The inside scoop—evaluating gene delivery methods

TECHNOLOGY FEATURE Exploiting mother nature 878 Bringing in the guns 880 BOX 1: Going the high-throughput way 876 BOX 2: Target: the endosome 878 BOX 3: RNAi craze 881 © 2005 Nature Publishing Group http://www.nature.com/naturemethods The inside scoop⎯evaluating gene delivery methods Techniques for delivering nucleic acids into mammalian cells have been around for decades. But tools and reagents continue to improve and target a broader range of cells and applications. Laura Bonetta reports. Pick a paper, any paper. Chances are that somewhere in the Methods section there is a description of a step for introducing DNA or RNA in cells. Although it has become a routine procedure, in vitro gene delivery can still be a challenge, especially when working with frail or scarce mammalian cells. The mammalian cell membrane is impenetrable to large molecules that, like DNA, carry an electrical charge. Researchers have thus come up with an arsenal of tricks⎯from using carrier molecules and viral vectors to pocking holes in the membrane⎯to sneak nucleic acids through. But no one method can be applied to all types of cells or experiments. “You look at the articles that have been published and find one that has done some comparison of different transfection methods for your cells. But if you don’t have any clues, you have to try different reagents, to find what will work best in your hands,” says Nina Iversen, a researcher at the University of Oslo. The good news is that there is no shortage of reagents to try! Solution revolution A popular way to get DNA and RNA inside cells is to use carrier molecules. Such methods do not require expensive equipment and, from a technical standpoint, are less difficult to use than viruses. They rely on the fact that nucleic acids can interact with positively charged molecules, such as cationic lipids and polymers, to form complexes that are more palatable to the cell. Carrier molecules on the market today owe their existence to the discovery, almost four decades ago, that coating DNA with DEAE-dextran would allow it to get past the cell membrane 1 ⎯a process dubbed ‘transfection’. In the early HeLa cells transduced with a self-inactivating retrovirus from Clontech’s pQX (retroQ) series, which expresses green fluorescent protein. (Courtesy of Clontech.) 1970s, researchers discovered that they could also transfect DNA using calcium phosphate, a less toxic chemical 2 . Some labs still use these reagents, which can be purchased as stand-alones or in kits. For example, Promega’s ProFection mammalian transfection systems offer optimized buffers and solutions for either calcium phosphate– or DEAE-dextran–mediated transfection. But for many researchers, the newer, lipid-based reagents offer greater ease of use and efficiency with lower toxicity. The first lipid-like molecules to come on the scene were mixtures of cationic and other lipids that would form artificial liposomes. Developed in the late 1980s, liposomal transfection reagents work by enveloping the DNA or RNA and then fusing with the cell membrane to deposit the nucleic acid cargo inside. Roche Applied Science sells liposome formulations based on the cationic lipids DOTAP and DOSPER. Qbiogene’s MegaFectin combines DOTAP with different lipids. Under optimized conditions, liposome-mediated methods yield high efficiencies and are much easier to use than calcium phosphate. Of lipids and polymers The latest generation of lipid-based transfection products includes multicomponent, nonliposomal reagents consisting of lipids, polymers and combinations thereof. Although their composition is proprietary, most of them work by forming a complex with DNA or RNA that interacts with the cell membrane. The complex is believed to be taken up by endocytosis and then released in the cytoplasm. “Unlike the liposomal agents which form spheres that vary greatly in size, nonliposomal lipids NATURE METHODS | VOL.2 NO.11 | NOVEMBER 2005 | 875 TECHNOLOGY FEATURE © 2005 Nature Publishing Group http://www.nature.com/naturemethods form micelles of uniform size resulting in more reproducible results,” says Jamuna Ramnath, technical service scientist at QIAGEN Inc. In the lipid arena researchers have a wealth of reagents to choose from. I n v i t r o g e n’s p r i m a r y p r o d u c t i s Lipofectamine 2000, which works with a variety of nucleic acids and cell lines. It is also applicable to high-throughput screens. “It is quite stable after it makes a complex with DNA, so samples can sit on the deck of a robot loader for a long time,” says Henry Chiou, manager for R&D at Invitrogen Corporation (Box 1). Roche Applied Sciences’ premier transfection reagent is the FuGENE 6 Transfection Reagent, a nonliposomal BOX 1 GOING THE HIGH-THROUGHPUT WAY Say you need to screen hundreds of cDNAs to search for genes that, when overexpressed, induce cell death. Or maybe you want to knock down several genes believed to function in the same pathway to better delineate the steps involved. The traditional way to transfect many cells at once is to place the cells in 96- or 384-well plates, add the DNA (or RNA) and transfection reagents, and then study the cells. Alternatively, the plates Magnetic beads coated with DNA encoding containing cells and DNA are loaded green fluorescent protein are used to direct the transfection of adjacent HEK293 cells. in a high-throughput electroporation (Courtesy of Mark Isalan.) instrument and processed. Given the amount of work involved, these protocols typically use robots to dispense the reagents and cells, as well as automated plate readers or microscopes to analyze the results. In an effort to simplify high-throughput transfection protocols Ziauddin and Sabatini3 developed a new method based on microarray technology. In a transfection microarray, plasmid DNA dissolved in a gelatin solution is printed on a glass slide and then covered with a lipid-based transfection reagent. After removing the excess reagent, the slide is placed in a culture dish and covered with cells in medium. Cells growing on the printed areas take up the DNA creating spots of localized transfection within a lawn of nontransfected cells. (In an alternative version of this method the lipid-based transfection reagent is added to the DNA prior to printing.) Because cells are added to the reagent, this approach was called ‘reverse transfection’. A recent study describes another procedure for conducting parallel cell transfections on microscope coverslip arrays, but using a ‘forward’ methodology4. “We achieved transfection of a variety of cell lines using magnetic beads coated with PCR products,” says Mark Isalan of the European Molecular Biology Laboratory and first author of the study. According to the protocol, cells are grown on a glass coverslip or slide to which magnetic microbeads coated with DNA are added. Using magnets, the beads are directed to the surface of individual cells where transfection occurs. “We can cont (...truncated)