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|Title:||Zinc finger nuclease-expressing baculoviral vectors mediate targeted genome integration of reprogramming factor genes to facilitate the generation of human induced pluripotent stem cells|
Gene delivery systems in vivo or in vitro
Induced pluripotent stem cells
|Source:||Phang, R.-Z., Tay, F.C., Goh, S.-L., Lau, C.-H., Zhu, H., Tan, W.-K., Liang, Q., Chen, C., Du, S., Li, Z., Tay, J.C.-K., Wu, C., Zeng, J., Fan, W., Toh, H.C., Wang, S. (2013). Zinc finger nuclease-expressing baculoviral vectors mediate targeted genome integration of reprogramming factor genes to facilitate the generation of human induced pluripotent stem cells. Stem Cells Translational Medicine 2 (12) : 935-945. ScholarBank@NUS Repository. https://doi.org/10.5966/sctm.2013-0043|
|Abstract:||Integrative gene transfer using retroviruses to express reprogramming factors displays high efficiency in generating induced pluripotent stem cells (iPSCs), but the value of the method is limited because of the concern over mutagenesis associated with random insertion of transgenes. Sitespecific integration into a preselected locus by engineered zinc-finger nuclease (ZFN) technology provides a potential way to overcome the problem. Here, we report the successful reprogramming of human fibroblasts into a state of pluripotency by baculoviral transduction-mediated, site-specific integration of OKSM (Oct3/4, Klf4, Sox2, and c-myc) transcription factor genes into the AAVS1 locus in human chromosome 19. Two nonintegrative baculoviral vectors were used for cotransduction, one expressing ZFNs and another as a donor vector encoding the four transcription factors. iPSC colonies were obtained at a high efficiency of 12% (the mean value of eight individual experiments). All characterized iPSC clones carried the transgenic cassette only at the ZFN-specified AAVS1 locus. We further demonstrated that when the donor cassette was flanked by heterospecific loxP sequences, the reprogramming genes in iPSCs could be replaced by another transgene using a baculoviral vector-based Cre recombinasemediated cassette exchange system, thereby producing iPSCs free of exogenous reprogramming factors. Although the use of nonintegrating methods to generate iPSCs is rapidly becoming a standard approach, methods based on site-specific integration of reprogramming factor genes as reported here hold the potential for efficient generation of genetically amenable iPSCs suitable for future gene therapy applications. © AlphaMed Press 2013.|
|Source Title:||Stem Cells Translational Medicine|
|Appears in Collections:||Staff Publications|
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