Validation of targets for
cancer drug discovery requires robust experimental models. Systems based on inducible gene expression are well suited to this purpose but are difficult to establish in several epithelial cell types. Using the recently discovered transcriptional
transactivator (rtTA2S-M2), we developed a strategy for fast and efficient generation of Tet On cells. Multiple clones of HCT116, SW480, and HT29 human
colon cancer cells for
doxycycline-regulated gene expression were constructed that constitutively express
green fluorescent protein (GFP) for selection/maintenance purposes. The cell lines displayed good fold inducibility (49-124xHCT116; 178-621xSW480; 261-787xHT29) and minimal leakiness after transient transfection with a
luciferase reporter or with vectors driving inducible expression of
red fluorescent protein (dsRed2), constitutively active c-Src or dominant negative K-Ras4B. The clones preserved their transformed phenotype as demonstrated by comparing their properties to respective wild type cells, in terms of growth in vitro and in vivo (as
tumor xenografts), cell cycle traverse, and sensitivity to drugs used in
chemotherapy. These engineered cell lines enabled tightly controlled inducible gene expression both in vitro and in vivo, and proved well suited for construction of double-stable cell lines inducibly expressing a
protein of interest. As such they represent a useful research tool for example, to dissect oncogene function(s) in
colon cancer. Supplementary material for this article be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_welman.doc.