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Simple and fast method to test the receptor for advanced glycosylated endproducts (RAGE) for its tumor suppressive
I Bendik1, P Schraml, C U Ludwig
1Department of Research, University Hospital Basel, Switzerland. Bendik@ubaclu.unibas.ch
Abstract:
The Tet gene expression system, that allows tightly controlled gene expression in response to doxycycline, was applied to analyze the influence of the receptor for advanced glycosylation endproducts (RAGE) on the growth of 293 cells in semi-solid medium. Establishing a Tet-On gene expression system involves two consecutive stable transfections. Here, we describe an alternative procedure to obtain a Tet-On gene expression system in a single transfection step for the use in tumor biology. The plasmids necessary for the regulated expression of RAGE together with the selectable marker plasmid were cotransfected in a molar ratio of 6:1. After aminoglycoside selection, 29 clones were analyzed using PCR revealing 8 colonies to be double stably transformed. Subsequent Western blot analysis showed inducible expression in 7 cell lines. Applying the one step protocol, the entire Tet-On expression system could be completed in half of the time required for the original two step method. The generated 293 double stable cells were used in the clonogenic assay for the testing of the tumor suppressive potential of RAGE.
Insights
This study introduces a faster method for creating Tet-On gene expression systems, enabling quicker analysis of how the receptor for advanced glycosylation endproducts (RAGE) affects cell growth and tumor potential.
Area of Science:
- Molecular Biology
- Cancer Biology
- Gene Expression Systems
Background:
- The Tet gene expression system offers doxycycline-inducible control over gene expression.
- Analyzing the role of the receptor for advanced glycosylation endproducts (RAGE) in cell growth requires stable gene expression systems.
- Traditional Tet-On system establishment involves two sequential stable transfections, which is time-consuming.
Purpose of the Study:
- To develop a streamlined, single-step transfection protocol for establishing Tet-On gene expression systems.
- To apply this improved system for investigating the influence of RAGE on 293 cell growth in semi-solid medium.
- To assess the tumor suppressive potential of RAGE using a clonogenic assay.
Main Methods:
- Co-transfection of plasmids for regulated RAGE expression and a selectable marker in a 6:1 molar ratio.
- Aminoglycoside selection followed by PCR screening of clones.
- Western blot analysis to confirm inducible RAGE expression.
- Clonogenic assays to evaluate cell growth and RAGE's tumor suppressive effects.
Main Results:
- A single-step co-transfection protocol successfully generated double stably transformed cell lines.
- Out of 29 analyzed clones, 8 were confirmed as double stably transformed via PCR.
- Inducible RAGE expression was validated in 7 of these cell lines through Western blot.
- The one-step protocol significantly reduced the time required for Tet-On system establishment.
Conclusions:
- The developed one-step protocol is an efficient alternative for creating Tet-On systems in tumor biology research.
- This method accelerates the generation of stable cell lines for studying gene function, such as RAGE's role in cancer.
- The study provides a foundation for further investigation into RAGE's tumor suppressive potential.