Courtesy of Qbiogene
Laboratories are loading mammalian cells and tissues with exogenous DNA more routinely and more successfully than ever before. The means available to deliver the DNA--lipofection, transduction, electroporation, and so on--seem to be increasing at a staggering rate, whether measured in terms of published protocols, commercially available kits and reagents, or supporting cell lines. This plethora of options may seem like a boon to researchers, but to someone approaching the field for the first time, the choices can seem daunting. Where to begin?
Most protocols generally use transfection at some stage, points out Garry Nolan, an associate professor at Stanford University, because even with viral transductions, the gene still needs to get into the packaging cell. Approximately 7,500 different labs use Nolan's virus-packaging cell lines. He cautions that many people entering the field have the mistaken impression that they can just walk into an experimental set of conditions, a manufacturer's kit or another lab's protocol, for instance, and get it to work.
Nolan advises users to set up a matrix of conditions, with varying concentrations of lipids and cationic polymers. "Buy a slew of them, as many as you can reasonably afford, and just try them all ... and then try mixtures of them," he says. "Usually in that space of iterations a pair exists, or a set, that works. But there's almost no predicting what the answer's going to be."
SOLUTION EVOLUTION Transduction reagents and protocols, like most things biological, have evolved over time. Most people now use some sort of lipid, says James Lu, director of marketing for Gene Therapy Systems, San Diego, who estimates the market for transfection reagents at $50 million.
Michelle Calos' Stanford lab favors FuGENE from Roche Diagnostics in Indianapolis as its main tool. Roche describes its product as "a blend of lipids and other, proprietary compounds." Carlos points out that "10 years ago, we used calcium phosphate all the time, and 20 years ago people used DEAE-Dextran. One has succeeded the other."
Courtesy of Qbiogene
DEAE-Dextran-mediated transfection is still used by some laboratories, though it retains only limited popularity. The toxicity of the polycation limits its utility to use in short-term assays (most often protein-expression studies) on select cell types.
When the more versatile calcium phosphate-mediated transfection was introduced in 1973, it rapidly became the reagent of choice. The method works for a wide variety of cell types, for both transient and stable transfections. And since it uses standard chemicals, most laboratories already have the necessary reagents on hand. Calcium phosphate is still used by laboratories that are very sensitive to cost, says Ferdinand Dabu, market segment manager for Netherlands-based Qiagen, who calls it "home brew."
But it's "very, very tricky," notes Dabu. The reactions are extremely dependent on pH, which can dramatically affect complex formation. "And that can affect reproducibility," he says. Nolan adds other drawbacks, including concerns about the effects of impurities in the formulation, as well as the potential of calcium to activate the cells. Ultimately, "postdocs gravitate to the easiest thing that works for them," Calos says. "They prefer to just mix the DNA with something and throw it on the cells."
LIPOSOMES AND THEIR KIN Lipid-based protocols and their kin have also evolved since their first description in 1987. Initially DNA was encapsulated within neutral lipid micelles, which then fused with the cell's plasma membrane. This has given way to a variety of competing technologies for transporting macromolecules into a cell. The methods generally are based on the formation of a complex between a cationic carrier and the negatively charged macromolecule.
Cationic carriers include lipids, polymers, and combinations thereof, some of which are modified to take advantage of one or another biological property. Many of these compounds are proprietary, but others are freely available. For example, Carlsbad, Calif.-based Qbiogene offers the liposomal transfection reagent MegaFectin 20, which is based on a cationic lipid called DOTAP. The company's MegaFectin 40 reagent combines DOTAP with a neutral lipid called DOPE, while its MegaFectin 60 is based on a DOTAP/cholesterol mixture. DOTAP is not unique to Qbiogene; a variety of formulations are sold by Roche Diagnostics and others. DOTAP, DOPE, and cholesterol are all available directly from chemical distributors such as Sigma-Aldrich in St. Louis.
Lipids such as DOPE are also incorporated into nonliposomal formulations. In these products the lipids form micelles, the cationic surface of which interacts with the DNA. The complex boasts a slight overall positive charge, allowing it to bind to the negatively charged plasma membrane. InvivoGen of San Diego and other companies employ DOPE as a "fusogenic lipid," in this case along with a proprietary lipid compound in its nonliposomal LipoGen product.
Qbiogene takes the idea of nonliposomal lipid transfection even further, by covalently attaching the polycationic carrier polyethylenimine to the human transferrin molecule. Thus, the reagent takes advantage of receptor-mediated endocytic uptake of the bound DNA. The company claims high transfection rates for a wide variety of primary and cultured cell types and species.
Akin to lipids, activated dendrimers are highly branched molecules in which each branch terminates in a charged group. "They're essentially spherical polyamines," Dabu explains. Qiagen's PolyFect and SuperFect reagents utilize dendrimers to condense and compact the DNA, allowing for easier entry into the cell. For the do-it-yourself lab, Dendritic Nanotechnologies of Mount Pleasant, Mich., manufactures and sells a wide variety of dendrimers.
Other technologies can also compact the DNA for ease of entry. Qiagen's Effectene includes a proprietary "enhancer" that is designed to "precondense the DNA before you complex it with the second lipid," says Dabu. Invitrogen of Carlsbad, Calif., employs the same idea in the "Plus" component of its Lipofectamine Plus reagent.
Courtesy of Bio-Rad Laboratories
VIRAL DELIVERY Some labs choose to deliver genes with viruses rather than lipids or other molecules. The advantage of using viral delivery, says Linnea Hager, senior product manager for expression systems at BD Biosciences Clontech of Palo Alto, Calif., "is that you can approach 100% gene-delivery efficiency."
Viral transduction introduces DNA by way of a viral infection, and the basic principle is deceptively simple: a gene is transfected into a cell. This cell then produces viral particles that are collected and used to infect the cells of interest, carrying the introduced gene with them. Researchers have adapted several different viral systems for this purpose.
Retroviral particles insert their nucleic acids into the host genome, so that all subsequent cell generations will carry the transfected gene. This approach has a drawback, however; the cells must be dividing. Lentiviruses (a subclass of retroviruses that includes HIV), on the other hand, can integrate into nondividing cells, and some lentiviral systems are now in use.
Unlike retroviruses, adenoviruses are episomal, that is, the nucleic acids they deliver don't integrate into the genome. But, they can transduce a wide variety of cell types, both quiescent and proliferating, and the episomes may remain in the cell indefinitely, often at high copy number. To construct the delivery vectors, the gene of interest is cloned into a plasmid containing an eviscerated adenoviral genome. This plasmid is delivered into a packaging cell, which provides the missing adenoviral genes in trans.
Another viral gene delivery system, adeno-associated virus (AAV), is gaining fast in popularity. Unlike adenoviruses, AAV appears to be harmless. It can also infect nondividing cells and integrates into the host genome at a specific location.
Calos says viral delivery systems are "overly complex." But labs don't have to do all the work themselves; several manufacturers now offer complete setups for the creation of viral vectors. Invitrogen, for example, offer adenoviral kits as well as lentivirus kits. Several companies offer retroviral kits, including BD Biosystems Clontech's Retro-X™ System, which come complete with vector and reporter plasmids, packaging cells, and PCR primers. And, some manufacturers market vectors, offering choices of tropism, promoter strength, and promoter control, either alone or integrated into kits.
AAV-based systems have been rapidly gaining ground with the help of kits such as La Jolla, Calif.-based Stratagene's AAV Helper-Free System. Adenovirus and (nonlenti) retrovirus currently each enjoy about 40% of the total viral vector market share, Hager says, with lentivirus and AAV sharing the remainder.
Courtesy of P. Furstenberger
SHOCK AND AWE When chemistry and viral methods fail, researchers then turn to their big guns. "If we strike out, we'd try the electroporator," Calos says, referring to her amaxa Nucleofector. The Cologne, Germany-based manufacturer claims that its technology "is unique in its ability to transfer DNA directly into the nucleus of a cell." "It works really well. For example, for human primary T cells, you can get over 60% transfection efficiency," Calos extols, noting that other, older electroporators have not given her similar success with hard-to-transfect cells.
Electroporation, the application to an electric shock to make the cell membrane transiently porous, is notoriously cytotoxic, and the Nucleofector is no exception: Calos achieves about 50% viability. This may be of little consequence if the cells are to be sorted or selected following transfection, or if they are easily grown and abundant. But it can be a major deterrent to using the technology on precious cells or for cultures whose viability is an issue.
If the cells are extremely precious, like the single-cell zygotes used to create transgenic animals, for example, researchers may opt to microinject each one individually. This can be achieved using a microscope and micromanipulators, such as Hercules, Calif.-based Bio-Rad's XenoWorks microinjection workstation.
Other techniques are used in more specialized situations. Plant biologists might gravitate toward a biolistic approach, shooting the genes into tissue at high velocity using a Helios or PDS-1000/He "gene gun" from Bio-Rad. This technique also has applications outside the plant kingdom; it can be used, for example, for adherent mammalian cells such as primary neurons that are resistant to lipid transfection, says Bio-Rad's Julie Moore.
Genetically modified crops are sometimes created using agrobacterial vectors or by transfecting protoplasts, which are cells from which the cell wall has been stripped. Other organisms have their own specialized tools. For example, BD Biosystems Clontech offers a series of baculovirus vectors that are tropic to insect cells.
Many parameters need to be considered when deciding which avenue to embrace, and which product or protocol to try first; each has its strong suits and shortcomings. Decide whether the gene needs to be integrated into the genome, with the potential of resulting positional effects, or episomal. Determine whether the cells can tolerate serum-free medium. Is there the time to optimize a protocol, or are results needed right away? Does the budget allow for pricey equipment and reagents; do you have the patience for tedious manipulations? Will the cells be sorted or selected after transfection? How robust, and how precious, are the cells? Is the lab equipped to accommodate biosafety precautions necessary for the protocol? Once these questions are answered, carefully consider manufacturers claims about their products, and researchers' claims about their protocols, and match them to your situation.
Many companies offer optimization kits to help with getting started. Kits are available that contain more than one transfection reagent, allowing researchers to determine which works best for their cells and culture conditions. Some manufacturers sell trial-sized aliquots of their reagents, while others send investigators product samples to test, in the hope of future sales.
Then set up a matrix of conditions, with varying concentrations of reagents, and dive in.
Josh P. Roberts is a freelance writer in Minneapolis.