Skip to main content

Advertisement

SpringerLink
Log in

Adipose-Depleted Mammary Epithelial Cells and Organoids

  • Published:
Journal of Mammary Gland Biology and Neoplasia Aims and scope Submit manuscript

Abstract

Analysis of genes and proteins involved in lipid biosynthesis in mammary epithelial cells (MECs) is complicated by the presence of adipose tissue in the mammary gland, which may be predominant in whole tissue lysates depending upon developmental stage. We have developed a method based on protocols used to establish primary mammary epithelial cell cultures that allows for analysis of MECs depleted of adipose. Adipose depletion yields enriched MECs that are suitable for gene expression profiling and protein analysis from a single mouse. Additionally, the phosphorylation of proteins is maintained, allowing investigation of signal transduction events. Application of this method to the analysis of MECs from genetically modified mice will aid in the identification of factors controlling tissue-specific events in the mammary gland. In contrast to other methods such as laser capture microdissection, the MEC enrichment method described here is performed using standard lab supplies, equipment, and techniques.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Figure 1
Figure 2

Similar content being viewed by others

Abbreviations

ADPH:

Adipophilin

ACLY:

ATP Citrate Lyase

Csn2:

β-casein

ChREBP:

Carbohydrate Responsive Element Binding Protein

C/EBPβ and C/EBPδ:

CCAAT/enhancer-binding protein beta and delta

Ker18, Ker19:

Cytokeratin 18 and 19

LCM:

Laser Capture Micro-dissection

MECs:

Mammary Epithelial Cells

PLIN:

Perilipin

qPCR:

quantitative Real Time PCR

THRSP, Spot14:

Thyroid Hormone Responsive Protein Spot 14

TAGs:

triglycerides

WTLs:

Whole mammary Tissue Lysates

References

  1. Rudolph MC, Neville MC, Anderson SM. Lipid synthesis in lactation: diet and the fatty acid switch. J Mammary Gland Biol Neoplasia. 2007;12(4):269–81.

    Article  PubMed  Google Scholar 

  2. Owens IS, Vonderhaar BK, Topper YJ. Concerning the necessary coupling of development to proliferation of mouse mammary epithelial cells. J Biol Chem. 1973;248(2):472–7.

    CAS  PubMed  Google Scholar 

  3. Harris A. Epithelial cell culture. Handbooks in practical animal cell biology. Cambridge: Cambridge University Press; 1996. xii, 182 p.

  4. Ip MM, Asch BB. Methods in mammary gland biology and breast cancer research. New York: Kluwer Academic/Plenum; 2000. xvi, 329 p., [4] leaves of col. plates.

  5. Schroeder A, et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol. 2006;7:3.

    Article  PubMed  Google Scholar 

  6. Pujuguet P, et al. Trichostatin A inhibits beta-casein expression in mammary epithelial cells. J Cell Biochem. 2001;83(4):660–70.

    Article  CAS  PubMed  Google Scholar 

  7. Berwick DC, et al. The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary adipocytes. J Biol Chem. 2002;277(37):33895–900.

    Article  CAS  PubMed  Google Scholar 

  8. Brown SB, Maloney M, Kinlaw WB. “Spot 14” protein functions at the pretranslational level in the regulation of hepatic metabolism by thyroid hormone and glucose. J Biol Chem. 1997;272(4):2163–6.

    Article  CAS  PubMed  Google Scholar 

  9. Uyeda K, Yamashita H, Kawaguchi T. Carbohydrate responsive element-binding protein (ChREBP): a key regulator of glucose metabolism and fat storage. Biochem Pharmacol. 2002;63(12):2075–80.

    Article  CAS  PubMed  Google Scholar 

  10. Zhu Q, et al. The Spot 14 protein is required for de novo lipid synthesis in the lactating mammary gland. Endocrinology. 2005;146(8):3343–50.

    Article  CAS  PubMed  Google Scholar 

  11. Rudolph M et al. Mammary epithelial specific deletion of escort protein SCAP reveals SREBF-dependent and independent fatty acid synthesis control during lactation. Submitted; 2009.

  12. Raught B, Liao WS, Rosen JM. Developmentally and hormonally regulated CCAAT/enhancer-binding protein isoforms influence beta-casein gene expression. Mol Endocrinol. 1995;9(9):1223–32.

    Article  CAS  PubMed  Google Scholar 

  13. Thangaraju M, et al. C/EBPdelta is a crucial regulator of pro-apoptotic gene expression during mammary gland involution. Development. 2005;132(21):4675–85.

    Article  CAS  PubMed  Google Scholar 

  14. Yeh WC, et al. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev. 1995;9(2):168–81.

    Article  CAS  PubMed  Google Scholar 

  15. Tanaka T, et al. Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene. EMBO J. 1997;16(24):7432–43.

    Article  CAS  PubMed  Google Scholar 

  16. Russell TD, et al. Transduction of the mammary epithelium with adenovirus vectors in vivo. J Virol. 2003;77(10):5801–9.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank the members of the Mammary Gland Biology Program Project Grant at the University of Colorado Denver for discussions during the development of this technique. SMA is supported by a grant from the Public Health Service PO1-HD38129. MCR is supported by a Department of Defense Predoctoral Fellowship BC 810596.

Author information

Authors and Affiliations

  1. Department of Pathology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, 80045, USA

    Michael C. Rudolph, Elizabeth A. Wellberg & Steven M. Anderson

  2. Program in Molecular Biology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, 80045, USA

    Michael C. Rudolph & Steven M. Anderson

  3. School of Medicine, Department of Pathology, University of Colorado Denver, MS 8104, 12801 East 17th Avenue, P.O. Box 6500, Aurora, CO, 80045, USA

    Steven M. Anderson

Authors
  1. Michael C. Rudolph
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elizabeth A. Wellberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Steven M. Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Steven M. Anderson.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rudolph, M.C., Wellberg, E.A. & Anderson, S.M. Adipose-Depleted Mammary Epithelial Cells and Organoids. J Mammary Gland Biol Neoplasia 14, 381–386 (2009). https://doi.org/10.1007/s10911-009-9161-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10911-009-9161-5

Keywords

Search

Navigation