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Transduction and transfection of difficult‐to‐transfect cells: Systematic attempts for the transfection of protozoa Leishmania
Author(s) -
Keller AndreaAnneliese,
Scheiding Berith,
Breitling Reinhard,
Licht Andreas,
Hemmerich Peter,
Lorkowski Stefan,
Reissmann Siegmund
Publication year - 2019
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.27463
Subject(s) - transfection , electroporation , internalization , microbiology and biotechnology , leishmania , biology , plasmid , dna , transduction (biophysics) , hek 293 cells , exogenous dna , cell , cell culture , gene , biochemistry , genetics , parasite hosting , world wide web , computer science
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.

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