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 The inside scoop⎯evaluating gene delivery methods Techniques for delivering nucleic acids into mammalian cells have been around for decades. But tools and methods 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 .com/nature e is a description of a step for introducing DNA or RNA in cells. Although it has .natur w become a routine procedure, in vitro gene delivery can still be a challenge, especially when working with frail or scarce mam- http://ww malian cells. The mammalian cell membrane is oup impenetrable to large molecules that, r G like DNA, carry an electrical charge. Researchers have thus come up with an arsenal of tricks⎯from using carrier lishing molecules and viral vectors to pocking b holes in the membrane⎯to sneak nucleic Pu acids through. But no one method can be applied to all types of cells or experi- Nature ments. “You look at the articles that have 5 been published and find one that has 200 done some comparison of different trans- © fection methods for your cells. But if you HeLa cells transduced with a self-inactivating retrovirus from Clontech’s pQX (retroQ) series, don’t have any clues, you have to try dif- which expresses green fluorescent protein. (Courtesy of Clontech.) ferent reagents, to find what will work best in your hands,” says Nina Iversen, a researcher at the University of Oslo. The 1970s, researchers discovered that they Science sells liposome formulations based good news is that there is no shortage of could also transfect DNA using calcium on the cationic lipids DOTAP and DOSPER. reagents to try! phosphate, a less toxic chemical2. Some Qbiogene’s MegaFectin combines DOTAP labs still use these reagents, which can be with different lipids. Under optimized con- Solution revolution purchased as stand-alones or in kits. For ditions, liposome-mediated methods yield A popular way to get DNA and RNA inside example, Promega’s ProFection mamma- high efficiencies and are much easier to use cells is to use carrier molecules. Such lian transfection systems offer optimized than calcium phosphate. methods do not require expensive equip- buffers and solutions for either calcium ment and, from a technical standpoint, are phosphate– or DEAE-dextran–mediated Of lipids and polymers less difficult to use than viruses. They rely transfection. But for many researchers, The latest generation of lipid-based trans- on the fact that nucleic acids can interact the newer, lipid-based reagents offer fection products includes multicompo- with positively charged molecules, such greater ease of use and efficiency with nent, nonliposomal reagents consisting of as cationic lipids and polymers, to form lower toxicity. lipids, polymers and combinations thereof. complexes that are more palatable to The first lipid-like molecules to come Although their composition is proprietary, the cell. on the scene were mixtures of cationic most of them work by forming a complex Carrier molecules on the market today and other lipids that would form artificial with DNA or RNA that interacts with the owe their existence to the discovery, liposomes. Developed in the late 1980s, cell membrane. The complex is believed almost four decades ago, that coating liposomal transfection reagents work by to be taken up by endocytosis and then DNA with DEAE-dextran would allow it enveloping the DNA or RNA and then fus- released in the cytoplasm. “Unlike the to get past the cell membrane1⎯a pro- ing with the cell membrane to deposit the liposomal agents which form spheres that cess dubbed ‘transfection’. In the early nucleic acid cargo inside. Roche Applied vary greatly in size, nonliposomal lipids NATURE METHODS | VOL.2 NO.11 | NOVEMBER 2005 | 875 TECHNOLOGY FEATURE form micelles of uniform size resulting in It is also applicable to high-throughput formulation of proprietary compounds, more reproducible results,” says Jamuna screens. “It is quite stable after it makes a which has been shown to successfully Ramnath, technical service scientist at complex with DNA, so samples can sit on transfect more than 700 different cell lines. QIAGEN Inc. the deck of a robot loader for a long time,” “One of the advantages of FuGENE 6 over In the lipid arena researchers have says Henry Chiou, manager for R&D at other transfection reagents is its broad a wealth of reagents to choose from. Invitrogen Corporation (Box 1). applicability. It can be used with a wide Invitrogen’s primary product is Roche Applied Sciences’ premier range of cell lines without optimization Lipofectamine 2000, which works with transfection reagent is the FuGENE 6 and is effective in serum-containing media a variety of nucleic acids and cell lines. Transfection Reagent, a nonliposomal allowing for a simple consistent method” says product manager, Jeffrey Emch. The reagent also seems to minimize the chance of nonspecific, off-target effects⎯in other methods BOX 1 GOING THE HIGH-THROUGHPUT WAY words, effects that are not caused by the transfected DNA or RNA. “We have done Say you need to screen hundreds of studies comparing up- and downregula- cDNAs to search for genes that, when tion of off-target gene expression, and we .com/nature e overexpressed, induce cell death. feel comfortable that FuGENE 6 limits off- Or maybe you want to knock down target effects,” says Emch. “In an internal .natur several genes believed to function in w study, off-target effects by FuGENE 6 were the same pathway to better delineate dramatically less than a leading competi- the steps involved. The traditional tive agent.” way to transfect many cells at once http://ww In addition to buffering the nega- is to place the cells in 96- or 384-well tive charge on the DNA many reagents plates, add the DNA (or RNA) and oup also make the cargo smaller to facilitate r transfection reagents, and then study G delivery into cells. InvivoGen’s LipoGen the cells. Alternatively, the plates Magnetic beads coated with DNA encoding combines a lipid that fuses with the cell containing cells and DNA are loaded green fluorescent protein are used to direct membrane with spermine, a divalent cat- in a high-throughput electroporation the transfection of adjacent HEK293 cells. lishing ionic polyamine that interacts with DNA b instrument and processed. Given (Courtesy of Mark Isalan.) and condenses it into compact lipid-DNA Pu the amount of work involved, these complexes. Similarly, QIAGEN’s Effectene protocols typically use robots to transfection reagent consists of a small dispense the reagents and cells, as well as automated plate readers or microscopes to Nature analyze the results. positively charged molecule (called ‘con- 5 In an effort to simplify high-throughput transfection protocols Ziauddin and densing enhancer’) that shrinks the DNA 200 Sabatini3 developed a new method based on microarray technology. In a transfection in a dense structure and a nonliposomal © microarray, plasmid DNA dissolved in a gelatin solution is printed on a glass slide lipid that coats the DNA allowing it to and then covered with a lipid-based transfection reagent. After removing the excess interact with the cell surface. Effectene reagent, the slide is placed in a culture dish and covered with cells in medium. Cells works in the presence of serum and shows growing on the printed areas take up the DNA creating spots of localized transfection low cytotoxicity. within a lawn of nontransfected cells. (In an alternative version of this method the Another line of products that compact lipid-based transfection reagent is added to the DNA prior to printing.) Because cells are DNA includes QIAGEN’s SuperFect and added to the reagent, this approach was called ‘reverse transfection’. PolyFect reagents. They consist of activa- A recent study describes another procedure for conducting parallel cell ted spherical molecules (7 nm in diame- transfections on microscope coverslip arrays, but using a ‘forward’ methodology4. “We ter) with positively charged tentacles. The achieved transfection of a variety of cell lines using magnetic beads coated with PCR DNA coils around these molecules into products,” says Mark Isalan of the European Molecular Biology Laboratory and first histone-like structures that can enter the author of the study. According to the protocol, cells are grown on a glass coverslip cell by endocytosis. “The dendrimer mol- or slide to which magnetic microbeads coated with DNA are added. Using magnets, ecules ensure reproducibility from one the beads are directed to the surface of individual cells where transfection occurs. experiment to the next,” says Ramnath. “We can control the position of the beads to determine which cells to transfect,” Once they have been taken up by endo- explains Isalan. “We can deliver DNA with micrometer resolution.” The efficacy of somes, the DNA-carrier complexes must the technique, which also works with 96-well plates, is greatly enhanced by adding a escape into the cytosol before being transfection reagent to the mix. degraded (Box 2). Some carrier molecules Isalan et al.5 used this technology to engineer an artificial version of a gene network are designed to help this process along. involved in embryonic patterning in the fruit fly. The scientists created a synthetic Metafectene, distributed by Biontex, is a embryo by using a tiny plastic chamber containing various purified genes, proteins, polycationic transfection reagent based metabolites and cell extracts. Some of the genes were attached to magnetic microbeads, on what the company calls “repulsive so that they could be directed to specific locations using magnets anchored to the membrane acidolysis” technology.