Classic transfection technologies have initially been developed for introducing plasmid DNA into cells, and plasmid DNA still remains the most common vector for transfection. DNA plasmids containing recombinant genes and regulatory elements can be transfected into cells to study gene function and regulation, mutational analysis and biochemical characterization of gene products, effects of gene expression on the health and life cycle of cells, as well as for large scale production of proteins for purification and downstream applications.
The topology (linear or supercoiled) and the size of the vector construct, the quality of the plasmid DNA, and the promoter choice are major factors that influence the efficiency of plasmid DNA transfection.
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Transient transfections are more efficient with highly supercoiled DNA compared to linear DNA, presumably because circular DNA is not vulnerable to exonucleases, while linear DNA fragments are quickly degraded by these enzymes (McLenachan et al., 2007; von Groll et al., 2006). In addition, atomic force microscopy analysis shows very different complexation patterns between cationic lipid reagents and circular and linear DNA topologies: while compact spherical or cylindrical condensates are observed with circular DNA, linear plasmids show extended pearl necklace-like structures. Although the cationic lipid-mediated transfection of the more compact circular plasmids is likely to go through endocytosis, the pathway of entry of extended linearized DNA structures might be quite different and less efficient (von Groll et al., 2006).
Stable transfections are more efficient when using linear DNA due to its optimal integration into the host genome. Linear DNA with free ends is more recombinogenic and more likely to be integrated into the host chromosome to yield stable transformants, even though it is taken up by the cell less efficiently.
Despite similar uptake efficiencies in cationic lipid-mediated transfection, nuclear delivery of large plasmids is compromised compared with small plasmid molecules. This effect is observed using equivalent mass or molar concentrations of different-sized constructs, suggesting that nuclear delivery of plasmids may be limited by the rate of intracellular transit and that small plasmids evade degradation by rapid transit through the cytoplasm, rather than through the saturation of cellular defenses (Lukacs, et al., 2000; McLenachan et al., 2007).
Quality of plasmid DNA
Purity and quality of the plasmid DNA is critical for a successful transfection. The best results are achieved with plasmid DNA of the highest purity that is free from phenol, sodium chloride, and endotoxins. Contaminants will kill the cells, and salt will interfere with lipid complexing, decreasing transfection efficiency. Endotoxins, also known as lipopolysaccharides, are released during the lysis step of plasmid preparations and are often co-purified with plasmid DNA. Their presence sharply reduces transfection efficiency in primary and other sensitive cells. We recommend isolating plasmid DNA using PureLink HiPure Plasmid Kits (Mini, Midi, Maxi, Mega, and Giga) that provide highest quality DNA for transfections.
Although cesium chloride banding also yields highly purified DNA, it is a labor intensive and time consuming process. Excess vortexing of DNA-lipid complexes or DNA solutions may result in some shearing, especially with larger molecules, thereby reducing transfection efficiency. The concentration of EDTA in the diluted DNA should not exceed 0.3 mM.
Gene product and promoter
Promoter choice is dependent on the host cell line, the protein to be expressed, and the level of expression desired. Many researchers use the strong CMV (cytomegalovirus) promoter because it provides the highest expression activity in the broadest range of cell types. Another strong promoter for high-level protein expression in mammalian cells is the EF-1α (human elongation factor-1α). However, using too strong a promoter to drive the expression of a potentially toxic gene can cause problems in transient transfection of plasmid DNA. For the potentially toxic gene products, use of weak promoters are recommended.
Toxic gene products are also a problem for selection of stably transfected cells. Cells expressing a gene for antibiotic resistance lose their growth advantage when such gene expression is detrimental to the health of the transfected cell, which makes it impossible to obtain stably transfected clones with a constitutive promoter. In such cases, an inducible promoter can be used to control the timing of gene expression, which will allow for the selection of stable transfectants. Inducible promoters normally require the presence of an inducer molecule (e.g., a metal ion, metabolite, or hormone) to function, but some inducible promoters function in the opposite manner, that is, gene expression is induced in the absence of a specific molecule.
Cell-type specific promoters, such as the polyhedrin promoter for insect cell expression, are also common. Literature searches are the best tool to determine which promoter will work best for your cell line or application.
Regardless of the transfection method used, it is important to perform control transfections to check for cell health, to determine whether the reported assay is working properly, and to establish any insert-related problems. To check for optimal cell growth conditions, include a negative control (no DNA, no transfection reagent). To establish that the reporter assay is working properly, include a positive control (parallel transfection with established transfection method). To determine whether there are insert-related problems, transfect a plasmid without the gene of interest.
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