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- Title
Fluid Phase Endocytosis Contributes to Transfection of DNA by PEI-25.
- Authors
Hufnagel, Hansjörg; Hakim, Parvez; Lima, Aline; Hollfelder, Florian
- Abstract
The understanding of internalization pathways of lipo- or polyplexes is crucial for engineering successful reagents for nonviral gene transfection. A known inhibitor of fluid phase endocytosis (FPE), rottlerin, was used to quantify the contribution of this pathway by flow cytometric and fluorescence assays. Rottlerin was shown to be a specific inhibitor of transfection by polyethylene imine (PEI-25)/DNA complexes, leading to a decrease in the amount of transfected HeLa and CHO-K1 cells and a decrease in the expression of enhanced green fluorescent protein (EGFP) reporter gene by up to 50%. Experiments using fluorescently labeled polyplexes result in a decrease of uptake by up to 40%. Additionally, rottlerin does not cross-inhibit clathrin- and caveolin-mediated endocytotic pathways of internalization, consistent with direct uptake inhibition by rottlerin. Nonspecific effects as a result of toxicity were ruled out by control experiments at concentrations where rottlerin inhibition was specific. These findings suggest that for CHO-K1 and HeLa cells, internalization of PEI-25/DNA complexes by FPE plays a decisive role in gene transfection. The establishment of an additional pathway that is independent of clathrin- and caveolin-mediated endocytotic uptake may have an impact on the design of future reagents of nonviral gene therapy and investigations of the uptake pathways and intracellular trafficking involved.Molecular Therapy (2009) 17 8, 1411–1417. doi:10.1038/mt.2009.121
- Subjects
ENDOCYTOSIS; GENETIC regulation; GENE transfection; GREEN fluorescent protein; GENE therapy; CANCER cells
- Publication
Molecular Therapy, 2009, Vol 17, Issue 8, p1411
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1038/mt.2009.121