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Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro.

Rebersek M, Faurie C, Kanduser M, Corović S, Teissié J, Rols MP, Miklavcic D - Biomed Eng Online (2007)

Bottom Line: Moreover, a new effect a shading effect was observed during our research.Namely, shading effect is observed during gene electrotransfer when cells are in clusters, where only cells facing negative electro-potential in clusters become transfected and other ones which are hidden behind these cells do not become transfected.On the basis of our results we can conclude that the new system can be used in in-vitro gene electrotransfer to improve cell transfection by changing electric field direction between electrical pulses, without affecting cell survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Ljubljana, Faculty of Electrical Engineering, TrZaska 25, Ljubljana, Slovenia. matej.rebersek@fe.uni-lj.si

ABSTRACT

Background: Gene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to a temporary increase of cell membrane permeability. This phenomenon, termed electroporation or electropermeabilization, allows various otherwise non-permeant molecules, including DNA, to cross the membrane and enter the cell. The aim of our research was to develop and test a new system and protocol that would improve gene electrotransfer by automatic change of electric field direction between electrical pulses.

Methods: For this aim we used electroporator (EP-GMS 7.1) and developed new electrodes. We used finite-elements method to calculate and evaluate the electric field homogeneity between these new electrodes. Quick practical test was performed on confluent cell culture, to confirm and demonstrate electric field distribution. Then we experimentally evaluated the effectiveness of the new system and protocols on CHO cells. Gene transfection and cell survival were evaluated for different electric field protocols.

Results: The results of in-vitro gene electrotransfer experiments show that the fraction of transfected cells increases by changing the electric field direction between electrical pulses. The fluorescence intensity of transfected cells and cell survival does not depend on electric field protocol. Moreover, a new effect a shading effect was observed during our research. Namely, shading effect is observed during gene electrotransfer when cells are in clusters, where only cells facing negative electro-potential in clusters become transfected and other ones which are hidden behind these cells do not become transfected.

Conclusion: On the basis of our results we can conclude that the new system can be used in in-vitro gene electrotransfer to improve cell transfection by changing electric field direction between electrical pulses, without affecting cell survival.

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Related in: MedlinePlus

Shading effect. Photos of phase contrast (a) and fluorescence (b) images were taken under inverted fluorescence microscope. Symbolic picture (c) was made for better representation of the observed shading effect. Drawn shapes represent cells and black areas represent regions of permeabilized membrane where DNA interacts with cell membrane. Symbols on the right represent electric field protocol used.
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Figure 8: Shading effect. Photos of phase contrast (a) and fluorescence (b) images were taken under inverted fluorescence microscope. Symbolic picture (c) was made for better representation of the observed shading effect. Drawn shapes represent cells and black areas represent regions of permeabilized membrane where DNA interacts with cell membrane. Symbols on the right represent electric field protocol used.

Mentions: To visualize interaction of DNA with cell membrane immediately after application of electric pulses, we stained plasmid DNA pEGFP-C1 with thiazole orange homodimer dye (TOTO-1, excitation 514 nm, emission 533 nm; Molecular Probes, USA). Plasmid DNA pEGFP-C1 was mixed with TOTO-1 by base pair to dye ratio of 5 and placed on ice for 1 hour [30]. Electropermeabilization procedure was the same as for gene electrotransfer, except that only two different electric field protocols were used as described previously in subsection 2.1: single polarity (SP) and both polarities (BP; Fig. 2), to determine areas of DNA interaction with cell membranes (Fig. 6a, b). Up to 5 minutes after electropermeabilization photos of phase contrast and fluorescence images of cells were taken under inverted fluorescence microscope (Fig. 7, 8). For fluorescence imaging excitation wavelength 480 nm (Polycome IV, Visitron Systems, Germany), dichroic mirror (Q505LP; Chroma, USA) and emission filter (HQ535/50m; Chroma, USA) were used.


Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro.

Rebersek M, Faurie C, Kanduser M, Corović S, Teissié J, Rols MP, Miklavcic D - Biomed Eng Online (2007)

Shading effect. Photos of phase contrast (a) and fluorescence (b) images were taken under inverted fluorescence microscope. Symbolic picture (c) was made for better representation of the observed shading effect. Drawn shapes represent cells and black areas represent regions of permeabilized membrane where DNA interacts with cell membrane. Symbols on the right represent electric field protocol used.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1941843&req=5

Figure 8: Shading effect. Photos of phase contrast (a) and fluorescence (b) images were taken under inverted fluorescence microscope. Symbolic picture (c) was made for better representation of the observed shading effect. Drawn shapes represent cells and black areas represent regions of permeabilized membrane where DNA interacts with cell membrane. Symbols on the right represent electric field protocol used.
Mentions: To visualize interaction of DNA with cell membrane immediately after application of electric pulses, we stained plasmid DNA pEGFP-C1 with thiazole orange homodimer dye (TOTO-1, excitation 514 nm, emission 533 nm; Molecular Probes, USA). Plasmid DNA pEGFP-C1 was mixed with TOTO-1 by base pair to dye ratio of 5 and placed on ice for 1 hour [30]. Electropermeabilization procedure was the same as for gene electrotransfer, except that only two different electric field protocols were used as described previously in subsection 2.1: single polarity (SP) and both polarities (BP; Fig. 2), to determine areas of DNA interaction with cell membranes (Fig. 6a, b). Up to 5 minutes after electropermeabilization photos of phase contrast and fluorescence images of cells were taken under inverted fluorescence microscope (Fig. 7, 8). For fluorescence imaging excitation wavelength 480 nm (Polycome IV, Visitron Systems, Germany), dichroic mirror (Q505LP; Chroma, USA) and emission filter (HQ535/50m; Chroma, USA) were used.

Bottom Line: Moreover, a new effect a shading effect was observed during our research.Namely, shading effect is observed during gene electrotransfer when cells are in clusters, where only cells facing negative electro-potential in clusters become transfected and other ones which are hidden behind these cells do not become transfected.On the basis of our results we can conclude that the new system can be used in in-vitro gene electrotransfer to improve cell transfection by changing electric field direction between electrical pulses, without affecting cell survival.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Ljubljana, Faculty of Electrical Engineering, TrZaska 25, Ljubljana, Slovenia. matej.rebersek@fe.uni-lj.si

ABSTRACT

Background: Gene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to a temporary increase of cell membrane permeability. This phenomenon, termed electroporation or electropermeabilization, allows various otherwise non-permeant molecules, including DNA, to cross the membrane and enter the cell. The aim of our research was to develop and test a new system and protocol that would improve gene electrotransfer by automatic change of electric field direction between electrical pulses.

Methods: For this aim we used electroporator (EP-GMS 7.1) and developed new electrodes. We used finite-elements method to calculate and evaluate the electric field homogeneity between these new electrodes. Quick practical test was performed on confluent cell culture, to confirm and demonstrate electric field distribution. Then we experimentally evaluated the effectiveness of the new system and protocols on CHO cells. Gene transfection and cell survival were evaluated for different electric field protocols.

Results: The results of in-vitro gene electrotransfer experiments show that the fraction of transfected cells increases by changing the electric field direction between electrical pulses. The fluorescence intensity of transfected cells and cell survival does not depend on electric field protocol. Moreover, a new effect a shading effect was observed during our research. Namely, shading effect is observed during gene electrotransfer when cells are in clusters, where only cells facing negative electro-potential in clusters become transfected and other ones which are hidden behind these cells do not become transfected.

Conclusion: On the basis of our results we can conclude that the new system can be used in in-vitro gene electrotransfer to improve cell transfection by changing electric field direction between electrical pulses, without affecting cell survival.

Show MeSH
Related in: MedlinePlus