title: Lentiviral vectors harboring a dual-gene system allow high and homogeneous transgene expression in selected polyclonal human embryonic stem cells.
abstract: Genetic modification of human embryonic stem cells (hESCs) is highly valuable for their exploitation in basic science and therapeutic applications. Here we developed lentiviral vectors (LVs) constitutively expressing a reporter and a selectable marker to enable high and homogeneous transgene expression within polyclonal hESCs. LVs carrying GFP and a downstream puromycin resistance gene, linked by the encephalomyocarditis virus (EMCV) or poliovirus internal ribosome entry sites (IRES), allowed homogeneous GFP expression after antibiotic selection. The GFP-expression levels were higher with the EMCV IRES. We also developed dual-promoter vectors harboring a reporter and an antibiotic resistance gene under the regulation of human EF1alpha and PGK1 promoters, respectively. Optimal efficiency was obtained when: (1) the reporter cassette was upstream rather than downstream of the selectable marker cassette, (2) the puromycin rather than the neomycin resistance gene was used, (3) a 5' deletion (314 bp) was created in the PGK promoter, and (4) two copies of a 120-bp element derived from the hamster Aprt CpG island were introduced upstream of the EF1alpha promoter. In summary, we developed bicistronic and novel dual-promoter LVs that enable high and homogeneous expression of transgenes by polyclonal hESCs after antibiotic selection. These vectors may provide important tools for basic and applied research on hESCs.
authors.affiliation: The Hadassah Human Embryonic Stem Cell Research Center, Hadassah University Medical Center, Jerusalem 91120, Israel.
authors.forename: Israel
authors.initials: I
authors.lastname: Ben-Dor
authors.forename: Pavel
authors.initials: P
authors.lastname: Itsykson
authors.forename: Daniel
authors.initials: D
authors.lastname: Goldenberg
authors.forename: Eithan
authors.initials: E
authors.lastname: Galun
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Reubinoff
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D006224:Cricetinae; D004351:Drug Resistance / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology; D004686:Encephalomyocarditis virus; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011691:Puromycin / Q000494:pharmacology; D013234:Stem Cells; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics*
chemical_list: D049452:Green Fluorescent Proteins; D011691:Puromycin