Dilution of reporter gene with stuffer DNA does not alter the transfection efficiency of polyethylenimines
Identifieur interne : 001E80 ( Main/Exploration ); précédent : 001E79; suivant : 001E81Dilution of reporter gene with stuffer DNA does not alter the transfection efficiency of polyethylenimines
Auteurs : Antoine Kichler [France] ; Christian Leborgne [France] ; Olivier Danos [France]Source :
- The Journal of Gene Medicine [ 1099-498X ] ; 2005-11.
English descriptors
- Teeft :
- Assay, Bioconjugate chem, Cell lines, Copyright, Dotap, Experimental conditions, Gene, Gene expression, Gene therapy, Gene transfer, Hepg2, Hepg2 cells, John wiley sons, Kichler, Lipid, Lipospermine dogs, Luciferase, Luciferase activity, Luciferase assay, Luciferase levels, Molecular weight, Monocationic lipid dotap, Negative charges, Optimal ratio, Pei, Plasmid, Polyglutamic, Polyglutamic acid, Polymer, Polyplexes, Polyplus transfection, Reporter gene, Reporter gene expression, Reporter genes, Reporter plasmid, Results show, Salmon sperm, Similar results, Sperm, Stuffer, Suboptimal amounts, Total amount, Transfected, Transfection, Transfection agent, Transfection experiments, Transfection medium.
Abstract
Background: Polyethylenimines (PEIs) are among the most efficient non‐viral gene transfer agents developed so far. However, transfections with these polymers were shown to require a very high copy number of plasmid DNA per cell to achieve gene expression. Here, we investigate whether it is possible to reduce the amount of plasmid DNA while keeping a high transfection efficiency. Methods: Transfection experiments were performed under various conditions in order to study the interdependence between the amount of reporter DNA, the amine‐to‐phosphate ratio and the transfection efficiency. Results: When suboptimal amounts of linear PEI 22 kDa/DNA complexes were used for transfection, a severe reduction in reporter gene expression was observed. On the other hand, for optimal amounts of PEI/DNA complexes more than half of the reporter gene can be replaced by carrier DNA or polyglutamic acid without substantially decreasing the transfection efficiency of the polymer both in cultured cells and after systemic administration in mice. When used under the same in vitro experimental conditions, the lipospermine DOGS, but not the monocationic lipid DOTAP, gave similar results. Conclusions: Taken together, our data suggest that the activity of compounds with endosome‐buffering capacities, such as PEIs and lipospermines, requires a threshold amount of transfection agent. In addition, our results indicate that, in many gene transfer situations, it will be possible to lower the dose of active plasmid thus reducing costs and the risk of immune stimulation triggered by bacterial DNA. Copyright © 2005 John Wiley & Sons, Ltd.
Url:
DOI: 10.1002/jgm.805
Affiliations:
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unit="page" from="1459">1459</biblScope><biblScope unit="page" to="1467">1467</biblScope><biblScope unit="page-count">9</biblScope><publisher>John Wiley & Sons, Ltd.</publisher><pubPlace>Chichester, UK</pubPlace><date type="published" when="2005-11">2005-11</date></imprint><idno type="ISSN">1099-498X</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1099-498X</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Assay</term><term>Bioconjugate chem</term><term>Cell lines</term><term>Copyright</term><term>Dotap</term><term>Experimental conditions</term><term>Gene</term><term>Gene expression</term><term>Gene therapy</term><term>Gene transfer</term><term>Hepg2</term><term>Hepg2 cells</term><term>John wiley sons</term><term>Kichler</term><term>Lipid</term><term>Lipospermine dogs</term><term>Luciferase</term><term>Luciferase activity</term><term>Luciferase assay</term><term>Luciferase levels</term><term>Molecular weight</term><term>Monocationic lipid dotap</term><term>Negative charges</term><term>Optimal ratio</term><term>Pei</term><term>Plasmid</term><term>Polyglutamic</term><term>Polyglutamic acid</term><term>Polymer</term><term>Polyplexes</term><term>Polyplus transfection</term><term>Reporter gene</term><term>Reporter gene expression</term><term>Reporter genes</term><term>Reporter plasmid</term><term>Results show</term><term>Salmon sperm</term><term>Similar results</term><term>Sperm</term><term>Stuffer</term><term>Suboptimal amounts</term><term>Total amount</term><term>Transfected</term><term>Transfection</term><term>Transfection agent</term><term>Transfection experiments</term><term>Transfection medium</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Background: Polyethylenimines (PEIs) are among the most efficient non‐viral gene transfer agents developed so far. However, transfections with these polymers were shown to require a very high copy number of plasmid DNA per cell to achieve gene expression. Here, we investigate whether it is possible to reduce the amount of plasmid DNA while keeping a high transfection efficiency. Methods: Transfection experiments were performed under various conditions in order to study the interdependence between the amount of reporter DNA, the amine‐to‐phosphate ratio and the transfection efficiency. Results: When suboptimal amounts of linear PEI 22 kDa/DNA complexes were used for transfection, a severe reduction in reporter gene expression was observed. On the other hand, for optimal amounts of PEI/DNA complexes more than half of the reporter gene can be replaced by carrier DNA or polyglutamic acid without substantially decreasing the transfection efficiency of the polymer both in cultured cells and after systemic administration in mice. When used under the same in vitro experimental conditions, the lipospermine DOGS, but not the monocationic lipid DOTAP, gave similar results. Conclusions: Taken together, our data suggest that the activity of compounds with endosome‐buffering capacities, such as PEIs and lipospermines, requires a threshold amount of transfection agent. In addition, our results indicate that, in many gene transfer situations, it will be possible to lower the dose of active plasmid thus reducing costs and the risk of immune stimulation triggered by bacterial DNA. Copyright © 2005 John Wiley & Sons, Ltd.</div></front></TEI><affiliations><list><country><li>France</li></country><region><li>Île-de-France</li></region><settlement><li>Évry (Essonne)</li></settlement></list><tree><country name="France"><region name="Île-de-France"><name sortKey="Kichler, Antoine" sort="Kichler, Antoine" uniqKey="Kichler A" first="Antoine" last="Kichler">Antoine Kichler</name></region><name sortKey="Danos, Olivier" sort="Danos, Olivier" uniqKey="Danos O" first="Olivier" last="Danos">Olivier Danos</name><name sortKey="Kichler, Antoine" sort="Kichler, Antoine" uniqKey="Kichler A" first="Antoine" last="Kichler">Antoine Kichler</name><name sortKey="Kichler, Antoine" sort="Kichler, Antoine" uniqKey="Kichler A" first="Antoine" last="Kichler">Antoine Kichler</name><name sortKey="Leborgne, Christian" sort="Leborgne, Christian" uniqKey="Leborgne C" first="Christian" last="Leborgne">Christian Leborgne</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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