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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
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]
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]
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]
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]
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
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]
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]
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]
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
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]
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
Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21(1):70–71. doi:10.1038/5007
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
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
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
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
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
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]
Laird PW, Zijderveld A, Linders K, Rudnicki MA, Jaenisch R, Berns A (1991) Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19(15):4293
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
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
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
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
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
Corresponding author
Editor information
Editors and Affiliations
Rights 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