title: A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells.
abstract: OBJECTIVE
  Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs.
  
  
  METHODS
  The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid.
  
  
  RESULTS
  Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts.
  
  
  CONCLUSIONS
  Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.
authors.affiliation: Primary and Stem Cell Systems, Life Technologies, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Thyagarajan
authors.forename: Kelly
authors.initials: K
authors.lastname: Scheyhing
authors.forename: Haipeng
authors.initials: H
authors.lastname: Xue
authors.forename: Andrew
authors.initials: A
authors.lastname: Fontes
authors.forename: Jon
authors.initials: J
authors.lastname: Chesnut
authors.forename: Mahendra
authors.initials: M
authors.lastname: Rao
authors.forename: Uma
authors.initials: U
authors.lastname: Lakshmipathy