MCF7 tet-off cells treated or not with 80 ng/ml doxycycline for 12d

Tet-on and tet-off systems are among the most popular vector systems for inducible transgene expression in mammalian cells. In tet-regulated systems the expression of the transgene depends on the presence or absence of the antibiotic tetracycline or its derivative doxycycline, which are added to the cell culture medium in concentrations considered to be far below cytotoxic levels. Therefore, potential effects on the treated cells, which are exerted by the antibiotic itself and unrelated to transgene expression, are often ignored. We examined the influence of low dose doxycycline on the transcriptional profile of two independent clones of MCF7 human breast carcinoma cells, transfected with tet-off regulator and response plasmids but not harboring any transgene. Treatment with 80 ng/ml doxycycline for 12 days markedly altered gene expression in these cells. Many genes associated with interferon-signaling were up-regulated while cell cycle-associated genes were down-regulated, which was also accompanied by a reduction of cell growth. These results highlight the importance of appropriate controls when working with tet-regulated gene expression systems, to allow distinction between the effects of transgene expression and potential “side effects” of the antibiotic used for its regulation. MCF7 human breast carcinoma cells were stably transfected with the pTet-Off-regulator plasmid and the pTRE2hyg response plasmid, not comprising any transgene. Two independent cell clones were grown in their normal growth medium for 12 days in the presence or absence of 80ng/ml doxycycline. The medium was renewed every second day. After 12 days, total RNA was extracted using the Trizol reagent.