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Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach


Wurst,  Wolfgang
Max Planck Institute of Psychiatry, Max Planck Society;
Institute of Developmental Genetics, Helmholtz Zentrum München;

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Pertek, A., Meier, F., Irmler, M., Beckers, J., Skylaki, S., Endele, M., et al. (2014). Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach. MOLECULAR BIOTECHNOLOGY, 56(8), 697-713. doi:10.1007/s12033-014-9748-y.

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.