http://hdl.handle.net/10033/118931 Wed, 22 May 2013 18:58:45 GMT 2013-05-22T18:58:45Z http://hdl.handle.net/10033/281672 Title: The ROSA26-iPSC mouse: a conditional, inducible, and exchangeable resource for studying cellular (De)differentiation. Authors: Haenebalcke, Lieven; Goossens, Steven; Dierickx, Pieterjan; Bartunkova, Sonia; D'Hont, Jinke; Haigh, Katharina; Hochepied, Tino; Wirth, Dagmar; Nagy, Andras; Haigh, Jody J Abstract: Control of cellular (de)differentiation in a temporal, cell-specific, and exchangeable manner is of paramount importance in the field of reprogramming. Here, we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming, these factors can be replaced by genes of interest-for example, to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that, similar to ESCs, Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon in vitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes. Thu, 21 Feb 2013 00:00:00 GMT http://hdl.handle.net/10033/281672 2013-02-21T00:00:00Z http://hdl.handle.net/10033/236995 Title: Lentivirus production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells. Authors: Gama-Norton, Leonor; Botezatu, Lacramioara; Herrmann, Sabrina; Schweizer, Matthias; Alves, Paula Marques; Hauser, Hansjoerg; Wirth, Dagmar Abstract: Currently, lentiviral vectors for research and gene therapy are produced from 293-T cells that are transiently transfected with plasmids encoding the vector and helper functions. However, transiently transfected vectors as well as the presence of SV40 virus large T-antigen (T-Ag) cause serious technical and safety considerations. We aimed to exploit single copy integration sites in the HEK293 genome supporting lentiviral vector production. We found that lentiviral vectors result in minimal infectious particle production from single copy integrants in HEK293. Moreover, once this cell line harbors single copy integrations of lentiviral vectors, its ability to transiently produce lentiviral vectors becomes strongly impaired. T-Ag has a dramatic effect on virus production. Low levels of constitutive T-Ag expression can overcome the production restriction imposed by integrated lentiviral vectors copies. Interestingly, T-Ag does not exert its role at the level of transcriptional activity of the vector; rather, it seems to impose an indirect effect on the cell thereby enabling lentiviral vector production. Altogether, our study highlights the restrictions for integrated lentiviral vectors that are relevant for the establishment of stable and safe producer cell lines. Sat, 01 Oct 2011 00:00:00 GMT http://hdl.handle.net/10033/236995 2011-10-01T00:00:00Z http://hdl.handle.net/10033/121272 Title: Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells. Authors: Sandhu, U; Cebula, M; Behme, S; Riemer, P; Wodarczyk, C; Metzger, D; Reimann, J; Schirmbeck, R; Hauser, H; Wirth, D Abstract: Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreER(T2), and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreER(T2). The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts. Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10033/121272 2011-01-01T00:00:00Z http://hdl.handle.net/10033/118966 Title: Toxin-antitoxin based transgene expression in mammalian cells. Authors: Nehlsen, K; Herrmann, S; Zauers, J; Hauser, H; Wirth, D Abstract: Long-term, recombinant gene expression in mammalian cells depends on the nature of the transgene integration site and its inherent properties to modulate transcription (epigenetic effects). Here we describe a method by which high transgene expression is achieved and stabilized in extensively proliferating cultures. The method is based on strict co-expression of the transgene with an antitoxin in cells that express the respective toxin. Since the strength of antitoxin expression correlates with an advantage for cell growth, the cells with strong antitoxin expression are enriched over time in cultures of heterogeneous cells. This principle was applied to CHO cell lines that conditionally express the toxin kid and that are transduced to co-express the antitoxin kis together with different transgenes of interest. Cultivation of pools of transfectants that express the toxin steadily increase their transgene expression within several weeks to reach a maximum that is up to 120-fold over the initial status. In contrast, average transgene expression drops in the absence of toxin expression. Together, we show that cells conditionally expressing kid can be employed to create overexpressing cells by a simple coupling of kis to the transgene of interest, without further manipulation and in absence of selectable drugs. Mon, 01 Mar 2010 00:00:00 GMT http://hdl.handle.net/10033/118966 2010-03-01T00:00:00Z