@article{MoradianLendleinGossen2020,
  author    = {Moradian, Hanieh and Lendlein, Andreas and Gossen, Manfred},
  title     = {Strategies for simultaneous and successive delivery of RNA},
  series = {Journal of molecular medicine},
  volume    = {98},
  journal   = {Journal of molecular medicine},
  number    = {12},
  publisher = {Springer},
  address   = {Heidelberg},
  issn      = {0946-2716},
  doi       = {10.1007/s00109-020-01956-1},
  pages     = {1767 -- 1779},
  year      = {2020},
  abstract  = {Advanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.},
  language  = {en}
}
@misc{MoradianRochLendleinetal.2020,
  author    = {Moradian, Hanieh and Roch, Toralf and Lendlein, Andreas and Gossen, Manfred},
  title     = {mRNA transfection-induced activation of primary human monocytes and macrophages},
  series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  journal   = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe},
  number    = {1},
  issn      = {1866-8372},
  doi       = {10.25932/publishup-51569},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-515694},
  pages     = {17},
  year      = {2020},
  abstract  = {Monocytes and macrophages are key players in maintaining immune homeostasis. Identifying strategies to manipulate their functions via gene delivery is thus of great interest for immunological research and biomedical applications. We set out to establish conditions for mRNA transfection in hard-to-transfect primary human monocytes and monocyte-derived macrophages due to the great potential of gene expression from in vitro transcribed mRNA for modulating cell phenotypes. mRNA doses, nucleotide modifications, and different carriers were systematically explored in order to optimize high mRNA transfer rates while minimizing cell stress and immune activation. We selected three commercially available mRNA transfection reagents including liposome and polymer-based formulations, covering different application spectra. Our results demonstrate that liposomal reagents can particularly combine high gene transfer rates with only moderate immune cell activation. For the latter, use of specific nucleotide modifications proved essential. In addition to improving efficacy of gene transfer, our findings address discrete aspects of innate immune activation using cytokine and surface marker expression, as well as cell viability as key readouts to judge overall transfection efficiency. The impact of this study goes beyond optimizing transfection conditions for immune cells, by providing a framework for assessing new gene carrier systems for monocyte and macrophage, tailored to specific applications.},
  language  = {en}
}
@article{MoradianRochLendleinetal.2020,
  author    = {Moradian, Hanieh and Roch, Toralf and Lendlein, Andreas and Gossen, Manfred},
  title     = {mRNA transfection-induced activation of primary human monocytes and macrophages},
  series = {Scientific reports},
  volume    = {10},
  journal   = {Scientific reports},
  number    = {1},
  publisher = {Springer Nature},
  address   = {London},
  issn      = {2045-2322},
  doi       = {10.1038/s41598-020-60506-4},
  pages     = {1 -- 15},
  year      = {2020},
  abstract  = {Monocytes and macrophages are key players in maintaining immune homeostasis. Identifying strategies to manipulate their functions via gene delivery is thus of great interest for immunological research and biomedical applications. We set out to establish conditions for mRNA transfection in hard-to-transfect primary human monocytes and monocyte-derived macrophages due to the great potential of gene expression from in vitro transcribed mRNA for modulating cell phenotypes. mRNA doses, nucleotide modifications, and different carriers were systematically explored in order to optimize high mRNA transfer rates while minimizing cell stress and immune activation. We selected three commercially available mRNA transfection reagents including liposome and polymer-based formulations, covering different application spectra. Our results demonstrate that liposomal reagents can particularly combine high gene transfer rates with only moderate immune cell activation. For the latter, use of specific nucleotide modifications proved essential. In addition to improving efficacy of gene transfer, our findings address discrete aspects of innate immune activation using cytokine and surface marker expression, as well as cell viability as key readouts to judge overall transfection efficiency. The impact of this study goes beyond optimizing transfection conditions for immune cells, by providing a framework for assessing new gene carrier systems for monocyte and macrophage, tailored to specific applications.},
  language  = {en}
}
@misc{ListekHoenowGossenetal.2020,
  author    = {Listek, Martin and H{\"o}now, Anja and Gossen, Manfred and Hanack, Katja},
  title     = {A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line},
  series = {Postprints der Universit{\"a}t Potsdam : Mathematisch Naturwissenschaftliche Reihe},
  journal   = {Postprints der Universit{\"a}t Potsdam : Mathematisch Naturwissenschaftliche Reihe},
  number    = {865},
  issn      = {1866-8372},
  doi       = {10.25932/publishup-45989},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-459893},
  pages     = {14},
  year      = {2020},
  abstract  = {The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.},
  language  = {en}
}
@article{ListekHoenowGossenetal.2020,
  author    = {Listek, Martin and H{\"o}now, Anja and Gossen, Manfred and Hanack, Katja},
  title     = {A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line},
  series = {Scientific Reports},
  volume    = {10},
  journal   = {Scientific Reports},
  publisher = {Macmillan Publishers Limited, part of Springer Nature},
  address   = {London},
  issn      = {2045-2322},
  doi       = {10.1038/s41598-020-58571-w},
  pages     = {12},
  year      = {2020},
  abstract  = {The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.},
  language  = {en}
}