Characterizing peptide-mediated DNA internalization in human cancer cells.
Cell penetrating peptides (CPPs) are currently used to deliver various macromolecular cargos to intracellular sites of action both in vitro and in vivo on an experimental basis. During the last few years, even more evidence has accumulated indicating that the main route of entry for most CPPs is through endocytosis rather than direct membrane penetration, as initially proposed. The specific endocytosis pathway utilized by CPPs is, however, still ill-defined and potentially varies depending on what CPPs, cargos, and cell lines are being studied. In this chapter, we provide detailed protocols for an initial characterization of the uptake mechanism involved in CPP-mediated delivery of DNA. Methods to both quantitatively and qualitatively study the uptake using fluorescence-assisted cell sorting (FACS) and confocal microscopy, respectively, are provided. Furthermore, methods to study the intracellular fate of the internalized cargo by co-localization studies between internalized DNA and established endosomal markers, e.g., transferrin, dextran as well as caveolin-1, are described. Finally, we provide a protocol to determine the dependence on dynamin, i.e., a central mediator of vesicle fission at the cell membrane, for DNA-peptide complex uptake using a dominant-negative construct of dynamin-2.
Wittrup
Anders
A
Department of Clinical Sciences, Section of Oncology, Lund University, Sweden.
Belting
Mattias
M
D002469:Cell Separation; D004247:DNA / Q000378:metabolism*; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis; D006801:Humans; D008854:Microscopy, Electron; D010455:Peptides / Q000378:metabolism*
D010455:Peptides; D004247:DNA; D034281:Dynamins