Transduction and transfection of difficult-to-transfect cells: Systematic attempts for the transfection of protozoa Leishmania.

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather "difficult" or "hard to transfect," including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.

Transfecting Macrophages.

Transfection is defined as the transfer of foreign nucleic acids into cells. In general, transfection may achieve either overexpression of a gene by the transfer of plasmid DNA or suppression of gene expression by RNA interference after transfer of small interfering RNA. Both approaches allow for the detailed investigation of the function of a particular gene product or mechanisms of gene regulation. Macrophages are considered as hard-to-transfect cells, as they have evolved to recognize foreign nucleic acids and to initiate an immune response to these molecules. The presented electroporation protocol provides an effective tool to efficiently transfect human THP-1 macrophages with siRNA or plasmid DNA while avoiding macrophage activation.