by Jeffrey M. Perkel
The entire foundation of modern molecular biology is predicated on a single supposition: That it is possible to deliver nucleic acids to cells.
From reporter gene assays to RNAi, knockout mice to gene therapy – all are based on the assumption that a DNA or RNA molecule, created or isolated in the lab, can be efficiently transferred into a cell and made to function. The problem is, cells don't really want to pick up any piece of DNA lying around, and the plasma membrane is a formidable barrier.
Researchers have devised a wide array of tools to circumvent that barrier, employing disciplines from chemistry to virology to physics. Many have been available for years, and the market is both well stocked and mature. Yet today's researchers still face new challenges in gene delivery, both in terms of nucleic acids (neither small RNAs nor massive plasmids work efficiently with many standard transfection tools) and cells. Non-dividing, primary, and stem cells, especially, are problematic – what the field calls hard-or difficult-to-transfect cells.
"The role of transfection has shifted because of market needs, regardless of technology," says Michelle Collins, global product manager for the gene expression division at Bio-Rad Laboratories. "The needs are driven by difficult-to-transfect cells."
Fundamentally, those technologies fall into two categories: Transfectionand transduction. The latter is a virally mediated process, co-opting virus particles to leverage what they do best: injecting nucleic acids into cells that would rather be left alone; the former is a non-viral method, and is instead accomplished by a variety of approaches, the simplest of which is chemical transfection.
Though classical chemical transfection protocols relied on reagents like calcium-phosphate and DEAE-dextran, most modern transfection reagents are lipid-based (though they may also include non-lipid components such as polymers and peptides). Perhaps the most popular, at least based on citation counts, is the Invitrogen Lipofectamine reagent from Life Technologies Corp.(producing more than 1,250 hits on PubMed).
A cationic lipid, Lipofectamine forms positively charged spheres to which negatively charged nucleic acids can adhere and which may enter cells either by membrane fusion or endocytosis. Actually, Lipofectamine is a family of reagents that includes Lipofectamine 2000 (applicable to all nucleic acids) and, more recently, Lipofectamine LTX and Lipofectamine RNAiMax, for plasmids and short RNAs, respectively.
That strategy – developing different formulations for different applications – is common in the nucleic acid delivery market. For instance, whereas Mirus Bio's TransIT-LT1 and Qiagen's Attractene work for plasmids, TransIT-siQUEST, X-tremeGENE reagent, and Hyperfect reagents (from Mirus, Roche, and Qiagen, respectively) work best for small RNAs. Mirus's TransIT-2020 specifically targets hard-to-transfect cells, while Qiagen's TransMessenger and Mirus' TransIT-mRNA formulations deliver larger, messenger RNAs.
According to Constanze Kindler, Qiagen's senior global product manager for transfection and microRNA reagents, TransMessenger arose from an unmet customer need.
"People who had difficulty with cells that don't divide, or that they had trouble getting DNA in, wondered if they could put in mRNA instead," Kindler says. But not just any reagent would do, she adds: "For mRNA transfection, you need a reagent that is free of RNAses."
Regardless of the nucleic acid or cell type, most transfection reagents follow a fairly straightforward protocol: Count and plate cells on day 0; the next day, mix nucleic acid and reagent, allow complexes to form, and apply to cells; then harvest 24 to 72 hours later. Yet there are alternatives. Qiagen, for instance, is advancing what it calls "Fast-Forward" protocols, in which, instead of applying transfection complexes to previously seeded cells, cells are plated on top of the preplated complexes.
"The advantage of that is, it speeds up the process," Kindler says. "You don't need to seed cells in advance."
Not all chemical transfection reagents are lipid-based. Sigma-Aldrich's N-TER, for instance, is a cationic peptide that complexes with the negatively charged backbone of an siRNA and packages it into a nanoparticulate
According to Supriya Shivakumar, Sigma's global commercial marketing manager for functional genomics, N-TER offers improved complex stability, decreased cytotoxicity, and faster expression than lipid-based reagents for difficult-to-transfect cells.
"Generally, you see effects earlier than what you'd see with a lipid-based method," she says.
Unlike chemical transfection reagents, which often are used to transfect attached adherent cells, electroporationis used for cells in suspension.
Electroporation exposes a cell to a high-intensity electric field that temporarily destabilizes the membrane. The membrane becomes highly permeable to exogenous molecules present in the media, thereby enabling DNA to enter the cell. Though thought to be harder on cells than transfection reagents, electroporation serves a vital role in the gene delivery market: some cells simply cannot be transfected any other way. It does, however, require a dedicated instrument.
One example is Bio-Rad Laboratories' Gene Pulser systems, which have been zapping cells for over 25 years. Bio-Rad's Gene Pulser MXcell™ (released in 2007), the fourth instrument to bear the GenePulser name, is an "open" system for both cuvette- and microtiter plate-based delivery to from one to 24 samples at a time, says Collins.
By "open," Collins means that, in addition to preset protocols, the system is fully programmable; in other words, optimal variables for each cell type (voltage and capacitance) can be applied to each sample independently. By contrast, Lonza's popular Amaxa® Nucleofector® Technology is "closed," meaning that programs are "black boxes" users can apply to their cells, but not modify – at least not on their own.
"There's always the possibility to do further optimization together with our Scientific Support Team," says Andrea Toell, senior product manager at Lonza Cologne AG.
According to Toell, the Nucleofector is not just another electroporator. Blending improved viability and efficiency with decreased nucleic acid requirements, the system also delivers nucleic acids directly into the nucleus. "You don't have to wait for any proliferation of cells to get expression of DNA you have transfected," she explains. "This is an advantage because you can transfect non-dividing cells like primary T cells or neurons."
In January Lonza will launch the first Nucleofector kit (the Basic Neuron 96-well Nucleofector AD Kit) specifically for finicky adherent cells, such as neurons, which don't like to be released from their solid supports prior to electroporation (cells are usually electroporated in suspension).
Another player in the electroporation arena is Life Technologies, which launched its Neon Transfection Device this past March. In place of a cuvette or microtiter plate, the Neon uses a pipette tip as an electroporation chamber. "The design of the vessel is such that you get a much more uniform current and generate a lot less heat than with a cuvette, and that helps the customer dial in the appropriate voltage without compromising the cells," says Keith Farnsworth, director of product development for the molecular biology systems division at Life Technologies.
Plant researchers, especially, favor yet another transfection approach, called "biolistics." A blend of the words biology and ballistics, biolistics uses helium gas to propel tiny, DNA-coated gold or tungsten macrocarriers towards target cells for penetration. Bio-Rad's Helios® GeneGun and PDS-1000/He™ both employ this approach, the former being a handheld device, the latter bench-mounted.
Other researchers, especially those who want to deliver nucleic acids to non-dividing cells or adherent cells that cannot tolerate electroporation (or the trypsinization process required to get them into suspension), prefer a more natural solution. Their approach: viral transduction, the use of genetically engineered virus particles as delivery devices.
The Invitrogen ViraPower Lentiviral system, for instance, provides all the reagents – plasmids, packaging cell lines, and so on – to create replication-incompetent virus for gene delivery in vitro and in vivo. Recent improvements to the system give users tighter control over expression of the transduced gene, says Farnsworth.
To use such the system, researchers must first create a recombinant virus genome, deliver it to a packaging cell line, harvest virus, and then use that virus to infect their cells of interest. It "requires a bit more upfront work [than transfection]," says Farnsworth. Alternatively, users can leverage a dedicated facility's expertise. Sigma-Aldrich offers 300,000 different pre-made lentiviral stocks for short-hairpin RNA delivery (150,000 each for human and mouse), and can also prepare virus particles for delivery of custom sequences, including cDNAs.
"It's not that simple to make virus," says Shivakumar. "You can make the virus, but you need certain a titer to get into the cells, and the harder the cells to transfect, the higher the required titer. It can be hard to get high titers reproducibly."
Whether you opt for transfection reagents, electroporation, or viral transduction, the bottom line is this: Whatever your experimental system, there absolutely is a way to get it to take up exogenous genetic material; the trick is figuring out how. Several tool providers (including Lonza and Qiagen) offer searchable databases of cell types and conditions. Alternatively, tech support teams can usually advise you on the proper reagents and conditions to make your experiments work, or at least how to get started.
In the meantime, as researchers continue to shift their focus away from domesticated cell lines towards primary and stem cells, expect new delivery tools to follow. Says Farnsworth, researchers "are always looking for better ways to deliver plasmids or siRNAs."