Tetracycline-Controlled Gene Expression System Achieves High-Level and Quantitative Control of Gene Expression

Yin, Dong; Zhu, Li; Schimke, Robert T.

doi:10.1006/abio.1996.0112

Cited by 87 publications

(64 citation statements)

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“…Interestingly, the tetracycline regulation of amber suppression in COS-1 cells is lower (5-to 17-fold) than that in HeLa cells (15-to 78-fold). This result is analogous to the finding that a tTA-regulated minimal promoter gave high basal levels of transcription of the target gene in Vero, BHK, and HEK293 cell lines but not in HeLa or GH3 (clonal cells derived from rat pituitary cells) cell lines (1,21,45). However, in contrast to our results, indicating 5-to 17-fold regulation of amber suppression by tetracycline in COS-1 cells, in BHK cells the background activity of the promoter was so high that there was essentially no tetracycline regulation of the target gene (1).…”

Section: Discussionsupporting

confidence: 81%

“…This level of regulation is similar to that seen in other transienttransfection systems (1,21,43,45) or in D. discoideum (10), where tetracycline is used to regulate expression of the suppressor tRNA rather than its function. The 15-to 78-fold regulation of amber suppression by tetracycline in HeLa cells is, however, substantially less than that in a stable HeLa cell line expressing the same tTA constitutively (17) or in animals using different transactivation systems induced by mifepristone (RU486) or ecdysone (31,43,44).…”

Section: Discussionsupporting

confidence: 78%

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Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

Park

RajBhandary

1998

Molecular and Cellular Biology

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As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyltRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.

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Section: Discussionsupporting

confidence: 81%

Section: Discussionsupporting

confidence: 78%

Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

Park

RajBhandary

1998

Molecular and Cellular Biology

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“…The Tet regulatory systems are currently the most widely used models for conditional gene expression. Maximal expression levels in these systems are very high and comparable with the maximal levels reachable from a constitutive and strong mammalian promoter such as CMV (24). Indeed, the level of CD24 protein following exposure to tetracycline was comparable with the level of the protein after transient expression (data not shown).…”

Section: Discussionmentioning

confidence: 62%

The CD24 Protein Inducible Expression System Is an Ideal Tool to Explore the Potential of CD24 as an Oncogene and a Target for Immunotherapy in Vitro and in Vivo

Shapira

Kazanov²,

Weisblatt

et al. 2011

Journal of Biological Chemistry

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Background: It is critical to tightly regulate gene expression to study its biological role. Results: A tetracycline-dependent CD24 expression system was successfully implemented. An efficient study of its potential as an oncogene and a target for immunotherapy was performed. Conclusion: This is a valuable tool to study CD24 pathogenesis and novel treatment options for CD24-expressing malignancies. Significance: The CD24 inducible expression system allows accurate analysis of its role and function.

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“…[29][30][31] Flow cytometric analysis of the representative clone D7 grown 96 h at varying tetracycline concentration confirmed a modulating effect of the MLL-AF4 expression levels on cell cycle progression. Cells cultured without tetracycline and therefore expressing MLL-AF4 at maximal level had one-half of the S phase redistributed in the G1 phase of the cell cycle then compared to nonexpressing cells grown at 1 mg/ml tetracycline (Figure 2).…”

Section: Modulation Of Cell Cycle and Differentiation By Graded Exprementioning

confidence: 75%

Modulation of cell cycle by graded expression of MLL-AF4 fusion oncoprotein

et al. 2004

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Acute lymphoblastic leukemia (ALLs) expressing MLL-AF4, the fusion product of t(4;11)(q21;q23), show marked leucocytosis and extramedullary disease in multiple organs, respond poorly to chemotherapy and have poor prognosis. In vitro, leukemic cells with the t(4;11) show resistance to serum deprivationinduced or interferon c-regulated CD95-mediated apoptosis. In addition, t(4;11) cells have prolonged doubling time and lower percentage of cells in cycle compared to non-t(4;11) B lineage cell lines. In this study, we examine the time-and leveldependent effects of MLL-AF4 conditional expression on cell cycle and differentiation of myelomonocytic leukemia cell line U937. By varying the concentration of tetracycline in growth media, we found that increasing levels of MLL-AF4 expression result in a progressive decrease in growth rate and fraction of S phase cells, paralleled by an increase in percentage of cells expressing CD11b. Our results demonstrate a dosage-dependent effect of MLL-AF4 fusion oncoprotein on cell cycle progression, with increasing expression levels resulting in the accumulation in G1, prolonged doubling time, both findings that might be responsible for the increased resistance to etoposide-mediated cytotoxicity. We propose the cell cycle control exerted by MLL-AF4 may be responsible of resistance to cell-death promoting stimuli in leukemia carrying the t(4;11) translocation.

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Tetracycline-Controlled Gene Expression System Achieves High-Level and Quantitative Control of Gene Expression

Cited by 87 publications

References 1 publication

Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

The CD24 Protein Inducible Expression System Is an Ideal Tool to Explore the Potential of CD24 as an Oncogene and a Target for Immunotherapy in Vitro and in Vivo

Modulation of cell cycle by graded expression of MLL-AF4 fusion oncoprotein

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