Dataset Information

Enhancing Electrotransfection Efficiency through Improvement in Nuclear Entry of Plasmid DNA.

ABSTRACT: The nuclear envelope is a physiological barrier to electrogene transfer. To understand different mechanisms of the nuclear entry for electrotransfected plasmid DNA (pDNA), the current study investigated how manipulation of the mechanisms could affect electrotransfection efficiency (eTE), transgene expression level (EL), and cell viability. In the investigation, cells were first synchronized at G2-M phase prior to electrotransfection so that the nuclear envelope breakdown (NEBD) occurred before pDNA entered the cells. The NEBD significantly increased the eTE and the EL while the cell viability was not compromised. In the second experiment, the cells were treated with a nuclear pore dilating agent (i.e., trans-1,2-cyclohexanediol). The treatment could increase the EL, but had only minor effects on eTE. Furthermore, the treatment was more cytotoxic, compared with the cell synchronization. In the third experiment, a nuclear targeting sequence (i.e., SV40) was incorporated into the pDNA prior to electrotransfection. The incorporation was more effective than the cell synchronization for enhancing the EL, but not the eTE, and the effectiveness was cell type dependent. Taken together, the data described above suggested that synchronization of the NEBD could be a practical approach to improving electrogene transfer in all dividing cells.

SUBMITTER: Cervia LD

PROVIDER: S-EPMC5992438 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Enhancing Electrotransfection Efficiency through Improvement in Nuclear Entry of Plasmid DNA.

Cervia Lisa D LD Chang Chun-Chi CC Wang Liangli L Mao Mao M Yuan Fan F

Molecular therapy. Nucleic acids 20180301

The nuclear envelope is a physiological barrier to electrogene transfer. To understand different mechanisms of the nuclear entry for electrotransfected plasmid DNA (pDNA), the current study investigated how manipulation of the mechanisms could affect electrotransfection efficiency (eTE), transgene expression level (EL), and cell viability. In the investigation, cells were first synchronized at G2-M phase prior to electrotransfection so that the nuclear envelope breakdown (NEBD) occurred before p ...[more]

PMID: 29858061

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Project description:Cationic polymers are commonly used to transfect mammalian cells, but their mechanisms of DNA delivery are unknown. This study seeks to decipher the mechanism by which plasmid DNA delivered by a class of cationic polymers traffics to and enters the nucleus. While studies have been performed to elucidate the mechanism of naked plasmid DNA (pDNA) import into the nuclei of mammalian cells, our objectives were to determine the effects of polymer complexation on pDNA nuclear import and the impact of polymer structure on that import. We have performed studies in whole cells and in isolated nuclei using flow cytometry and confocal microscopy to characterize how polymer-DNA complexes (polyplexes) are able to deliver their pDNA cargo to the nuclei of their target cells. The polymers tested herein include (i) linear poly(ethylenimine) (JetPEI), a polyamine, and (ii) two poly(glycoamidoamine)s (PGAAs), polyamines that contain carbohydrate moieties (meso-galactarate, Glycofect (G4), and L-tartarate, T4) within their repeat units. Our results indicate that, when complexed with the PGAAs, pDNA association with the nuclei was severely hampered in isolated nuclei compared to whole cells. When the pDNA was complexed with JetPEI, there was slight inhibition of pDNA-nuclear interaction in isolated nuclei compared to whole cells. However, even in the case of PEI, the amount of pDNA imported into the nucleus increases in the presence of cytosolic extract, thus indicating that intracellular components also play a role in pDNA nuclear import for all polymers tested. Interestingly, PEI and G4 exhibit the highest reporter gene expression as well as inducing higher envelope permeability compared to T4, suggesting that the ability to directly permeabilize the nuclear envelope may play a role in increasing expression efficiency. In addition, both free T4 and G4 polymers are able to cross the nuclear membrane without their pDNA cargo in isolated nuclei, indicating the possibility of different modes of nuclear association for free polymers vs polyplexes. These results yield insight to how the incorporation of carbohydrate moieties influences intracellular mechanisms and will prove useful in the rational design of safe and effective polymer-based gene delivery vehicles for clinical use.

Dataset Information

Enhancing Electrotransfection Efficiency through Improvement in Nuclear Entry of Plasmid DNA.

Publications

Enhancing Electrotransfection Efficiency through Improvement in Nuclear Entry of Plasmid DNA.

Similar Datasets

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