Transcription of plasmid DNA: Influence of plasmid DNA/polyethylenimine complex formation

Honoré, Isabelle; Grosse, Stéphanie; Frison, Natacha; Favatier, Florence; Monsigny, Michel; Fajac, Isabelle

doi:10.1016/j.jconrel.2005.06.018

Cited by 75 publications

(54 citation statements)

References 24 publications

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“…The highest GFP expression could be detected at N/P ratio 40 as already seen by others (16). It is known that the N/P ratio plays a crucial role for maximization of the transfection efficiency by influencing the size and the charge of the PEI/DNA complexes (17). Therefore, higher N/P ratios increase DNA condensation and endocytotic uptake by the cells (18).…”

Section: Discussionmentioning

confidence: 55%

Plasmid linearization changes shape and efficiency of transfection complexes

Lehner¹,

Wang²,

Hunziker³

2013

European Journal of Nanomedicine

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Abstract:The ability to efficiently transfect plasmid DNA (pDNA) into eukaryotic cells has exerted major impact on scientific research in recent years, and translation to clinical application is ongoing, but challenging. In addi tion to the choice of the delivery vector, the topology of the DNA seems to be a key factor for efficient transfection. The nanostructured DNA/Vector complexes may differ in size, charge, and shape, for example. This study therefore investigated the transfection efficiency of circular versus linearized plasmid DNA using a GFP expressing vector with Lipofectamine2000 and linear 25 kDA polyethylen imine (PEI). Transfection efficiency and cytotoxicity were measured by flow cytometry and fluorescence micro scopy. Shape was determined by transmission electron microscopy. Transfection agent concentrations were cho sen below the toxicity level. We determined the optimal N/P ratio over 48 h by using two different concentrations of plasmid DNA. With the increase of DNA concentra tion and increasing N/P ratio, transfection efficiency also increased. Our results showed a better transfection effi ciency with the circular compared to the linearized DNA, under the same experimental conditions for both Lipo fectamine and PEI. In electron microscopy, there was a notable difference in the shape of the complexes: circu lar DNA had random coil appearance in well compacted, roughly spherical shape, while linearized DNA appeared as wormlike strands, both, when complexed with Lipo fectamine or with polyethyleneimine. This generates the hypothesis that the shape of the transfection particle may be an important factor for successful gene transfer.

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Section: Discussionmentioning

confidence: 55%

Plasmid linearization changes shape and efficiency of transfection complexes

Lehner¹,

Wang²,

Hunziker³

2013

European Journal of Nanomedicine

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“…Several previous reports comment on a possible influence of the compactness of the polyplex structures on the performance of polyplexes as transfection agents. In particular, the particle size has been shown to affect the internalization pathway whereas compact structures positively influence DNA protection and cytoplasmic transportation [23,27,28]. Figure 5.…”

Section: Influence Of the Transfection Matrixmentioning

confidence: 99%

Influence of Polyplex Formation on the Performance of Star-Shaped Polycationic Transfection Agents for Mammalian Cells

et al. 2016

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Genetic modification ("transfection") of mammalian cells using non-viral, synthetic agents such as polycations, is still a challenge. Polyplex formation between the DNA and the polycation is a decisive step in such experiments. Star-shaped polycations have been proposed as superior transfection agents, yet have never before been compared side-by-side, e.g., in view of structural effects. Herein four star-shaped polycationic structures, all based on (2-dimethylamino) ethyl methacrylate (DMAEMA) building blocks, were investigated for their potential to deliver DNA to adherent (CHO, L929, HEK-293) and non-adherent (Jurkat, primary human T lymphocytes) mammalian cells. The investigated vectors included three structures where the PDMAEMA arms (different arm length and grafting densities) had been grown from a center silsesquioxane or silica-coated γ-Fe 2 O 3 -core and one micellar structure self-assembled from poly(1,2-butadiene)-block PDMAEMA polymers. All nano-stars combined high transfection potential with excellent biocompatibility. The micelles slightly outperformed the covalently linked agents. For method development and optimization, the absolute amount of polycation added to the cells was more important than the N/P-ratio (ratio between polycation nitrogen and DNA phosphate), provided a lower limit was passed and enough polycation was present to overcompensate the negative charge of the plasmid DNA. Finally, the matrix (NaCl vs. HEPES-buffered glucose solution), but also the concentrations adjusted during polyplex formation, affected the results.

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“…Polyplexes of polymeric gene carriers and pDNA have to be recognized and transcribed after entering the nuclei. It has been reported that polymeric carriers inhibit transcription, and the extent of pDNA condensation may be a major factor determining the transcription efficiency [16]. Since polyplexes are not well transcribed in a cell-free system using reticulocyte lysates due to their strong compaction, it is difficult to quantitatively estimate their transcription efficiencies in living cells.…”

Section: Introductionmentioning

confidence: 99%

Quantitative comparison between poly(L-arginine) and poly(L-lysine) at each step of polyplex-based gene transfection using a microinjection technique

Hashimoto

Kawazu

Nagasaki

et al. 2012

Science and Technology of Advanced Materials

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Among the well-studied polypeptide-type gene carriers, transfection efficiency is empirically known to be higher for poly(L-arginine) (PR) than poly(L-lysine) (PK). The big difference between PR and PK should be determined at one of the intracellular trafficking steps based on the different charge densities, structures or PKa values. However, the endosomal escape and the intranuclear transcription efficiency in living cells have not been clarified yet. In this study, a novel method for quantifying the intranuclear transcription efficiency and the nuclear transport of the polyplex is established based on the nuclear and the cytosolic microinjection technique, and the results for PK and PR with different molecular weights (MWs) are compared in living cells. The intranuclear transcription efficiency is the same in PR and PK and it decreases rapidly with increasing MW, in spite of the commonly measured transfection efficiency. The transcription efficiency is strongly suppressed at high MW and strongly correlates with the polyplex forming ability expressed as a critical ratio of the number of polypeptide cationic groups to the number of pDNA anionic groups. When considered with the results of the cellular uptake and in vitro transfection with or without chloroquine, the rate-limiting step for their gene transfer is the buffering effect-independent endosomal escape.

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Transcription of plasmid DNA: Influence of plasmid DNA/polyethylenimine complex formation

Cited by 75 publications

References 24 publications

Plasmid linearization changes shape and efficiency of transfection complexes

Plasmid linearization changes shape and efficiency of transfection complexes

Influence of Polyplex Formation on the Performance of Star-Shaped Polycationic Transfection Agents for Mammalian Cells

Quantitative comparison between poly(L-arginine) and poly(L-lysine) at each step of polyplex-based gene transfection using a microinjection technique

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