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.
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. 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.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Rui-Zhe
authors.initials: RZ
authors.lastname: Phang
authors.forename: Felix Chang
authors.initials: FC
authors.lastname: Tay
authors.forename: Sal-Lee
authors.initials: SL
authors.lastname: Goh
authors.forename: Cia-Hin
authors.initials: CH
authors.lastname: Lau
authors.forename: Haibao
authors.initials: H
authors.lastname: Zhu
authors.forename: Wee-Kiat
authors.initials: WK
authors.lastname: Tan
authors.forename: Qingle
authors.initials: Q
authors.lastname: Liang
authors.forename: Can
authors.initials: C
authors.lastname: Chen
authors.forename: Shouhui
authors.initials: S
authors.lastname: Du
authors.forename: Zhendong
authors.initials: Z
authors.lastname: Li
authors.forename: Johan Chin-Kang
authors.initials: JC
authors.lastname: Tay
authors.forename: Chunxiao
authors.initials: C
authors.lastname: Wu
authors.forename: Jieming
authors.initials: J
authors.lastname: Zeng
authors.forename: Weimin
authors.initials: W
authors.lastname: Fan
authors.forename: Han Chong
authors.initials: HC
authors.lastname: Toh
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
mesh_terms: D016367:Baculoviridae / Q000235:genetics*; D002454:Cell Differentiation; D002460:Cell Line; D065150:Cellular Reprogramming; D002888:Chromosomes, Human, Pair 19; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D005838:Genotype; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D014162:Transfection; D016335:Zinc Fingers
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D014157:Transcription Factors; C045073:Cre recombinase; D019426:Integrases; D004720:Endonucleases
keywords: Cre-loxP system; Gene delivery systems in vivo or in vitro; Gene expression; Induced pluripotent stem cells