Inducible and Reversible Transgene Expression in Human Stem Cells after Efficient and Stable Gene Transfer

¹ Stem Cell Program, Institute for Cell Engineering and Department of Gynecology & Obstetrics, Johns Hopkins University School of Medicine, Baltimore, Maryland
² Stem Cell Program, Institute for Cell Engineering and Department of Gynecology & Obstetrics, Johns Hopkins University School of Medicine, Baltimore, Maryland; Graduate Program in Immunology, Johns Hopkins University, Baltimore, Maryland
³ Neuro-Oncology Branch, NCI/NINDS, National Institutes of Health, Bethesda, Maryland
⁴ Xuanwu Hospital, The Capital University of Medical Sciences, Beijing, China
⁵ Stem Cell Program, Institute for Cell Engineering and Department of Gynecology & Obstetrics, Johns Hopkins University School of Medicine, Baltimore, Maryland; Graduate Program in Immunology, Johns Hopkins University, Baltimore, Maryland; Departments of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland

^* To whom correspondence should be addressed. E-mail: lcheng{at}welch.jhu.edu.

	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 EF1 or UBC is suppressed by the tTS bound to the nearby tetO site. In the presence of a 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 GFP reporter or a cellular gene. We observed a tight suppression in the un-induced 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.

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