Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer

Betty Ying Zhou, Zhaohui Ye, Guibin Chen, Zhigang Peter Gao, Yu A. Zhang, Linzhao Cheng

Research output: Contribution to journalArticle

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 EF1α 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.

Original languageEnglish (US)
Pages (from-to)779-789
Number of pages11
JournalStem Cells
Volume25
Issue number3
DOIs
StatePublished - Mar 2007

Fingerprint

Transgenes
Stem Cells
Doxycycline
Genes
Tetracycline
Ubiquitin C
HEK293 Cells
Hematopoietic Stem Cells
Green Fluorescent Proteins
Binding Sites
Cell Line

Keywords

  • Gene transfer
  • Human embryonic stem cells
  • Human hematopoietic progenitor cells
  • Inducible gene expression
  • Lentiviral vectors

ASJC Scopus subject areas

  • Cell Biology

Cite this

Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer. / Zhou, Betty Ying; Ye, Zhaohui; Chen, Guibin; Gao, Zhigang Peter; Zhang, Yu A.; Cheng, Linzhao.

In: Stem Cells, Vol. 25, No. 3, 03.2007, p. 779-789.

Research output: Contribution to journalArticle

Zhou, Betty Ying ; Ye, Zhaohui ; Chen, Guibin ; Gao, Zhigang Peter ; Zhang, Yu A. ; Cheng, Linzhao. / Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer. In: Stem Cells. 2007 ; Vol. 25, No. 3. pp. 779-789.
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