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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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Non-confocal DIC (A, C and inserts in E till G) and confocal images (B and D till G) of Vero cells after cytosolic microinjection of 158 kDa TRITC-dextran. Images (A) and (B) represent a microinjected cell 60 min after microinjection. Images (C) till (G) represent another microinjected cell after 10 s (D), 40 s (E), 5 min (F) and 10 min (G) incubation with 1% (w/v) TCHD.
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Figure 1: Non-confocal DIC (A, C and inserts in E till G) and confocal images (B and D till G) of Vero cells after cytosolic microinjection of 158 kDa TRITC-dextran. Images (A) and (B) represent a microinjected cell 60 min after microinjection. Images (C) till (G) represent another microinjected cell after 10 s (D), 40 s (E), 5 min (F) and 10 min (G) incubation with 1% (w/v) TCHD.

Mentions: In a first approach we microinjected 158 kDa TRITC-dextrans in the cytoplasm of Vero cells and followed their nuclear influx in the absence and presence of TCHD by confocal laser scanning microscopy (CLSM). In the absence of TCHD, no TRITC-dextran could be detected in the nucleus, not even after 1 h of incubation (Figure 1A and B). This is as expected, since it is well-known that molecules larger than ∼70 kDa cannot move passively through the NPC network (41). When TRITC-dextran microinjected cells were incubated with 1% (w/v) TCHD-containing medium, a rapid nuclear localization of the TRITC-dextrans was detected (Figure 1C till G). Indeed, as soon as 10 s after addition of TCHD to the cells, TRITC-dextran was already detected in the nucleus. After 10 min the TRITC-dextran fluorescence was homogeneously distributed throughout the cell. These data clearly demonstrate that TCHD opens the NPCs what results in nuclear passage of macromolecules that otherwise are excluded from the nucleus. We also co-injected TCHD (2% w/v) and TRITC-dextran (158 kDa) in the cytosol, but under these conditions we could not observe nuclear localization (data not shown). One likely explanation is that TCHD, which is an amphiphilic compound and contains a polar ethylene glycol moiety and an apolar butylene moiety, can rapidly cross cell membranes and thus becomes rapidly diluted in the surrounding medium (38).Figure 1.


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)

Non-confocal DIC (A, C and inserts in E till G) and confocal images (B and D till G) of Vero cells after cytosolic microinjection of 158 kDa TRITC-dextran. Images (A) and (B) represent a microinjected cell 60 min after microinjection. Images (C) till (G) represent another microinjected cell after 10 s (D), 40 s (E), 5 min (F) and 10 min (G) incubation with 1% (w/v) TCHD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Non-confocal DIC (A, C and inserts in E till G) and confocal images (B and D till G) of Vero cells after cytosolic microinjection of 158 kDa TRITC-dextran. Images (A) and (B) represent a microinjected cell 60 min after microinjection. Images (C) till (G) represent another microinjected cell after 10 s (D), 40 s (E), 5 min (F) and 10 min (G) incubation with 1% (w/v) TCHD.
Mentions: In a first approach we microinjected 158 kDa TRITC-dextrans in the cytoplasm of Vero cells and followed their nuclear influx in the absence and presence of TCHD by confocal laser scanning microscopy (CLSM). In the absence of TCHD, no TRITC-dextran could be detected in the nucleus, not even after 1 h of incubation (Figure 1A and B). This is as expected, since it is well-known that molecules larger than ∼70 kDa cannot move passively through the NPC network (41). When TRITC-dextran microinjected cells were incubated with 1% (w/v) TCHD-containing medium, a rapid nuclear localization of the TRITC-dextrans was detected (Figure 1C till G). Indeed, as soon as 10 s after addition of TCHD to the cells, TRITC-dextran was already detected in the nucleus. After 10 min the TRITC-dextran fluorescence was homogeneously distributed throughout the cell. These data clearly demonstrate that TCHD opens the NPCs what results in nuclear passage of macromolecules that otherwise are excluded from the nucleus. We also co-injected TCHD (2% w/v) and TRITC-dextran (158 kDa) in the cytosol, but under these conditions we could not observe nuclear localization (data not shown). One likely explanation is that TCHD, which is an amphiphilic compound and contains a polar ethylene glycol moiety and an apolar butylene moiety, can rapidly cross cell membranes and thus becomes rapidly diluted in the surrounding medium (38).Figure 1.

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