Isolation and Fluorescence-Activated Cell Sorting of Mouse Keratinocytes Expressing β-Galactosidase

  • Maria Kasper
  • Rune Toftgård
  • Viljar JaksEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1453)


During the past decade, the rapid development of new transgenic and knock-in mouse models has propelled epidermal stem-cell research into “fast-forward mode”. It has become possible to identify and visualize defined cell populations during normal tissue maintenance, and to follow their progeny during the processes of homeostasis, wound repair, and tumorigenesis. Moreover, these cells can be isolated using specific labels, and characterized in detail using an array of molecular and cell biology approaches. The bacterial enzyme, β-galactosidase (β-gal), the product of the LacZ gene, is one of the most commonly used in vivo cell labels in genetically-engineered mice. The protocol described in this chapter provides a guideline for the isolation of viable murine epidermal cells expressing β-gal, which can then be subjected to further characterization in vivo or in vitro.

Key words

Beta-galactosidase (β-galactosidase) Genetically engineered reporter mice Keratinocytes Skin Epidermis Stem cells FACS sorting 



V.J. is supported by an EMBO Installation Grant and two grants, nos. ETF8932 and PUT4 from the Estonian Research Council. M.K. is supported by grants from the Swedish Research Council, the Swedish Cancer Society, the Karolinska Institutet, the Harald and Greta Jeanssons Foundation, the Swedish Foundation for Strategic Research, the Center for Innovative Medicine, and the Ragnar Söderberg Foundation. R.T. is supported by grants from the Swedish Research Council and the Swedish Cancer Society.


  1. 1.
    Mansour SL, Thomas KR, Deng CX et al (1990) Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination. Proc Natl Acad Sci U S A 87:7688–7692CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Morrey JD, Bourn SM, Bunch TD et al (1991) In vivo activation of human immunodeficiency virus type 1 long terminal repeat by UV type A (UV-A) light plus psoralen and UV-B light in the skin of transgenic mice. J Virol 65:5045–5051PubMedPubMedCentralGoogle Scholar
  3. 3.
    Ikawa M, Kominami K, Yoshimura Y et al (1995) Green fluorescent protein as a marker in transgenic mice. Dev Growth Differ 37:455–459CrossRefGoogle Scholar
  4. 4.
    Snippert HJ, van der Flier LG, Sato T et al (2010) Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143:134–144CrossRefPubMedGoogle Scholar
  5. 5.
    Bickenbach JR, Chism E (1998) Selection and extended growth of murine epidermal stem cells in culture. Exp Cell Res 244:184–195CrossRefPubMedGoogle Scholar
  6. 6.
    Trempus CS, Morris RJ, Bortner CD et al (2003) Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol 120:501–511PubMedGoogle Scholar
  7. 7.
    Tani H, Morris RJ, Kaur P (2000) Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci U S A 97:10960–10965CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Jensen KB, Collins CA, Nascimento E et al (2009) Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell 4:427–439CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Nijhof JG, Braun KM, Giangreco A et al (2006) The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells. Development 133:3027–3037CrossRefPubMedGoogle Scholar
  10. 10.
    Tumbar T, Guasch G, Greco V et al (2004) Defining the epithelial stem cell niche in skin. Science 303:359–363CrossRefPubMedGoogle Scholar
  11. 11.
    Takeda N, Jain R, Leboeuf MR et al (2013) Hopx expression defines a subset of multipotent hair follicle stem cells and a progenitor population primed to give rise to K6+ niche cells. Development 140:1655–1664CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Snippert HJ, Haegebarth A, Kasper M et al (2010) Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327:1385–1389CrossRefPubMedGoogle Scholar
  13. 13.
    Barker N, van Es JH, Kuipers J et al (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003–1007CrossRefPubMedGoogle Scholar
  14. 14.
    Jaks V, Barker N, Kasper M et al (2008) Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 40:1291–1299CrossRefPubMedGoogle Scholar
  15. 15.
    Wu WY, Morris RJ (2005) Method for the harvest and assay of in vitro clonogenic keratinocytes stem cells from mice. Methods Mol Biol 289:79–86PubMedGoogle Scholar
  16. 16.
    Philpott MP, Green MR, Kealey T (1992) Rat hair follicle growth in vitro. Br J Dermatol 127:600–607CrossRefPubMedGoogle Scholar
  17. 17.
    Rendl M, Lewis L, Fuchs E (2005) Molecular dissection of mesenchymal-epithelial interactions in the hair follicle. PLoS Biol 3:e331CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Rogers G, Martinet N, Steinert P et al (1987) Cultivation of murine hair follicles as organoids in a collagen matrix. J Invest Dermatol 89:369–379CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biosciences and Nutrition and Center for Innovative MedicineKarolinska InstitutetHuddingeSweden
  2. 2.Institute of Molecular and Cell BiologyUniversity of TartuTartuEstonia

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