TY - GEN A1 - Listek, Martin A1 - Hönow, Anja A1 - Gossen, Manfred A1 - Hanack, Katja T1 - A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 865 KW - Antibody generation KW - Assay systems Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-459893 SN - 1866-8372 IS - 865 ER - TY - GEN A1 - Moradian, Hanieh A1 - Roch, Toralf A1 - Lendlein, Andreas A1 - Gossen, Manfred T1 - mRNA transfection-induced activation of primary human monocytes and macrophages BT - Dependence on carrier system and nucleotide modifcation T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1403 KW - sirna transfection KW - mediated delivery KW - gene delivery KW - efficient KW - immunogenicity KW - lipoplexes KW - cells KW - therapeutics KW - polarization KW - pathways Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-515694 SN - 1866-8372 IS - 1 ER -