title: Transgene expression after stable transfer of a mammalian artificial chromosome into human hematopoietic cells.
abstract: OBJECTIVE
  The transfer of mammalian artificial chromosomes (MACs) to hematopoietic stem and progenitor cells (HSPCs) presents a promising new strategy for ex vivo gene therapy that alleviates numerous concerns surrounding viral transduction along with a unique platform for the systematic study of stem cell biology and fate. Here we report the transfer of a satellite DNA-based artificial chromosome (an ACE), made in mouse cells, into human cord blood hematopoietic cells.
  
  
  METHODS
  A GFP-Zeo-ACE encoding the genes for humanized Renilla green fluorescence protein (hrGFP) and zeomycin resistance (zeo) was transferred into CD34 positively selected cord blood cells using cationic reagents.
  
  
  RESULTS
  Post ACE transfer, CFU-GM-derived colonies were generated in methylcellulose in the presence or absence of bleomycin. Bleomycin-resistant cells expressed GFP and contained intact autonomous ACEs, as demonstrated by fluorescent in situ hybridization. Moreover, when the cells from these plates were replated in methylcellulose, we observed secondary bleomycin-resistant CFU-GM-derived colonies, demonstrating stable chromosome retention and transgene function in a CFU-GM progenitor.
  
  
  CONCLUSIONS
  To our knowledge this is the first report demonstrating the transfer of a mammalian artificial chromosome and the stable expression of an encoded transgene in human hematopoietic cells.
authors.affiliation: Chromos Molecular Systems, Inc., British Columbia, Canada.
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Vanderbyl
authors.forename: Brent
authors.initials: B
authors.lastname: Sullenbarger
authors.forename: Nicole
authors.initials: N
authors.lastname: White
authors.forename: Carl F
authors.initials: CF
authors.lastname: Perez
authors.forename: G Neil
authors.initials: GN
authors.lastname: MacDonald
authors.forename: Tom
authors.initials: T
authors.lastname: Stodola
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Bunnell
authors.forename: Harry C
authors.initials: HC
authors.lastname: Ledebur
authors.forename: Larry C
authors.initials: LC
authors.lastname: Lasky
mesh_terms: D000818:Animals; D001761:Bleomycin / Q000494:pharmacology; D022221:Chromosomes, Artificial, Mammalian / Q000235:genetics*; D004351:Drug Resistance; D005312:Fetal Blood / Q000166:cytology; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D019076:Transgenes / Q000235:genetics
chemical_list: D001761:Bleomycin; D049452:Green Fluorescent Proteins