Abstract
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.
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References
Contag, C. H. and Bachmann, M. H. (2002) Advances in in vivo bioluminescence imaging of gene expression. Annu Rev Biomed Eng 4, 235–60.
Gross, S. and Piwnica-Worms, D. (2005) Spying on cancer: molecular imaging in vivo with genetically encoded reporters. Cancer Cell 7, 5–15.
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.
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.
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.
Shaner, N. C., Patterson, G. H., and Davidson, M. W. (2007) Advances in fluorescent protein technology. J Cell Sci 120, 4247–60.
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.
Tumbar, T., Guasch, G., Greco, V., et al. (2004) Defining the epithelial stem cell niche in skin. Science 303, 359–63.
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.
Romano, R. A. and Sinha, S. (2010) Tetracycline-regulated gene expression in transgenic mouse epidermis. Methods Mol Biol 585, 287–302.
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.
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Zaidi, M.R., Day, CP., Merlino, G. (2011). Fluorescent Protein-Assisted Purification for Gene Expression Profiling. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 699. Humana Press. https://doi.org/10.1007/978-1-61737-950-5_19
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DOI: https://doi.org/10.1007/978-1-61737-950-5_19
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Online ISBN: 978-1-61737-950-5
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