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Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore.

Vandenbroucke RE, Lucas B, Demeester J, De Smedt SC, Sanders NN - Nucleic Acids Res. (2007)

Bottom Line: Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success.Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes.In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.

ABSTRACT
One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

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Effect of TCHD on the nuclear entry of microinjected Cy5-pDNA microinjected in the cytoplasm of Vero cells. A schematic overview of the experimental set up is shown in (B). (A) Represents a microinjected cell with arrow (1) representing the place of cytoplasmic microinjection and arrow (2) the place were the z-scan was performed. The black squares in (C) represent the z-scan through the cytosol and nucleus of a microinjected Vero at time point zero, so just after microinjection and before addition of TCHD. The gray circles represent the z-scan 60 min after addition of 1% (w/v) TCHD. In image (C), the center of the cell nucleus is located at z-value –0.048. The time-dependent increase of Cy5-pDNA fluorescence in the nucleus after cytoplasmic injection of Cy5-pDNA without (gray squares) and with (black circles) TCHD is shown in (D). The black circle in (A) indicates the nuclear membrane.
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Figure 2: Effect of TCHD on the nuclear entry of microinjected Cy5-pDNA microinjected in the cytoplasm of Vero cells. A schematic overview of the experimental set up is shown in (B). (A) Represents a microinjected cell with arrow (1) representing the place of cytoplasmic microinjection and arrow (2) the place were the z-scan was performed. The black squares in (C) represent the z-scan through the cytosol and nucleus of a microinjected Vero at time point zero, so just after microinjection and before addition of TCHD. The gray circles represent the z-scan 60 min after addition of 1% (w/v) TCHD. In image (C), the center of the cell nucleus is located at z-value –0.048. The time-dependent increase of Cy5-pDNA fluorescence in the nucleus after cytoplasmic injection of Cy5-pDNA without (gray squares) and with (black circles) TCHD is shown in (D). The black circle in (A) indicates the nuclear membrane.

Mentions: Next, we tested whether TCHD can also facilitate the nuclear uptake of pDNA, since pDNAs are much larger (2–10 MDa) than 158 kDa TRITC-dextran and have dimensions in the range of the inner diameter of the channels formed by the NPCs. When Cy5-pDNA was microinjected in the cytoplasm of Vero cells, a fluorescent spot was visible at the injection site (Figure 2A, position 1). After 1 h incubation with TCHD, we could not observe accumulation of the pDNA inside the nucleus by CLSM (data not shown). Importantly, during that time the fluorescent microinjection spot became more diffuse but remained visible, indicating a restricted mobility of pDNAs in the cytoplasm. This is in agreement with the observations by Lukacs et al. (42) who showed that the diffusion of pDNA in the cytoplasm may be an important rate-limiting barrier in gene delivery utilizing non-viral vectors. Hence, after 1 h only a small fraction of the microinjected pDNA will have reached the nuclear membrane. Additionally, as stated earlier the inner diameter of the NPC is in the size range of pDNA. Therefore, even in the presence of TCHD the number of pDNA molecules that enter the nucleus is probably low and beyond the detection limit of the CLSM.Figure 2.


Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore.

Vandenbroucke RE, Lucas B, Demeester J, De Smedt SC, Sanders NN - Nucleic Acids Res. (2007)

Effect of TCHD on the nuclear entry of microinjected Cy5-pDNA microinjected in the cytoplasm of Vero cells. A schematic overview of the experimental set up is shown in (B). (A) Represents a microinjected cell with arrow (1) representing the place of cytoplasmic microinjection and arrow (2) the place were the z-scan was performed. The black squares in (C) represent the z-scan through the cytosol and nucleus of a microinjected Vero at time point zero, so just after microinjection and before addition of TCHD. The gray circles represent the z-scan 60 min after addition of 1% (w/v) TCHD. In image (C), the center of the cell nucleus is located at z-value –0.048. The time-dependent increase of Cy5-pDNA fluorescence in the nucleus after cytoplasmic injection of Cy5-pDNA without (gray squares) and with (black circles) TCHD is shown in (D). The black circle in (A) indicates the nuclear membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919477&req=5

Figure 2: Effect of TCHD on the nuclear entry of microinjected Cy5-pDNA microinjected in the cytoplasm of Vero cells. A schematic overview of the experimental set up is shown in (B). (A) Represents a microinjected cell with arrow (1) representing the place of cytoplasmic microinjection and arrow (2) the place were the z-scan was performed. The black squares in (C) represent the z-scan through the cytosol and nucleus of a microinjected Vero at time point zero, so just after microinjection and before addition of TCHD. The gray circles represent the z-scan 60 min after addition of 1% (w/v) TCHD. In image (C), the center of the cell nucleus is located at z-value –0.048. The time-dependent increase of Cy5-pDNA fluorescence in the nucleus after cytoplasmic injection of Cy5-pDNA without (gray squares) and with (black circles) TCHD is shown in (D). The black circle in (A) indicates the nuclear membrane.
Mentions: Next, we tested whether TCHD can also facilitate the nuclear uptake of pDNA, since pDNAs are much larger (2–10 MDa) than 158 kDa TRITC-dextran and have dimensions in the range of the inner diameter of the channels formed by the NPCs. When Cy5-pDNA was microinjected in the cytoplasm of Vero cells, a fluorescent spot was visible at the injection site (Figure 2A, position 1). After 1 h incubation with TCHD, we could not observe accumulation of the pDNA inside the nucleus by CLSM (data not shown). Importantly, during that time the fluorescent microinjection spot became more diffuse but remained visible, indicating a restricted mobility of pDNAs in the cytoplasm. This is in agreement with the observations by Lukacs et al. (42) who showed that the diffusion of pDNA in the cytoplasm may be an important rate-limiting barrier in gene delivery utilizing non-viral vectors. Hence, after 1 h only a small fraction of the microinjected pDNA will have reached the nuclear membrane. Additionally, as stated earlier the inner diameter of the NPC is in the size range of pDNA. Therefore, even in the presence of TCHD the number of pDNA molecules that enter the nucleus is probably low and beyond the detection limit of the CLSM.Figure 2.

Bottom Line: Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success.Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes.In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.

ABSTRACT
One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

Show MeSH
Related in: MedlinePlus