Intracellular route and transcriptional competence of polyethylenimine-DNA complexes.
Polyethylenimine (PEI) is a cationic polymer which can be complexed with DNA. PEI-DNA complexes can be used for in vitro and in vivo gene delivery approaches. The excess of positive surface charges enhances the association of the complex with the plasmamembrane of cells and facilitates their uptake by endocytosis. The intracellular transport pathway from the endosome to the nucleus is not understood. Here we show that PEI-DNA complexes are taken up by all cells which are treated with these complexes, indicating, that the uptake is not the rate limiting step in the final transfection efficiency. We reveal by fluorescent microscopy, cell fractionation studies and electron microscopy, that PEI-DNA complexes accumulate in the lysosomal compartment, from where they are released through small local membrane damages. However, the cytoplasmic pool of PEI-DNA complexes is small and with the applied morphological approaches PEI aggregates could not be detected in the nucleus. This indicates, that only a small fraction of the complexes reach their final destiny. To test whether the association of DNA with PEI might be the critical step for transfection, we performed in vitro transcription assays with PEI-DNA complexes. These experiments revealed, that the transcription is not impaired when PEI is closely attached to the template DNA. Our results thus point to the transfer of PEI-DNA complexes from the lysosomal compartment to the nucleus as the rate limiting step in cell transfection.
Bieber
Thorsten
T
University of Marburg, Institut für Cytologie und Cytopathologie, Robert-Koch Strasse 5, D-35033, Marburg, Germany
Meissner
Wolfgang
W
Kostin
Sawa
S
Niemann
Axel
A
Elsasser
Hans-Peter
HP
D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D004274:DNA, Recombinant / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D010190:Pancreatic Neoplasms; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D011094:Polyethyleneimine