You might think of transfection as breaking into a car. Considering the fancy locks and computer chips that come standard in many modern vehicles, gaining entry without a key may necessitate the kind of ingenuity required in penetrating a cell membrane. Its hydrophilic exterior of negatively charged phosphate groups and hydrophobic interior of hydrocarbon chains limit passage to only a select group of substances. But, decades of research have uncovered the details of the membrane’s makeup and function, and companies have leveraged this understanding to develop products that facilitate transfection with efficiency. Unlike thieves stymied by theft-deterrent technology, our ability to crack the cell membrane code has improved considerably.
Increased emphasis on genomics and proteomics has driven the improvement of transfection protocols, and the development of new ones. Companies also face higher standards. Researchers need transfection techniques that are gentle enough to keep cells and tissues healthy for subsequent observation. Other scientists require products that enable transfection at a high-throughput pace. And the future of medicine depends upon finding reliable means for in vivo transfection.
Of all physical methods, electroporation is among the most popular. The procedure involves the application of a high-voltage current that opens pores in the cell membrane through which nucleic acids and other molecules can pass. Because of the size of the pores, electroporation has been especially useful to transfect large items, such as plasmids, into the cell. However, even more than 25 years after the protocol’s development, “we don’t know much about the molecular mechanism,” said says Justin Teissie, director of research at the Institute of Pharmacology and Structural Biology at CNRS in France. The electroporation pioneer explained that “to understand the molecular mechanism of permeabilization” requires observing the pores themselves, a feat that has never been achieved. This lack of insight and the potential for adverse effects keeps some researchers from using electroporation.
Fortunately, they have plenty of other options. Chemical approaches have blossomed during the last several years. The selection of reagents consists of cationic liposomes, polymers, a hybrid of liposomes and polymers, calcium phosphate, and DEAE dextran, according to a paper published in a 2005 issue of the American Journal of Physiology – Cell Physiology1. “In almost all of these chemical methods, the reagents promote transfection by complexing with the DNA to neutralize the charge, condensing the DNA, mediating interaction and attachment to the cell membrane, and promoting entry into the cell, typically via endocytosis and subsequent endosomal escape.”
Whether you prefer chemical or physical means for transfection, you can avail yourself of the many products packaged for high-throughput applications. These include 96-well plates that come with wells already filled with transfection reagent; you can choose plates with different amounts of reagent, depending on the optimal conditions for your project. Or, you can bypass the plates by purchasing transfection reagents that can be directly added to the cell culture media. You can speed through transfection with new 25-well and 96-well electroporation plates; allowing quick optimization, the plates also provide the platform to efficiently screen large libraries of small molecules or nucleic acids. Plate handlers can free your hands by automatically loading plates into the electroporation instrument.
The list below represents some of the products that are sure to give you the edge in transfection—without the possible felony for breaking and entering.
Reference:
1 DA Dean, “Nonviral gene transfer to skeletal, smooth, and cardiac muscle in living animals,” Am J Physiol Cell Physiol 289:C233-C245, 2005
Based on the well-established Nucleofector technology amaxa has developed a system for the highly efficient transfection of primary cells and difficult-to-transfect cell lines in 96-well format. The Nucleofector 96-well Shuttle System consists of three components, the 96-well Shuttle, the Nucleofector II Device and a laptop. Driven by the Nucleofector II, the 96-well Shuttle can deliver DNA directly into the nucleus, allowing even non-dividing primary cells (such as resting T cells or neurons) to be efficiently transfected. siRNA delivery into the cytoplasm, on the other hand, also occurs with up to 99% efficiency. Optimal nucleofection conditions depend upon the individual cell type, not on the substrate being transfected. This means that identical conditions are used for the nucleofection of DNA, siRNA or shRNA vectors. So it is a simple matter to switch back and forth between substrates or to perform co-transfections with DNA and RNA together.
BTX, the leader in electroporation products and specialty electrodes, has developed a new and innovative product line for experiment optimization and large volume electroporation. The HT Electroporation System is a breakthrough in Molecular Delivery. Try out many electroporation conditions (cell density, buffer, pulse voltage and width, etc.) to find the highest transfection efficiency, quickly and easily, with the NEW BTX® HT 96 Well Electroporation System.
