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Tet-On lentiviral transductants lose inducibility when silenced for extended intervals in mammary epithelial cells
Yang Yu1,2, Michelle Montag Lowy3,2, Randolph C Elble3,2
1Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL 62794, United States.
Abstract:
Silencing of virally transduced genes by promoter methylation and histone deacetylation has been a chronic problem both experimentally and therapeutically. We observed frequent silencing of the tetracycline-inducible Tet-On promoter borne by the Tripz lentivirus in mammary epithelial cell lines. We found that silencing could be prevented by continuous induction, but uninduced Tet-On gradually became uninducible, suggesting promoter modification. Accordingly, silencing was reversible by a common inhibitor of histone deacetylases, sodium butyrate. The effect was cell-line dependent, as HEK293 cells exhibited only moderate silencing that could be partly reversed by extended induction. These results indicate the need to test individual cell lines prior to using this system for studies that require induction after long periods of repression such as in animal models or RNA interference screens.
Insights
Gene silencing in lentivirus vectors is a common issue. Continuous induction prevents silencing of the Tet-On promoter, which is reversible with sodium butyrate, but requires cell-line specific testing.
Area of Science:
- Molecular Biology
- Gene Therapy
- Epigenetics
Background:
- Virally transduced gene silencing via promoter methylation and histone deacetylation presents challenges in experimental and therapeutic applications.
- The tetracycline-inducible Tet-On promoter in Tripz lentivirus vectors is prone to silencing in mammary epithelial cells.
Purpose of the Study:
- To investigate the silencing mechanisms of the Tet-On promoter in lentiviral vectors.
- To identify strategies for preventing or reversing Tet-On promoter silencing.
- To assess the cell-line dependency of Tet-On promoter silencing and reversibility.
Main Methods:
- Utilized Tripz lentivirus vectors carrying the Tet-On promoter in mammary epithelial cell lines and HEK293 cells.
- Investigated the effect of continuous induction versus repression on promoter activity.
- Administered sodium butyrate, a histone deacetylase inhibitor, to assess reversibility of silencing.
- Evaluated cell-line specific responses to induction and repression protocols.
Main Results:
- Frequent silencing of the Tet-On promoter was observed in mammary epithelial cell lines.
- Continuous induction prevented promoter silencing, while uninduced promoters became gradually uninducible.
- Silencing was reversible with sodium butyrate, demonstrating the role of histone deacetylation.
- HEK293 cells showed moderate silencing, partially reversible with extended induction, indicating cell-line specificity.
Conclusions:
- The Tet-On promoter's silencing is an epigenetic phenomenon influenced by promoter methylation and histone deacetylation.
- Continuous induction or histone deacetylase inhibition can prevent or reverse Tet-On promoter silencing.
- Cell-line specific testing is crucial before employing Tet-On systems for long-term repression studies, particularly in animal models or RNA interference screens.
