title: Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells.
abstract: Human pluripotent stem cells offer a powerful system to study human biology and disease. Here, we report a system to both express transgenes specifically in ES cell derived hematopoietic cells and knockdown gene expression stably throughout the differentiation of ES cells. We characterize a CD43 promoter construct that when inserted into the AAVS1 "safe harbor" locus utilizing a zinc finger nuclease specifically drives GFP expression in hematopoietic cells derived from a transgenic ES cell line and faithfully recapitulates endogenous CD43 expression. In addition, using the same gene targeting strategy we demonstrate that constitutive expression of short hairpin RNAs within a microRNA backbone can suppress expression of PU.1, an important regulator of myeloid cell development. We show that PU.1 knockdown cell lines display an inhibition in myeloid cell formation and skewing towards erythroid development. Overall, we have generated a powerful system to track hematopoietic development from pluripotent stem cells and study gene function through hematopoietic specific gene expression and constitutive gene knockdown.
authors.affiliation: School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA; Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: Amita
authors.initials: A
authors.lastname: Tiyaboonchai
authors.affiliation: Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: Helen
authors.initials: H
authors.lastname: Mac
authors.affiliation: Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: Razveen
authors.initials: R
authors.lastname: Shamsedeen
authors.affiliation: Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Mills
authors.affiliation: Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: Siddarth
authors.initials: S
authors.lastname: Kishore
authors.affiliation: 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.forename: Deborah L
authors.initials: DL
authors.lastname: French
authors.affiliation: 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. Electronic address: gaduep@email.chop.edu.
authors.forename: Paul
authors.initials: P
authors.lastname: Gadue
mesh_terms: D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000295:instrumentation / Q000379:methods*; D018390:Gene Targeting; D017930:Genes, Reporter; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051917:Leukosialin / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D019076:Transgenes
chemical_list: D051917:Leukosialin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins