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Merck
  • Simple derivation of transgene-free iPS cells by a dual recombinase approach.

Simple derivation of transgene-free iPS cells by a dual recombinase approach.

Molecular biotechnology (2014-03-29)
Anna Pertek, Florian Meier, Martin Irmler, Johannes Beckers, Stavroula Skylaki, Max Endele, Wolfgang Wurst, Nilima Prakash, Ralf Kühn
摘要

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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DAPI, for nucleic acid staining
Sigma-Aldrich
Anti-PITX3 antibody produced in rabbit, affinity isolated antibody