Simple and fast method to test the receptor for advanced glycosylated endproducts (RAGE) for its tumor suppressive potential using the Tet-On system

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 semisolid 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.