Fluorescent Protein-Assisted Purification for Gene Expression Profiling

  • M. Raza Zaidi
  • Chi-Ping Day
  • Glenn Merlino
Part of the Methods in Molecular Biology book series (MIMB, volume 699)


Cell type-specific expression of fluorescent proteins allows the purification of rare cells from complex ­tissues by flow cytometry. This strategy is especially useful for molecular analysis of cancer cells because these cells can be effectively purified away from the noncancerous tumor stroma. Coexpression of bioluminescence with fluorescence makes further allows in vivo tracking of cancer cells, which can then be purified at specific tumorigenic stages. Here, we describe protocols for purifying rare skin stem cells, and for in vivo monitoring and purification of cancer cells from lung metastases. Also described is a protocol for the isolation of total RNA from the purified cells for the purpose of performing gene expression profiling.

Key words

Tetracycline-induced system Green fluorescent protein Luciferase Skin stem cells Label-retaining cells Pol2 promoter Syngeneic mice Lung metastases 


  1. 1.
    Contag, C. H. and Bachmann, M. H. (2002) Advances in in vivo bioluminescence imaging of gene expression. Annu Rev Biomed Eng 4, 235–60.CrossRefGoogle Scholar
  2. 2.
    Gross, S. and Piwnica-Worms, D. (2005) Spying on cancer: molecular imaging in vivo with genetically encoded reporters. Cancer Cell 7, 5–15.Google Scholar
  3. 3.
    Spergel, D. J., Kruth, U., Shimshek, D. R., Sprengel, R., and Seeburg, P. H. (2001) Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies. Prog Neurobiol 63, 673–86.CrossRefGoogle Scholar
  4. 4.
    Day, C. P., Carter, J., Bonomi, C., et al. (2009) Lentivirus-mediated bifunctional cell labeling for in vivo melanoma study. Pigment Cell Melanoma Res 22, 283–95.CrossRefGoogle Scholar
  5. 5.
    Haas, D. L., Case, S. S., Crooks, G. M., and Kohn, D. B. (2000) Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors. Mol Ther 2, 71–80.CrossRefGoogle Scholar
  6. 6.
    Shaner, N. C., Patterson, G. H., and Davidson, M. W. (2007) Advances in fluorescent protein technology. J Cell Sci 120, 4247–60.CrossRefGoogle Scholar
  7. 7.
    Wolnicka-Glubisz, A., King, W., and Noonan, F. P. (2005) SCA-1+ cells with an adipocyte phenotype in neonatal mouse skin. J Invest Dermatol 125, 383–5.CrossRefGoogle Scholar
  8. 8.
    Tumbar, T., Guasch, G., Greco, V., et al. (2004) Defining the epithelial stem cell niche in skin. Science 303, 359–63.CrossRefGoogle Scholar
  9. 9.
    Zhu, Z., Zheng, T., Lee, C. G., Homer, R. J., and Elias, J. A. (2002) Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling. Semin Cell Dev Biol 13, 121–8.CrossRefGoogle Scholar
  10. 10.
    Romano, R. A. and Sinha, S. (2010) Tetracycline-regulated gene expression in transgenic mouse epidermis. Methods Mol Biol 585, 287–302.CrossRefGoogle Scholar
  11. 11.
    Ventura, A., Meissner, A., Dillon, C. P., et al. (2004) Cre-lox-regulated conditional RNA interference from transgenes. Proc Natl Acad Sci USA 101, 10380–5.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • M. Raza Zaidi
    • 1
  • Chi-Ping Day
    • 1
  • Glenn Merlino
    • 1
  1. 1.Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer InstituteNational Institutes of HealthBethesdaUSA

Personalised recommendations