Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach |
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Authors: | Anna Pertek Florian Meier Martin Irmler Johannes Beckers Stavroula Skylaki Max Endele Wolfgang Wurst Nilima Prakash Ralf Kühn |
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Affiliation: | 1. Institute of Developmental Genetics, Helmholtz Zentrum München, 85764, Munich, Germany 2. Institute of Stem Cell Research, Helmholtz Zentrum München, 85764, Munich, Germany 4. Chair of Developmental Genetics, Technical University Munich, 85350, Freising, Weihenstephan, Germany 3. Institute of Experimental Genetics, Helmholtz Zentrum München, 85764, Munich, Germany 5. Chair of Experimental Genetics, Technical University Munich, 85350, Freising, Weihenstephan, Germany 11. Institute of Stem Cell Research, Helmholtz Zentrum München, 85764, Munich, Germany 6. Department of Biosystems Science and Engineering, ETH Zurich, 4058, Basel, Switzerland 7. Deutsches Zentrum für Neurodegenerative Erkrankungen e. V, 80336, Munich, Germany 8. Max-Planck-Institute of Psychiatry, 80804, Munich, Germany 9. Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-Universit?t München, 80336, Munich, Germany 10. Helmholtz Center Munich, Ingolst?dter Landstr. 1, 85764, Munich, Germany
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Abstract: | Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies. |
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