title: High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors.
abstract: Here we describe the sustained expression of transgenes introduced into human embryonic stem (ES) cells using self-inactivating lentiviral vectors. At low multiplicity of infection, vesicular stomatitis virus-pseudotyped vectors containing a green fluorescent protein (GFP) transgene under the control of a human elongation factor 1alpha promoter transduced human ES cells at high efficiency. The majority of the transduced ES cells, which harbored low numbers of integrated vectors, continued to express GFP after 60 days of culture. Incorporation of a scaffold attachment region (SAR) from the human interferon-beta gene into the lentiviral vector backbone increased the average level of GFP expression, and inclusion of the SAR together with a chromatin insulator from the 5' end of the chicken beta-globin locus reduced the variability in GFP expression. When the transduced ES cells were induced to differentiate into CD34(+) hematopoietic precursors in vitro, GFP expression was maintained with minimal silencing. The ability to efficiently introduce active transgenes into human ES cells will facilitate gain-of-function studies of early developmental processes in the human system. These results also have important implications for the possible future use of gene-modified human ES cells in transplantation and tissue regeneration applications.
authors.affiliation: National Primate Research Center, School of Medicine, University of Wisconsin, Madison, Wisconsin, USA.
authors.forename: Yue
authors.initials: Y
authors.lastname: Ma
authors.forename: Ali
authors.initials: A
authors.lastname: Ramezani
authors.forename: Rachel
authors.initials: R
authors.lastname: Lewis
authors.forename: Robert G
authors.initials: RG
authors.lastname: Hawley
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D001854:Bone Marrow Cells / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D004622:Embryo, Mammalian / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D013234:Stem Cells / Q000737:chemistry* / Q000378:metabolism* / Q000821:virology; D017154:Stromal Cells / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes
chemical_list: D018952:Antigens, CD34; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins