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Manipulation of gene expression by an ecdysone-inducible gene switch in tumor xenografts
L R Karns1, A Kisielewski, K M Gulding
11Upstate Biotechnology Inc., Charlottesville, Virginia, USA. lkarns@argonex.com
Background:
Rapid, robust and reversible induction of transgene expression would significantly facilitate cancer gene therapy as well as allow the in vivo functional study of newly discovered genes in tumor formation and progression. The popularity of the ecdysone inducible gene switch system has led us to investigate whether such a system can successfully regulate gene expression in a syngeneic tumor system in vivo.
Results:
MBT-2 and Panc02 carcinoma cells were transfected with components of a modification of the ecdysone switch system driving firefly luciferase (F-Luc). In vitro luciferase expression +/- ecdysone analog GS-E indicated a robust induction with minimal baseline activity and complete decay after 24 hours without drug. In vitro selection of MBT-2 transfected cell clones which had complete absence of F-Luc expression in the absence of stimulation but which expressed this gene at high levels in response to GS-E were chosen for in vivo evaluation. Tumors from engineered MBT-2 cells were grown to 5 mm in diameter prior to GS-E administration, animals euthanized and tumors removed at 6, 12 and 24 hours after GS-E administration and assayed for F-Luc activity. GS-E resulted in a maximal induction of F-Luc activity at 6 hours in tumor tissue with almost complete reversion to control levels by 12 hours.
Conclusions:
This study is the first demonstration that robust and reversible transgene expression in tumors is feasible using the ecdysone system, allowing future rapid in vivo functional characterization of gene function or gene therapy applications.
Insights
The ecdysone gene switch system enables robust and reversible transgene expression in tumors. This breakthrough facilitates in vivo gene function studies and cancer gene therapy applications.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Developing controllable gene expression systems is crucial for cancer gene therapy and studying gene function in tumors.
- The ecdysone inducible gene switch system is a popular tool for regulating gene expression.
Purpose of the Study:
- To investigate the efficacy of a modified ecdysone gene switch system for regulating transgene expression in a syngeneic tumor model in vivo.
- To assess the robustness and reversibility of transgene induction in tumor tissue.
Main Methods:
- Carcinoma cells (MBT-2 and Panc02) were transfected with a modified ecdysone switch system driving firefly luciferase (F-Luc).
- In vitro validation confirmed robust F-Luc induction with GS-E and complete decay without the drug.
- Selected MBT-2 clones with stringent on/off F-Luc expression were used for in vivo studies.
- Tumor-bearing animals received GS-E, and F-Luc activity was measured at various time points post-administration.
Main Results:
- In vitro, the ecdysone system showed strong F-Luc induction with minimal baseline activity and rapid decay.
- In vivo, GS-E administration led to maximal F-Luc activity induction in tumor tissue by 6 hours.
- Transgene expression returned to near-baseline levels within 12 hours, demonstrating reversibility.
Conclusions:
- This study demonstrates the feasibility of using the ecdysone system for robust and reversible transgene expression in tumors.
- This system offers a powerful tool for rapid in vivo functional characterization of genes in cancer research.
- The findings support the potential application of this system in future cancer gene therapy strategies.