Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation

Gödecke, Natascha; Zha, Lisha; Spencer, Shawal; Behme, Sara; Riemer, Pamela; Rehli, Michael; Hauser, Hansjörg; Wirth, Dagmar

doi:10.1093/nar/gkx601

Cited by 32 publications

(48 citation statements)

References 72 publications

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“…This indicates that transgene expression in tumor cells might be epigenetically silenced, which is a known phenomenon for tetracycline‐inducible cytomegalovirus (CMV) promoters. ( 22‐26 )…”

Section: Resultsmentioning

confidence: 99%

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E2F7 Is a Potent Inhibitor of Liver Tumor Growth in Adult Mice

et al. 2021

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Upregulation of the E2F-dependent transcriptional network has been identified in nearly every human malignancy and is an important driver of tumorigenesis. Two members of the E2F family, E2F7 and E2F8, are potent repressors of E2F-dependent transcription. They are atypical in that they do not bind to dimerization partner (DP) proteins and are not controlled by pRb. The physiological relevance of E2F7 and E2F8 remains incompletely understood, largely because tools to manipulate their activity in vivo have been lacking. Here, we generated transgenic mice with doxycycline-controlled transcriptional activation of E2f7 and E2f8 and induce their expression during postnatal development, in adulthood, and in the context of cancer. Systemic induction of E2f7 and to lesser extendE2f8 transgenes in juvenile mice impaired cell proliferation, caused replication stress, DNA damage and apoptosis, and inhibited animal growth. In adult mice, however, E2F7 and E2F8 induction was well tolerated, yet profoundly interfered with DNA replication, DNA integrity and cell proliferation in DEN-induced liver tumors. Conclusion: Collectively, our findings demonstrate that atypical E2Fs can override cellcycle entry and progression governed by other E2F-family members, and suggest that this property can be exploited to inhibit proliferation of neoplastic hepatocytes when growth and development have subsided during adulthood.

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Section: Resultsmentioning

confidence: 99%

“…epigenetically silenced, which is a known phenomenon for tetracycline-inducible CMV promoters (23)(24)(25)(26)(27).…”

Section: Accepted Articlementioning

confidence: 99%

E2F7 Is a Potent Inhibitor of Liver Tumor Growth in Adult Mice

et al. 2021

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“…EGFP was assessed using microscopy and results show that ES colonies that are cultured beyond three weeks significantly lose their ability to express EGFP in the presence of IPTG compared to cells in earlier time points ( Figure 3C-D'). Altogether, these data suggest that the decrease in gene expression strength is likely due to the genetic circuit being susceptible to epigenetic modifications, specifically to methylation and/or histone acetylation (33,34,58).…”

Section: Assessing the Stability Of Genetic Circuits In Rosa26 Locusmentioning

confidence: 90%

“…These model cell lines are useful for enhancing our understanding of synthetic gene circuits that underscore the potential of synthetic biology tools, however, these cell lines may not be good predictors of the challenges that arise in stem cells (31). Plasmid and viral gene delivery systems have shown to lose expression over weeks of cell culture, which is thought to be a consequence of epigenetic modifications of the inserted DNA (32)(33)(34). The current understanding of transgene silencing suggests that silencing of genes can occur through the methylation of the expression cassette and/or the formation of heterochromatin, both of which facilitate changes in gene expression; however, the circumstances that trigger these mechanisms are still being elucidated.…”

Section: Introductionmentioning

confidence: 99%

Rosa26 docking sites for investigating genetic circuit silencing in stem cells

Fitzgerald

Livingston

Gibbs

et al. 2019

Preprint

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Approaches in mammalian synthetic biology have transformed how cells can be programmed to have reliable and predictable behaviour. The majority of mammalian synthetic biology has been accomplished using immortalized cell lines that are easy to grow, and easy to transfect. Additionally, genetic circuits that integrate into the genome of these immortalized lines remain functional for many generations, often for the lifetime of the cells. However, when genetic circuits are integrated into the genome of stem cells, gene silencing is observed within a few generations. To investigate the stability of genetic circuits and their propensity to silence in stem cells, the Rosa26 locus of mouse pluripotent stem cells was modified to contain docking sites for site-specific integration of genetic circuits. We show that the silencing of genetic circuits can be reversed with the addition of sodium butyrate, a histone deacetylase inhibitor. These findings demonstrate an approach to stabilize the function of genetic circuits in pluripotent stem cells to ensure robust function over many generations. Altogether, this work introduces an approach to overcome silencing of genetic circuits in pluripotent stem cells that enable the possibility of leveraging synthetic biology in pluripotent stem cells to meet therapeutic needs.

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“…CARLIN barcoding diversity is in the range of 44.000. However, CARLIN is based on the Tet-O system, which is known to be silenced epigenetically once cells differentiate (Godecke et al, 2017). In adult tissues, 7 days of continuous induction with Dox, reached editing ratios close to 0% in brain, heart, and muscle and 31-88% in other organs.…”

Section: Higher Resolution Lineage Barcoding With Cre or Cas9mentioning

confidence: 99%

Genetic Tools to Study Cardiovascular Biology

et al. 2020

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Progress in biomedical science is tightly associated with the improvement of methods and genetic tools to manipulate and analyze gene function in mice, the most widely used model organism in biomedical research. The joint effort of numerous individual laboratories and consortiums has contributed to the creation of a large genetic resource that enables scientists to image cells, probe signaling pathways activities, or modify a gene function in any desired cell type or time point, à la carte. However, as these tools significantly increase in number and become more sophisticated, it is more difficult to keep track of each tool's possibilities and understand their advantages and disadvantages. Knowing the best currently available genetic technology to answer a particular biological question is key to reach a higher standard in biomedical research. In this review, we list and discuss the main advantages and disadvantages of available mammalian genetic technology to analyze cardiovascular cell biology at higher cellular and molecular resolution. We start with the most simple and classical genetic approaches and end with the most advanced technology available to fluorescently label cells, conditionally target their genes, image their clonal expansion, and decode their lineages.

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Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation

Cited by 32 publications

References 72 publications

E2F7 Is a Potent Inhibitor of Liver Tumor Growth in Adult Mice

E2F7 Is a Potent Inhibitor of Liver Tumor Growth in Adult Mice

Rosa26 docking sites for investigating genetic circuit silencing in stem cells

Genetic Tools to Study Cardiovascular Biology

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