• Stem Cells Transl Med · Dec 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.

    • Rui-Zhe Phang, Felix Chang Tay, Sal-Lee Goh, Cia-Hin Lau, Haibao Zhu, Wee-Kiat Tan, Qingle Liang, Can Chen, Shouhui Du, Zhendong Li, Johan Chin-Kang Tay, Chunxiao Wu, Jieming Zeng, Weimin Fan, Han Chong Toh, and Shu Wang.
    • Department of Biological Sciences, National University of Singapore, Singapore.
    • Stem Cells Transl Med. 2013 Dec 1; 2 (12): 935-45.

    AbstractIntegrative 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. Site-specific 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 recombinase-mediated 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.

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