Article abstract
Nature Methods 6, 363 - 369 (2009)
Published online: 31 March 2009 | doi:10.1038/nmeth.1323
Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon
Kosuke Yusa1, Roland Rad1, Junji Takeda2 & Allan Bradley1
Abstract
Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4 (Pou5f1), Sox2, Klf4 and Myc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we describe an efficient piggyBac transposon–based approach to generate integration-free iPSCs. Transposons carrying 2A peptide–linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. We removed transposons from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be identified by negative selection. piggyBac excised without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as pluripotency gene expression, teratoma formation and contribution to chimeras. piggyBac transposon–based reprogramming may be used to generate therapeutically applicable iPSCs.
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
- Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan.
Correspondence to: Allan Bradley1 e-mail: abradley@sanger.ac.uk
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