4 results for Aneja, MK

  • Transgene expression of transfected supercoiled plasmid DNA concatemers in mammalian cells

    Maucksch, Christof; Bohla, A; Hoffmann, F; Schleef, M; Aneja, MK; Elfinger, M; Hartl, D; Rudolph, C (2009)

    Journal article
    The University of Auckland Library

    Background Supercoiled topology of transfected plasmid DNA (pDNA) is critical for transgene expression in mammalian cells. In the present study, we analysed transgene expression of transfected supercoiled pDNA concatemers. Methods Jurkat T cells were transfected with a supercoiled 4.7-kb monomeric and, in parallel, a 9.4-kb dimeric pEGFP plasmid concatemer using electroporation. The absolute amounts of pDNA delivered into the cytoplasm and the nucleus were quantified by quantitative real-time polymerase chain reaction. Further, the number and mean fluorescent intensity (MFI) of enhanced green fluorescent protein (EGFP) expressing cells and the relative amounts of TOTO-1 fluorescently-labeled pDNA associated with the cell, located in the cytoplasm, and in the nucleus, were analysed by flow cytometry. Results For both constructs, significantly higher amounts of pDNA were detected in the cytoplasm compared to the nucleus. Furthermore, from FACS analysis, we could infer the relative gene copy (Egene) and plasmid expression efficiency (Eplasmid) by determining the ratio of the EGFP MFI of the transfected cells to TOTO-1 MFI per nucleus on the single cell level. Egene and Eplasmid were significantly 1.6-and 3.5-fold higher for EGFP-dimer than for EGFP-monomer, although the transfection rates considering the number of transfected cells were significantly lower for EGFP-dimer than for EGFPmonomer. Together with hydrodynamic plasmid diameter measurements, these observations suggest that concatemer arrangement increases relative gene expression efficiency, whereas plasmid size is important for cell and nucleus entry after electroporation. Conclusions We propose using preferably small supercoiled plasmid concatemers as the ideal plasmid vectors to maximize both transgene expression and the number of transfected target cells.

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  • Cell type differences in activity of the Streptomyces bacteriophage phi C31 integrase

    Maucksch, Christof; Aneja, MK; Hennen, E; Bohla, A; Hoffmann, F; Elfinger, M; Rosenecker, J; Rudolph, C (2008)

    Journal article
    The University of Auckland Library

    Genomic integration by the Streptomyces bacteriophage ΦC31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the ΦC31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34+ haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of rC31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stemcells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in ΦC31 integrase transfected A549 lung than Jurkat T cells. When the rC31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the ΦC31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of ΦC31 integrase activity.

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  • Self-Assembly of Ternary Insulin-Polyethylenimine (PEI)-DNA Nanoparticles for Enhanced Gene Delivery and Expression in Alveolar Epithelial Cells

    Elfinger, M; Pfeifer, C; Uezguen, S; Golas, MM; Sander, B; Maucksch, Christof; Stark, H; Aneja, MK; Rudolph, C (2009-10)

    Journal article
    The University of Auckland Library

    Enhancing gene delivery and expression in alveolar epithelial cells could offer the opportunity for the treatment of acquired and inherited lung diseases. Here, we show that particle adsorption of human insulin (INS) is capable of increasing plasmid DNA (pDNA) delivery from polyethylenimine (PEI) nanoparticles specifically in alveolar epithelial cells. INS receptors were predominantly detected on alveolar but not on bronchial epithelial cells. INS was adsorbed on the surface of PEI gene vectors by spontaneous self-assembly resulting in ternary PEI−pDNA−INS nanoparticles. Surface adsorption was confirmed by particle size, surface charge, and fluorescence resonance energy transfer (FRET) measurements. INS adsorption significantly increased gene expression of PEI−pDNA nanoparticles up to 16-fold on alveolar epithelial cells but not on bronchial epithelial cells. This increased gene expression was INS receptor specific. Our results demonstrate that targeting INS receptor for gene delivery in alveolar epithelial cells represents a promising approach for enhanced gene delivery and expression.

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  • Optimization of Streptomyces bacteriophage phi C31 integrase system to prevent post integrative gene silencing in pulmonary type II cells

    Aneja, MK; Geiger, J; Imker, R; Uzgun, S; Kormann, M; Hasenpusch, G; Maucksch, Christof; Rudolph, C (2009-12-31)

    Journal article
    The University of Auckland Library

    φC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of φC31 integrase system for alveolar type II cells. Luciferase and β-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1α (EF1α) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1α promoter when combined with φC31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with φC31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.

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