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 express ion in response to doxycycline, was applied to analyze the influence of the receptor for advanced glycosylation endproducts (RAGE) on the growth of 29 3 cells in semisolid medium. Establishing a Tet-On gene expression system i nvolves two consecutive stable transfections. Here, we describe an alternat ive procedure to obtain a Tet-On gene expression system in a single transfe ction step for the use in tumor biology. The plasmids necessary for the reg ulated expression of RAGE together with the selectable marker plasmid were cotransfected in a molar ratio of 6:1. After aminoglycoside selection, 29 c lones were analyzed using PCR revealing 8 colonies to be double stably tran sformed. Subsequent Western blot analysis showed inducible expression in 7 cell lines. Applying the one step protocol, the entire Tet-On expression sy stem could be completed in half of the time required for the original two s tep method. The generated 293 double stable cells were used in the clonogen ic assay for the testing of the tumor suppressive potential of RAGE.