A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGFß signaling in the mammary gland.

ABSTRACT

INTRODUCTION: Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference (RNAi)-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFß/Smad pathway as an example. METHODS: We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse) which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. A panel of conditionally immortalized mammary epithelial cell (IMEC) cultures were derived from the virgin mammary glands of offspring of these crosses and used to assess the Smad dependency of different biological responses to TGFß. RESULTS: IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFß. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFß-induced apoptotic, growth inhibitory and EMT responses, whereas either Smad can support TGFß-induced invasion as long as a threshold level of total Smad is exceeded. CONCLUSIONS: This work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium.