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Noncoding DNA in lipofection of HeLa cells: a few insights

Nathalie Symens (UGent) , Joanna Rejman (UGent) , Bart Lucas (UGent) , Jo Demeester (UGent) , Stefaan De Smedt (UGent) and Katrien Remaut (UGent)
(2013) MOLECULAR PHARMACEUTICS. 10(3). p.1070-1079
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Abstract
In cationic carrier-mediated gene delivery, the disproportional relationship between the quantity of delivered DNA and the amount of encoded protein produced is a well-known phenomenon. The numerous intracellular barriers which need to be overcome by pDNA to reach the nucleoplasm play a major role in it. In contrast to what one would expect, a partial replacement of coding pDNA by noncoding DNA does not lead to a decrease in transfection efficiency. The mechanism underlying this observation is still unclear. Therefore, we investigated which constituents of the transfection process might contribute to this phenomenon. Our data reveal that the topology of the noncoding plasmid DNA plays a major role. Noncoding pDNA can be used only in a supercoiled form to replace coding pDNA in Lipofectamine lipoplexes, without a loss in transfection levels. When noncoding pDNA is linearized or partly digested, it diminishes the transfection potential of coding pDNA, as does noncoding salmon DNA. The difference in transfection efficiencies could not be attributed to diverse physicochemical characteristics of the Lipofectamine lipoplexes containing different types of noncoding DNA or to the extent of their internalization. At the level of endosomal release, however, nucleic acid release from the endosomal compartment proceeds faster when lipoplexes contain noncoding salmon DNA. Since the half-life of pDNA in the cytosol hardly exceeds 90 min, it is conceivable that prolonged release of coding pDNA from complexes carrying supercoiled noncoding pDNA may explain its positive effect on transfection, while this depot effect does not exist when noncoding salmon DNA is used.
Keywords
NUCLEAR, TOPOLOGY, TRANSGENE EXPRESSION, MAMMALIAN-CELLS, NONLINEAR PHARMACODYNAMICS, PLASMID DNA, TRANSFECTION EFFICIENCY, TRANSPORT, IN-VITRO TRANSCRIPTION, endosomal depot, endosomal release, mRNA, cationic lipids, Lipofectamine, NONVIRAL GENE DELIVERY, transfection, salmon DNA, intact coding plasmid DNA

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Chicago
Symens, Nathalie, Joanna Rejman, Bart Lucas, Jo Demeester, Stefaan De Smedt, and Katrien Remaut. 2013. “Noncoding DNA in Lipofection of HeLa Cells: a Few Insights.” Molecular Pharmaceutics 10 (3): 1070–1079.
APA
Symens, N., Rejman, J., Lucas, B., Demeester, J., De Smedt, S., & Remaut, K. (2013). Noncoding DNA in lipofection of HeLa cells: a few insights. MOLECULAR PHARMACEUTICS, 10(3), 1070–1079.
Vancouver
1.
Symens N, Rejman J, Lucas B, Demeester J, De Smedt S, Remaut K. Noncoding DNA in lipofection of HeLa cells: a few insights. MOLECULAR PHARMACEUTICS. 2013;10(3):1070–9.
MLA
Symens, Nathalie, Joanna Rejman, Bart Lucas, et al. “Noncoding DNA in Lipofection of HeLa Cells: a Few Insights.” MOLECULAR PHARMACEUTICS 10.3 (2013): 1070–1079. Print.
@article{3197624,
  abstract     = {In cationic carrier-mediated gene delivery, the disproportional relationship between the quantity of delivered DNA and the amount of encoded protein produced is a well-known phenomenon. The numerous intracellular barriers which need to be overcome by pDNA to reach the nucleoplasm play a major role in it. In contrast to what one would expect, a partial replacement of coding pDNA by noncoding DNA does not lead to a decrease in transfection efficiency. The mechanism underlying this observation is still unclear. Therefore, we investigated which constituents of the transfection process might contribute to this phenomenon. Our data reveal that the topology of the noncoding plasmid DNA plays a major role. Noncoding pDNA can be used only in a supercoiled form to replace coding pDNA in Lipofectamine lipoplexes, without a loss in transfection levels. When noncoding pDNA is linearized or partly digested, it diminishes the transfection potential of coding pDNA, as does noncoding salmon DNA. The difference in transfection efficiencies could not be attributed to diverse physicochemical characteristics of the Lipofectamine lipoplexes containing different types of noncoding DNA or to the extent of their internalization. At the level of endosomal release, however, nucleic acid release from the endosomal compartment proceeds faster when lipoplexes contain noncoding salmon DNA. Since the half-life of pDNA in the cytosol hardly exceeds 90 min, it is conceivable that prolonged release of coding pDNA from complexes carrying supercoiled noncoding pDNA may explain its positive effect on transfection, while this depot effect does not exist when noncoding salmon DNA is used.},
  author       = {Symens, Nathalie and Rejman, Joanna and Lucas, Bart and Demeester, Jo and De Smedt, Stefaan and Remaut, Katrien},
  issn         = {1543-8384},
  journal      = {MOLECULAR PHARMACEUTICS},
  keyword      = {NUCLEAR,TOPOLOGY,TRANSGENE EXPRESSION,MAMMALIAN-CELLS,NONLINEAR PHARMACODYNAMICS,PLASMID DNA,TRANSFECTION EFFICIENCY,TRANSPORT,IN-VITRO TRANSCRIPTION,endosomal depot,endosomal release,mRNA,cationic lipids,Lipofectamine,NONVIRAL GENE DELIVERY,transfection,salmon DNA,intact coding plasmid DNA},
  language     = {eng},
  number       = {3},
  pages        = {1070--1079},
  title        = {Noncoding DNA in lipofection of HeLa cells: a few insights},
  url          = {http://dx.doi.org/10.1021/mp300569j},
  volume       = {10},
  year         = {2013},
}

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