Skip to main content
SpringerLink
Log in

Lineage Tracing in the Mammary Gland Using Cre/lox Technology and Fluorescent Reporter Alleles

  • Protocol
Mammary Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1293))

Abstract

Lineage tracing using Cre/lox technology has become a well-established technique to study the contribution of different (stem) cell populations to organ development and function. When used in the mammary gland, it forms a valuable addition to the already existing experimental toolbox and an important alternative to other readouts measuring stem cell potential, such as the fat pad transplantation assay.

Here I describe how to set up and analyze an in vivo lineage tracing experiment using tamoxifen-inducible Cre/lox technology, highlighting the specific challenges that the investigator faces when employing this method and interpreting the results in the mammary gland.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Watson CJ, Khaled WT (2008) Mammary development in the embryo and adult: a journey of morphogenesis and commitment. Development 135(6):995–1003. doi:10.1242/dev.005439, 135/6/995 [pii]

    Article  CAS  PubMed  Google Scholar 

  2. Smith BA, Welm AL, Welm BE (2012) On the shoulders of giants: a historical perspective of unique experimental methods in mammary gland research. Semin Cell Dev Biol 23(5):583–590, 10.1016/j.semcdb.2012.03.005 S1084-9521(12)00047-X [pii]

    Article  PubMed Central  PubMed  Google Scholar 

  3. Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, Wu L, Lindeman GJ, Visvader JE (2006) Generation of a functional mammary gland from a single stem cell. Nature 439(7072):84–88. doi:10.1038/nature04372, nature04372 [pii]

    Article  CAS  PubMed  Google Scholar 

  4. Stingl J, Eirew P, Ricketson I, Shackleton M, Vaillant F, Choi D, Li HI, Eaves CJ (2006) Purification and unique properties of mammary epithelial stem cells. Nature 439(7079):993–997. doi:10.1038/nature04496, nature04496 [pii]

    CAS  PubMed  Google Scholar 

  5. Deome KB, Faulkin LJ Jr, Bern HA, Blair PB (1959) Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res 19(5):515–520

    CAS  PubMed  Google Scholar 

  6. van Amerongen R, Bowman AN, Nusse R (2012) Developmental stage and time dictate the fate of Wnt/beta-catenin-responsive stem cells in the mammary gland. Cell Stem Cell 11(3):387–400. doi:10.1016/j.stem.2012.05.023, S1934-5909(12)00342-6 [pii]

    Article  PubMed  Google Scholar 

  7. Van Keymeulen A, Rocha AS, Ousset M, Beck B, Bouvencourt G, Rock J, Sharma N, Dekoninck S, Blanpain C (2011) Distinct stem cells contribute to mammary gland development and maintenance. Nature 479(7372):189–193, 10.1038/nature10573, nature10573 [pii]

    Article  PubMed  Google Scholar 

  8. Akagi K, Sandig V, Vooijs M, Van der Valk M, Giovannini M, Strauss M, Berns A (1997) Cre-mediated somatic site-specific recombination in mice. Nucleic Acids Res 25(9):1766–1773, gka289 [pii]

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A 85(14):5166–5170

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Smedley D, Salimova E, Rosenthal N (2011) Cre recombinase resources for conditional mouse mutagenesis. Methods 53(4):411–416, 10.1016/j.ymeth.2010.12.027. S1046-2023(10)00310-5 [pii]

    Article  CAS  PubMed  Google Scholar 

  11. Murray SA, Eppig JT, Smedley D, Simpson EM, Rosenthal N (2012) Beyond knockouts: cre resources for conditional mutagenesis. Mamm Genome 23(9–10):587–599. doi:10.1007/s00335-012-9430-2

    Article  PubMed Central  PubMed  Google Scholar 

  12. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21(1):70–71. doi:10.1038/5007

    Article  CAS  PubMed  Google Scholar 

  13. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double-fluorescent Cre reporter mouse. Genesis 45(9):593–605. doi:10.1002/dvg.20335

    Article  CAS  PubMed  Google Scholar 

  14. Snippert HJ, van der Flier LG, Sato T, van Es JH, van den Born M, Kroon-Veenboer C, Barker N, Klein AM, van Rheenen J, Simons BD, Clevers H (2010) Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143(1):134–144, S0092-8674(10)01064-0 [pii]. 10.1016/j.cell.2010.09.016

    Article  CAS  PubMed  Google Scholar 

  15. Metzger D, Clifford J, Chiba H, Chambon P (1995) Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc Natl Acad Sci U S A 92(15):6991–6995

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P (1996) Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci U S A 93(20):10887–10890

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Feil R, Wagner J, Metzger D, Chambon P (1997) Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun 237(3):752–757, S0006-291X(97)97124-2 [pii] 10.1006/bbrc.1997.7124

    Article  CAS  PubMed  Google Scholar 

  18. Indra AK, Warot X, Brocard J, Bornert JM, Xiao JH, Chambon P, Metzger D (1999) Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res 27(22):4324–4327, gkc656 [pii]

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Laird PW, Zijderveld A, Linders K, Rudnicki MA, Jaenisch R, Berns A (1991) Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19(15):4293

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, Clevers H (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449(7165):1003–1007, nature06196 [pii]. 10.1038/nature06196

    Article  CAS  PubMed  Google Scholar 

  21. Nakamura E, Nguyen MT, Mackem S (2006) Kinetics of tamoxifen-regulated Cre activity in mice using a cartilage-specific CreER(T) to assay temporal activity windows along the proximodistal limb skeleton. Dev Dyn 235(9):2603–2612. doi:10.1002/dvdy.20892

    Article  CAS  PubMed  Google Scholar 

  22. Lim X, Tan SH, Koh WL, Chau RM, Yan KS, Kuo CJ, van Amerongen R, Klein AM, Nusse R (2013) Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling. Science 342(6163):1226–1230. doi:10.1126/science.1239730

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Shehata M, van Amerongen R, Zeeman AL, Giraddi RR, Stingl J (2014) The influence of tamoxifen on normal mouse mammary gland homeostasis Breast Cancer Res 16:411

    Google Scholar 

Download references

Acknowledgements

RvA is supported by the Dutch Cancer Society (KWF cancer research career award, 2013-6057) and by a MacGillavry fellowship from the University of Amsterdam. I thank my ex-colleagues and Roel Nusse at Stanford University (USA) for discussions and feedback during the experimental stages of this work, Lauran Oomen and Lenny Brocks at the Netherlands Cancer Institute in Amsterdam (the Netherlands) for help with the Leica SP5, Ronald Breedijk, and Erik Manders for help with analyzing samples on the Nikon A1, Ji-Ying Song for the image depicted in Fig. 1b and Amber Zeeman for comments on the manuscript and the image depicted in Fig. 1a.

Author information

Authors and Affiliations

  1. Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands

    Renée van Amerongen

Authors
  1. Renée van Amerongen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Renée van Amerongen .

Editor information

Editors and Affiliations

  1. CIC bioGUNE, Technological Park of Bizkaia, Derio, Spain

    Maria del Mar Vivanco

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media LLC New York

About this protocol

Cite this protocol

van Amerongen, R. (2015). Lineage Tracing in the Mammary Gland Using Cre/lox Technology and Fluorescent Reporter Alleles. In: Vivanco, M. (eds) Mammary Stem Cells. Methods in Molecular Biology, vol 1293. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2519-3_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2519-3_11

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2518-6

  • Online ISBN: 978-1-4939-2519-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation