Transduction and transfection of difficult‐to‐transfect cells: Systematic attempts for the transfection of protozoa Leishmania. Issue 1 (14th September 2018)

• Record Type: Journal Article
• Authors: Keller, Andrea‐Anneliese; Scheiding, Berith; Breitling, Reinhard; Licht, Andreas; Hemmerich, Peter; Lorkowski, Stefan; Reissmann, Siegmund
• Is Part Of: Journal of cellular biochemistry. Volume 120:Issue 1(2019)
• Page Start: 14
• Page End: 27
• Publication Date: 2018-09-14
• Subjects: cell‐penetrating peptides (CPPs) -- cytotoxicity -- DNA‐binding proteins -- electropermeabilization -- particle sizes and zeta potentials of complexes -- promastigotes of Leishmania tarentolae -- transfection with plasmids; Cytochemistry -- Periodicals; 572
• DOI: 10.1002/jcb.27463
• Languages: English
• ISSNs: 0730-2312

Abstract

Abstract: 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. Abstract : Cells of the protozoa Leishmania tarantolae are difficult to transfect with plamids. Despite successful internalization of proteins with cell‐penetrating peptides we had to fight with many different problems by the transfection with plasmids. Systematic attempts with chemical, biochemical and mechanical and electrical methods were performed. We try to find reasons for easy transduction and difficult transfection and to give more general recommendations for improving internalization. The micrographs show one important reason for unsuccessful uptake of complexes formed from fluorescent DNA and cell‐penetrating peptides: Agglomerates can be larger than cells. A: MPGα; B: CAD‐2; C: Proteoducin; Bar size 20 µm.