Transfection Connection: Methods of Nucleic Acid Delivery Into Eukaryotic Cells

Date: September 29, 1997 Table Comparing Products Perhaps the most fundamental question when one has a nucleic acid or gene sequence in hand is "So, what does it do?" Whether these sequences encode a protein, or are promoter/enhancer regulatory elements, the general goal is to determine function and to understand the regulation and interaction of these genes and gene products with other cellular components. These questions are, of course, best addressed within a cellular context. The ability to introduce DNA into cultured cells is therefore both powerful and necessary for studying the function and control of mammalian genes. A wide variety of techniques is available that permit the delivery of molecules into eukaryotic cells. Unfortunately, no single method can be identified as ideal for all cell types or systems. Each system and, indeed, the specific experimental goals or requirements may require different gene delivery methods. There are two general approaches for introducing or delivering nucleic acids into mammalian cells. Transfection is the process whereby the nucleic acid sequences (as well as proteins and oligonucleotides) are introduced by either biochemical or physical processes. Biochemical methods include DEAE-dextran, calcium phosphate, and liposome-mediated transfection methods. Physical transfection methods include the direct micro-injection of materials, biolistic particle delivery (both methods deliver materials by mechanically perforating the cell membrane), and electroporation. Electroporation exposes target cells to brief, defined electrical pulses to create transient pores that allow nucleic acids and proteins to cross the cell membrane. The second general approach for delivering nucleic acids into cells is infection. Infection is a viral mediated process whereby target cells are infected with a virus whose genome carries an inserted cloned sequence. While this can be an efficient method for some cell lines and tissues, the limitation of this technique is that it requires host cells to possess a viral receptor in order to achieve high efficiency transfer. This requirement limits the broad utility of this method of transfection to only select systems. For nearly 30 years, biochemical procedures have been the most commonly used methods for transfecting cells; these are now being somewhat supplanted by methods such as electroporation. While biochemical methods do not obtain the efficiencies of newer technologies, they offer the following advantages: published protocols are available for numerous cell lines; these methods do not require removal of adherent cells for transfection to occur; and laboratories incur minimal expenditures for equipment. Additionally, while the traditional methods of transfection were useful only on adherent cell lines, lipid-based methods work well with suspension cultures. With each new generation of transfection reagents, efficiencies have also increased, while cellular cytotoxicity has decreased. Vaheri and Pagano, Virology, 27:424-436, 1965 described this method, the first nucleic acid transfection technique. With DEAE-dextran mediated cell transfection, positively charged DEAE-dextran binds to the negatively charged phosphate groups of the DNA, forming aggregates. These complexes, when applied to cells, subsequently bind to the negatively charged plasma membrane. Cellular uptake is believed to be mediated by endocytosis, further assisted by an osmotic shock. Key features and considerations for DEAE-dextran transfection include This method is generally useful for transient expression only. High concentrations of DEAE-dextran can be toxic to cells. Different cell lines show different sensitivity toward DEAE-dextran and osmotic shock. Transfection efficiencies will vary with cell type. Preparation is quick: DEAE-dextran is prepared, and DNA is added; cells are rinsed; DEAE-dextran/DNA mixture is added to cells, cells are incubated for 15 minutes, cells are washed and growth medium can be immediately added. Calcium phosphate-mediated transfection, described in 1973 by Graham and Van der Eb, Virology, 52:456, 1973, quickly became the most common method of cell transfection. Based on the formation of small, insoluble calcium phosphate-DNA precipitates, this procedure requires that calcium ions first bind to the negatively charged phosphate groups of the DNA. When phosphate is subsequently added to the mixture, it bonds ionically to the calcium ions to form a crystalline complex, which precipitates. The size of these precipitate complexes affects the efficiency of transfection. It is believed that the precipitate attaches to the cell surface and is absorbed by endocytosis. Key features and considerations for calcium phosphate transfection include: This method is useful for both stable and transient transfection. It is useful with a wide range of cell types, although efficiencies will vary between each type. Size and quality of the precipitate are crucial to the success of transfection. Reagent consistency is critical for reproducibility. Preparation is reasonably quick; a slightly longer preparation of DNA mixture is required compared to DEAE dextran. Cells are typically incubated for 6 to 12 hours, washed, and followed with a brief glycerol/HEPES or DMSO shock treatment. Cells are finally washed and complete growth medium is added. Lipid-mediated transfection was described in 1987 by Felgner et al. (PNAS, 84:7413). This method relies on cationic lipids to form small unilamellar liposomes whose surfaces are positively charged. Liposomes have an inherent attraction to both the phosphate groups of DNA and the negatively charged plasma membranes of cells. First-generation reagents used neutral lipids as transfection vehicles and relied on a strategy that attempted to capture DNA within the liposomes themselves. Today's generation of cationic lipids attempts to bind DNA in the form of lipid-DNA complexes rather than physically capture DNA within the liposomes. It is thought that between two to four liposomes appear to associate with a single DNA 5 kb vector molecule. With higher efficiencies, lower toxicity, and a broad host cell range, lipid-mediated transfection has become the most versatile biochemical-based transfection method available. Two types of lipids are commonly used for lipid-mediated transfection: monocationic and polycationic lipids. Polycationic reagents yield higher transfection efficiencies than their monocationic cousins. It is believed that this is due to the presence of the highly positively charged amine head-groups of these reagents (such as the spermine head-group on DOSPA). Both the monocationic and polycationic lipids form unilamellar vesicles in solution. These vesicles interact spontaneously with DNA to form stable cationic complexes that subsequently adhere to and fuse with the negatively charged cell membrane. Key features and considerations for lipid-mediated transfection include Consistency and reproducibility Higher transfection efficiency Lower toxicity Useful on a broad range of cell lines and tissues Capability to be used with adherent or suspension cultures Ease of use and speed Stability of reagents Selecting the appropriate lipid for transfecting a specific cell line may require some fine-tuning on your part. Of course, you should always contact the company's technical service department for protocols that may already be available. This should significantly reduce the time required to optimize experimental parameters. Most companies providing support during the writing of this article were very helpful and provided extensive lists of cell lines for which methods have been established. With that said and done, for a given cell line, different lipid reagents will have varying transfection efficiencies. An initial strategy may therefore include trying different reagents and determining which has the greatest efficiency for your system. (Surely meant to help you obtain the highest transfection possible for your system, some companies provide kits with multiple lipid formulations for optimizing protocols. Invitrogen, for example, has a kit with eight different lipids, while PanVera and Promega each market a kit with three.) Identifying which transfection agent is ideal for your system is easily accomplished with a reporter assay, either by indirectly evaluating activities in an extract or by direct histochemical staining for activity in transfected cells. Here again, most companies can provide control reporter vectors for this purpose. (Some companies such as Invitrogen and Stratagene provide reporter vector controls with their kits.) For those fellow impatient souls, RT-PCR-based assays are quite helpful in getting out of the efficiency-determination starting blocks in a hurry. Establishing a reliable reporter assay can also be useful in comparing efficiencies between transfections, thereby standardizing expression levels. Remember, as well, that the condition and maintenance of the cells to be transfected will have a significant effect on the transfectability and repeatability of your experiments. Along with the level of confluency, the number of passages can affect transfection. In addition, it is best to limit variation of media and serum during these experiments. Finally, excessive exposure to transfection reagents will eventually result in cell death. Once you have determined which reagent works best, your next task will be to optimize your protocol. The most important variables to concentrate on are lipid concentration (lipid:DNA ratio), duration of transfection, DNA concentration (starting with the highest purity DNA is always advisable), and cell density of your culture. In general, with increasing amounts of DNA, there will be an increase in the amount (or ratio) of lipid required. The optimal amount of lipid required for a given concentration of DNA, however, appears to vary with the density of the culture. In all instances, whether DEAE-dextran, calcium phosphate or lipid methodologies, in no case should cultures be greater than 80% confluent. For most lipid-mediated transfection reagents, including or omitting serum has little or no effect on transfection efficiencies. It is recommended, however, that this be empirically determined for your specific system. The world of transfection doesn't end here. There are yet a few more products to consider before heading to the phone. Described as a "specifically designed polycation of defined shape and diameter," QIAGEN's SuperFect Reagent is a solution of specifically designed activated dendrimers. Dendrimers are spherical polyamindoamine molecules with branches radiating from a central core and terminating at charged amino groups. Chemical activation promotes efficient uptake of DNA by eukaryotic cells. This reagent is thought to assemble DNA into compact structures, thereby optimizing the entry of DNA into cells. To stabilize the SuperFect-DNA complexes during their transport to the nucleus, the SuperFect reagent is designed to buffer the lysosome after fusion with the endosome, leading to pH inhibition of lysosomal nucleases. SuperFect is claimed to provide greater efficiencies than many commercially available liposome-based reagents with less cytotoxicity. New from Life Technologies (GIBCO/BRL) is the LipofectAMINE PLUSTM Reagent, a proprietary reagent that is used in conjunction with Life Technologies' original LipofectAMINE Reagent. The PLUS Reagent confers a two to five times increase in transfection efficiencies over the standard LipofectAMINE reagent. Use of the PLUS reagent also reduces the amount of cationic lipid reagent and DNA required and minimizes protocol optimization procedures. Boehringer Mannheim, in addition to its line of liposome-based reagents, provides the FuGENETM 6 Transfection Reagent. An optimized blend of lipids and other proprietary compounds, FuGENE 6 is claimed to provide high transfection efficiencies without the need for the optimization steps required with some cationic liposome-based reagents. Because of the low cytotoxicity of the reagent blend, for most applications, the reagent does not need to be removed before the reporter gene assay, and can function in the presence or absence of serum. When HMG-1, -2 Mixture is added to DNA samples prior to the use of cationic lipids, Wako BioProducts claims that expression increases up to ten times over the rate achieved when untreated DNA or oligonucleotide samples are used. This product seems particularly suited for transfecting tissues. HMG-1, -2 (for high mobility group -1 and -2) are the major non-histone nuclear proteins found in eukaryotic cells. In the nucleus, these proteins are thought to function in the formation of chromatin and the regulation of gene expression. In the application of transfection, it is speculated that HMG-1, -2 stabilizes and assists in the active transport of DNA into the nucleus, thereby increasing transfection efficiencies. If retroviral transfection is an option available to your lab, PanVera provides a recombinant human fibronectin fragment (manufactured by TaKaRa Shuzo Co.) which when coated on the surface of plates, dishes, or flasks significantly enhances retroviral-mediated transfection. The 63-kDa RetroNectin¦ protein contains a central cell-binding domain, a high affinity heparin-binding domain, and the CS1 site of human fibronectin. It is believed that the heparin-binding domain of the chimeric protein assists in co-localizing retroviral particles and target cells. The DNA world delivered to your cell door. A number of products are available to either ease your current transfection methods or introduce you to even higher efficiencies and host cell selection. Luckily, many manufacturers continue to support the bread-and-butter techniques of calcium phosphate and DEAE-dextran-based methods, providing kits that allow consistencies between lots and experiments. In addition, there is now a new generation of liposome reagents that are specifically designed to speed and maximize your experiments. Expression is the name of the game for most experiments, and these products are meant to get you there quicker and with less cytotoxic effects on cells. While liposome methods seem to predominate, some novel methodologies seem to further the ability of delivering nucleic acids into cells. Which method is best will be difficult to decide. Nevertheless, there are options, that until a few years ago did not exist. Table Comparing Products Thomas Unger, Vice President of Research and Development for Miragen Inc., a biotech company located in Irvine, CA, can be reached at tfunger@aol.com

Transfection Connection: Methods of Nucleic Acid Delivery Into Eukaryotic Cells

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