title: A Doxycycline-Inducible System for Genetic Correction of iPSC Disease Models.
abstract: Patient-derived induced pluripotent stem cells (iPSCs) are valuable tools for the study of developmental biology and disease modeling. In both applications, genetic correction of patient iPSCs is a powerful method to understand the specific contribution of a gene(s) in development or diseased state(s). Here, we describe a protocol for the targeted integration of a doxycycline-inducible transgene expression system in a safe harbor site in iPSCs. Our gene targeting strategy uses zinc finger nucleases (ZFNs) to enhance homologous recombination at the AAVS1 safe harbor locus, thus increasing the efficiency of the site-specific integration of the two targeting vectors that make up the doxycycline-inducible system. Importantly, the use of dual-drug selection in our system increases the efficiency of positive selection for double-targeted clones to >50 %, permitting a less laborious screening process. If desired, this protocol can also be adapted to allow the use of tissue-specific promoters to drive gene expression instead of the doxycycline-inducible promoter (TRE). Additionally, this protocol is also compatible with the use of Transcription-Activator-Like Effector Nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system in place of ZFNs.
authors: School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.|Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors: Xiuli
authors: X
authors: Sim
authors: Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA.
authors: Fabian L
authors: FL
authors: Cardenas-Diaz
authors: Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors: Deborah L
authors: DL
authors: French
authors: Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA. gaduep@email.chop.edu.|Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA, USA. gaduep@email.chop.edu.
authors: Paul
authors: P
authors: Gadue
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D004318:Doxycycline / Q000494:pharmacology*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D056426:Genetic Loci; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination / Q000187:drug effects; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008954:Models, Biological; D011401:Promoter Regions, Genetic / Q000187:drug effects; D019076:Transgenes; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D049452:Green Fluorescent Proteins; D004720:Endonucleases; D004318:Doxycycline
keywords: Disease modeling; Doxycycline-inducible expression; Gene targeting; Genetic correction; Homologous recombination; Zinc finger nucleases
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