Yusa Kosuke, Rad Roland, Takeda Junji, Bradley Allan
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
Nat Methods. 2009 May;6(5):363-9. doi: 10.1038/nmeth.1323. Epub 2009 Mar 31.
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.
通过Oct4（Pou5f1）、Sox2、Klf4和Myc的转基因表达，已从体细胞中产生了诱导多能干细胞（iPSC）。然而，这项技术应用于再生医学的一个主要困难是重编程因子的递送。逆转录病毒转导会增加致瘤风险，而瞬时表达方法的重编程效率则低得多。在此，我们描述了一种基于piggyBac转座子的高效方法来生成无整合的iPSC。携带2A肽连接的重编程因子的转座子诱导小鼠胚胎成纤维细胞重编程，其效率与逆转录病毒转导相当。我们通过重新表达转座酶从这些原代iPSC中去除了转座子。无转基因的iPSC可通过阴性选择来鉴定。piggyBac切除时无足迹，使iPSC基因组没有任何基因改变。iPSC满足多能性的所有标准，如多能性基因表达、畸胎瘤形成和对嵌合体的贡献。基于piggyBac转座子的重编程可用于生成具有治疗应用价值的iPSC。
