Numerous chemical and physical methods have been used to introduce DNA expression vectors into mammalian cells both in vitro
and in vivo
, including, but not limited to, calcium phosphate precipitation, microinjection, electroporation, receptor-mediated gene transfer, particle guns, viral vectors, polyfection and lipofection [1
The use of cationic liposome/DNA complexes (lipoplexes) and cationic polymers/DNA (polyplexes) for the transfer of genes into somatic cells has become very popular due to its limited toxicity and relative effectiveness in vitro
. The ionic interaction between cationic lipids and DNA leads to the formation of lipoplexes that are generally slightly cationic. The resulting DNA/lipid complexes fuse with the anionic cytoplasmic membrane and/or are introduced into the cells via an endocytic pathway [2
]. The delivery of the DNA into the nucleus is still not fully understood. While transfection with cationic lipids and polymers offers some advantages over viral transduction, such as simplicity of production, low toxicity, and low immunogenicity; it has yet to reach the levels observed with viral transduction. Furthermore, the adherence of the cationic complexes to the nucleic acid can interfere with its accessibility to enzymes required for processing the DNA [3
One of the most effective and accessible physical transfection methods, electroporation (also known as electrotransfer, electropermeabilization, or nucleofection), involves the application of brief electric pulses to cells or tissues to increase the permeability of cells to macromolecules [1
]. The recent development of the nucleofection system has been a significant advance over standard electroporation systems that have been limited by high toxicity and a requirement for large numbers of cells. A number of cell lines have already been tested for their compatibility with the nucleofection system [5
]. However, there have been no systematic studies comparing nucleofection to chemical transfection systems in various cell types across species.
In this study, chemical reagent-mediated transfection was compared to nucleofection using a number of primary and immortalized cell systems in three different mammalian species (human, rabbit, and pig) to evaluate the efficiency and toxicity. The results presented here indicate that nucleofection is more effective than chemical transfection reagents from several different cationic categories (dendrimer, polyethylenimine, lipid) at delivering DNA into a variety of different cell types. These studies also provided useful insight into transfection optimization conditions and relative cell viability for the various cells tested.
Previous studies indicated that the ratio of DNA to lipid is an important variable that determines the efficiency of transfection and the cellular toxicity [1
]. To evaluate the effect of varying the ratio of DNA to transfection reagent, the cells were transfected with a constant quantity of plasmid DNA in a complex with a variable amount of a given transfection reagent. One to three different DNA/reagent ratios were evaluated for each cell system. In each case, the optimum charge ratio for a given reagent was used for the comparison with nucleofection. The nucleofection buffer and program are critical parameters for nucleofection, so different programs and buffers were tested to obtain the optimal transfection efficiency.