannot be performed any other way: If you want to measure transcription, your reporter must be capable of being transcribed—in other words, a gene. And there are countless other applications in which DNA is appropriate, as well. But there are a handful of applications in which researchers have the choice of delivering DNA, mRNA or protein. In such cases, which macromolecule should you choose? Here, we review the pros and cons of each.
DNA
There are few applications in which researchers can choose from among DNA, RNA and protein—when creating induced pluripotent stem cells, for instance, as well as genome editing with the Cas9 protein and intracellular antibody delivery, applications in which the protein itself is key.
Given the choice, most researchers still opt for DNA, says Xavier de Mollerat du Jeu, a senior scientist at Thermo Fisher Scientific. “DNA is the method of choice so far, because it’s easy to handle, we have all the tools for cloning, and it’s stable and easy to propagate.”
Researchers have plenty of experience working with DNA, of course, and no shortage of reagents to deliver it to cells, including Thermo’s Lipofectamine® 3000 and Promega’s ViaFect™ and FuGENE® reagents. DNA is also relatively inexpensive to make, compared with protein or RNA, and can continue pumping out mRNA and protein long after delivery.
On the other hand, DNA must be transcribed to be effective, meaning it must get into the nucleus, whereas mRNA and protein need only cross one cellular membrane. Also, researchers using DNA-based vectors run the theoretical risk of genomic integration, a problem especially for clinical applications of iPS cell and genome editing; this issue doesn’t exist with mRNA- and protein-based strategies.
RNA
According to du Jeu, mRNA tends to transfect more efficiently than DNA—that is, more cells in a population typically will take up mRNA than DNA. And it yields protein more rapidly than DNA, as it doesn’t need to be transcribed first. There also are dedicated mRNA delivery reagents, such as Thermo’s Lipofectamine MessengerMAX™.
But each transfected cell will produce less protein than with DNA, all things being equal, as protein production will last only as long as the mRNA does, and mRNA is relatively unstable. Also, with no DNA to churn out additional transcripts, the amount of protein a cell can make is limited by the number of transcripts the cell takes up in the first place.
And mRNA poses some other disadvantages, says Kevin Kopish, strategic marketing manager at Promega. For long-term expression work (such as during iPS cell generation), for instance, mRNA may need to be delivered repeatedly for days. (EMD Millipore’s Simplicon™, a “self-replicating RNA” construct for iPS cell reprogramming, can circumvent that limitation.) Researchers also may not be well versed in working with RNA, which is far more nuclease-sensitive than DNA, or have the appropriate reagents available (for instance, mRNA requires different transfection reagents than DNA, as it is used in the cytoplasm rather than the nucleus).
Then there’s the question of the biomolecule’s source. To deliver mRNA, you need to make it, either by synthesizing it chemically (an expensive proposition) or by transcribing it from a DNA template.
That said, mRNA can yield better results than DNA in some contexts, for instance, when transfecting primary or terminally differentiated cells. With primary cells, says du Jeu, DNA transfection efficiency can be 30% or lower. “But with mRNA, you can get 80% transfection efficiency, a drastic improvement,” he says.
Protein
Delivering protein, such as purified Cas9 enzyme or iPS cell transcription factors, is perhaps the most straightforward approach to using the molecules in the cell. The molecules are ready to go immediately upon cellular entry, and it is easier to control dosage than with nucleic acids, says Kyle Hondorp, product manager at Active Motif, which sells a protein-delivery reagent called Chariot™.
“Chariot tends to be a very polarizing product,” Hondorp says. “People who have used it and get it to work love it to death. But not all do.”
One problem is that to deliver protein, you must make it and purify enough to use, or buy it from a commercial supplier, which can be time consuming and expensive. But perhaps most significantly, unlike nucleic acids, each protein has unique biochemical properties of size, shape, charge, hydrophobicity and more. Thus, conditions that work for one protein may not work for another. According to Hondorp, Active Motif’s reagent works for GFP tetramers, but there is a size ceiling. Large multiprotein complexes “will be too large to deliver with the Chariot method,” she says. (Other protein transfection reagents include Clontech’s Xfect reagent and Thermo Scientific’s Pierce Protein Transfection Reagent.)
One potential alternative, says du Jeu, is electroporation, the process by which holes are punched in a cell membrane using a jolt of electricity. “Electroporation doesn’t care what kind of protein it is, it just pokes holes in the cell,” he says. “It works really well for protein delivery, especially for Cas9.”
According to du Jeu, the first question researchers should ask when weighing DNA, RNA and protein delivery is: “What cells are you working with?” Some cells are amenable to DNA transfection, and others are not. Non-replicating cells typically are tougher to crack with DNA and may be good candidates for mRNA or protein.
Vendors typically list cell lines and cell types on which their reagents work, and a quick call to tech support—plus a review of the literature—can go a long way. If nothing else, you can always run a quick test by asking your vendor for trial sizes of its reagents. “We always offer samples of transfection reagents,” Promega’s Kopish says. “Customers can try them in their hands and see which ones they are most satisfied with.”
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