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01.10.2010 | Research article | Ausgabe 5/2010 Open Access

Breast Cancer Research 5/2010

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

Zeitschrift:
Breast Cancer Research > Ausgabe 5/2010
Autoren:
Ethan A Kohn, Zhijun Du, Misako Sato, Catherine MH Van Schyndle, Michael A Welsh, Yu-an Yang, Christina H Stuelten, Binwu Tang, Wenjun Ju, Erwin P Bottinger, Lalage M Wakefield
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​bcr2728) contains supplementary material, which is available to authorized users.
Ethan A Kohn, Zhijun Du contributed equally to this work.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

EAK, ZD and LMW developed ideas and designed experiments. EAK and LMW wrote the manuscript. EAK and ZD performed the majority of the experiments, with assistance from CMHVS, MAW and Y-aY. BT, MS, and CHS contributed expertise and helped perform fluorescence-activated cell sorting analyses, immunofluorescence and zymography. WJ and EPB provided intellectual input and expertise, and contributed to breeding of mice with mixed Smad genotypes and manuscript editing.

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-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 (TGFβ)/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. Conditionally immortalized mammary epithelial cell cultures were derived from the virgin mammary glands of offspring of these crosses and were used to assess the Smad dependency of different biological responses to TGFβ.

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 TGFβ. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFβ-induced apoptotic, growth inhibitory and epithelial-to-mesenchymal transition responses, whereas either Smad2 or Smad3 can support TGFβ-induced invasion as long as a threshold level of total Smad is exceeded.

Conclusions

The present 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.
Zusatzmaterial
Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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Authors’ original file for figure 5
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Authors’ original file for figure 6
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Authors’ original file for figure 7
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Literatur
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