title: Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors.
abstract: Generation of stable transgenic embryonic stem (ES) cell lines by classic transfection is still a difficult task, requiring time-consuming clonal selection, and hampered by clonal artifacts and gene silencing. Here we describe a novel system that allows construction of lentivectors and generation of stable ES cell lines with > 99% transgene expression within a very short time frame. Rapid insertion of promoters and genes of interest is obtained through a modular recombinational cloning system. Vectors contain central polypurine tract from HIV-1 element and woodchuck hepatitis virus post-transcriptional regulatory element as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system 1) is functional in mouse and human ES cells, 2) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue-specific promoters, and 3) allows ES cells expressing two constructs through selection with different antibiotics to be obtained. The technology described herein should become a useful tool in stem cell research.
authors.affiliation: Biology of Aging Laboratory, Department of Rehabilitation and Geriatrics, University of Geneva Medical School, Switzerland. david.suter@hcuge.ch
authors.forename: David M
authors.initials: DM
authors.lastname: Suter
authors.forename: Laetitia
authors.initials: L
authors.lastname: Cartier
authors.forename: Esther
authors.initials: E
authors.lastname: Bettiol
authors.forename: Diderik
authors.initials: D
authors.lastname: Tirefort
authors.forename: Marisa E
authors.initials: ME
authors.lastname: Jaconi
authors.forename: Michel
authors.initials: M
authors.lastname: Dubois-Dauphin
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Krause
mesh_terms: D000818:Animals; D002460:Cell Line; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus; D051379:Mice; D011995:Recombination, Genetic / Q000235:genetics; D050436:Regulatory Elements, Transcriptional / Q000235:genetics; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes / Q000235:genetics