Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach
Mol Biotechnol. 2014 Mar 28. [Epub ahead of print]
|Authors/Editors:||Pertek A, Meier F, Irmler M, Beckers J, Skylaki S, Endele M, Wurst W, Prakash N, Kühn R.|
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.