Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon

Abstract

Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4, Sox2, Klf4, and cMyc. 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 show a highly 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. Transposons were removed from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be easily identified by HSVtk-FIAU selection. piggyBac excises without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as expression of embryonic stem cell-specific markers, teratoma formation and contribution to chimeras. piggyBac transposon-based reprogramming may be used to generate therapeutically applicable iPSCs.

Mechanism and methods to induce pluripotency

“…To meet this need, several nonintegration systems and removable systems have been developed. These include episomal vectors (Yu et al, 2009), the piggyBac transposon system (Kaji et al, 2009;Woltjen et al, 2009;Yusa et al, 2009), and minicircle vectors (Jia et al, 2010).…”

“…To achieve simultaneous expression of reprogramming factors, cDNA encoding as many as five reprogramming factors connected by the 2A peptide sequence from the foot-and-mouth disease virus was cloned in a single vector. Individual proteins were produced through a "ribosomal skipping" mechanism mediated by the 2A peptide (Szymczak et al, 2004;Yusa et al, 2009). In the case of the piggyBac system, after successful generation of iPS cells, the reprogramming cassette can be removed by piggyBac transposase without footprint and transgene-free iPS cells were obtained (Yusa et al, 2009).…”

“…Individual proteins were produced through a "ribosomal skipping" mechanism mediated by the 2A peptide (Szymczak et al, 2004;Yusa et al, 2009). In the case of the piggyBac system, after successful generation of iPS cells, the reprogramming cassette can be removed by piggyBac transposase without footprint and transgene-free iPS cells were obtained (Yusa et al, 2009). However, the removal efficiency of piggyBac transposon was only 1 in 10 5 (Yusa et al, 2009), and such a low efficiency limits wider application of this technology in reprogramming and gene therapy.…”

Induced Pluripotent Stem Cell Technology and Direct Conversion: New Possibilities to Study and Treat Parkinson’s Disease

“…by eliminating Myc transduction [9], but also the generation of iPSCs with nonintegrating adenoviruses and temperature sensitive Sendai viruses has been tested and proven possible [10,11]. In a next logical follow up step, the use of viruses has been abandoned completely by utilization of direct repeated transfection with 'Yamanaka factor' containing plasmids [12] or piggyback transposons [13]. Another interesting approach is the use of microRNAs (miRs) to drive induction of pluripotency.…”

Non-viral iPSCs: a safe way for therapy?

“…Various new approaches have been employed to generate genetically unmodified or non-integrative human iPSCs: (1) non-integrative vectors, including episomal vectors, adenoviral vectors, and sendai viral vectors (Yu et al, 2009;Zhou and Freed, 2009;Jia et al, 2010;Ban et al, 2011;Chou et al, 2011;Hiratsuka et al, 2011;Okita et al, 2011); (2) excisable integrating vectors, such as Cre-recombinase excisable viruses, piggyBac transposon (Kaji et al, 2009;Soldner et al, 2009;Woltjen et al, 2009;Yusa et al, 2009;Sommer et al, 2010); (3) DNA-free materials, such as pluripotency-associated recombinant proteins, RNA, and microRNA (Kim et al, 2009;Warren et al, 2010;Miyoshi et al, 2011); (4) small molecules that can facilitate reprogramming (Feng et al, 2009;Li and Ding, 2010;Efe and Ding, 2011). Here we will briefly summarize recent literatures on episomal vectors-or small molecules-based technologies for generation of iPSCs ( Fig.…”