title: Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer.
abstract: We report here a lentiviral vector system for regulated transgene expression. We used the tetracycline repressor fused with a transcriptional suppression domain (tTS) to specifically suppress transgene expression. Human cells were first transduced with a tTS-expressing vector and subsequently transduced with a second lentiviral vector-containing transgene controlled by a regular promoter adjacent to a high-affinity tTS-binding site (tetO). After optimizing the location of the tetO site in the latter vector, we achieved a better inducible transgene expression than the previous lentiviral vectors using the tetracycline repressor systems. In this new system, the transgene transcription from a cellular promoter such as EF1alpha or ubiquitin-C promoter is suppressed by the tTS bound to the nearby tetO site. In the presence of the tetracycline analog doxycycline (Dox), however, the tTS binding is released from the transgene vector and transcription from the promoter is restored. Thus, this system simply adds an extra level of regulation, suitable for any types of promoters (ubiquitous or cell-specific). We tested this tTS-suppressive, Dox-inducible system in 293T cells, human multipotent hematopoietic progenitor cells, and three human embryonic stem cell lines, using a dual-gene vector containing the green fluorescent protein reporter or a cellular gene. We observed a tight suppression in the uninduced state. However, the suppression is reversible, and transgene expression was restored at 5 ng/ml Dox. The lentiviral vectors containing the tTS-suppressive, Dox-inducible system offer a universal, inducible, and reversible transgene expression system in essentially any mammalian cell types, including human embryonic stem cells.
authors.affiliation: Stem Cell Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Broadway Research Building, Room 747, 733 North Broadway, Baltimore, MD 21205, USA.
authors.forename: Betty Ying
authors.initials: BY
authors.lastname: Zhou
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Ye
authors.forename: Guibin
authors.initials: G
authors.lastname: Chen
authors.forename: Zhigang Peter
authors.initials: ZP
authors.lastname: Gao
authors.forename: Yu A
authors.initials: YA
authors.lastname: Zhang
authors.forename: Linzhao
authors.initials: L
authors.lastname: Cheng
mesh_terms: D045744:Cell Line, Tumor; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D049452:Green Fluorescent Proteins