Related Experiment Videos
Zinc improves gene transfer mediated by DNA/cationic polymer complexes.
Chantal Pichon1, Brigitte Guérin, Matthieu Réfrégiers
1Centre de Biophysique Moléculaire, CNRS UPR 4301, rue Charles-Sadron, F-45071 Orléans Cedex 02, France.
The Journal of Gene Medicine
|September 11, 2002
Summary
Zinc ions significantly enhance DNA transfection efficiency, particularly with histidine-rich molecules. This improvement in polyfection is achieved by promoting endosomal fusion, leading to increased plasmid delivery into the cell cytosol and nucleus.
Area of Science:
- Biotechnology
- Molecular Biology
- Cell Biology
Background:
- Plasmid transfer into the cytosol limits transfection efficiency with DNA/cationic polymer complexes.
- Divalent metal ions, specifically Zn2+, can improve polyfection efficiency.
Purpose of the Study:
- To investigate the effect of Zn2+ on polyfection efficiency using DNA/cationic polymer complexes.
- To determine the mechanism by which Zn2+ enhances transfection, particularly with DNA/histidylated polylysine (His-pLK) complexes.
Main Methods:
- Supplementation of transfection medium with ZnCl2.
- Transfection of HepG2 cells with EGFP plasmid complexed with pLK, polyethyleneimine, and His-pLK.
- Flow cytometry and confocal microscopy.
- Resonance energy transfer studies with H5WYG peptide.
Main Results:
- 250 micro M ZnCl2 increased EGFP-positive cells from 1% to over 40% with DNA/His-pLK complexes.
- Zn2+ effect is selective and cell-dependent.
- Zinc ions promote endosomal membrane fusion, enhancing plasmid delivery to the cytosol and nucleus.
- Zinc inhibits membrane permeabilization but promotes membrane fusion.
Conclusions:
- Addition of zinc ions to transfection medium increases endosomal fusion of polyplexes, especially with histidine-rich molecules.
- Enhanced fusion leads to increased cytosolic plasmid availability, improving polyfection efficiency.
- Zn2+ is a promising additive for improving gene delivery systems.