Abstract
A considerable number of transgenic or knockout mice in which epidermal keratinocytes have been targeted die shortly after birth due to barrier defects. In this case, recovery and cultivation of keratinocytes from these animals provide an opportunity for in vitro studies. Working with isolated keratinocytes is also interesting for certain experiments which cannot be performed in live animals.
Primary human keratinocytes can be kept in culture for a variable number of passages and then senesce. Immortalization can be achieved by transduction with constructs encoding viral genes. Murine keratinocytes can be kept in culture as primary cells. Naturally the numbers of cells obtained by direct isolation from mouse epidermis is restricted and sometimes not sufficient for certain biochemical analyses. To overcome this restriction some permanent murine keratinocyte lines have been generated by transfection with SV40T or HPV E6E7 genes. This is, however, not suitable if established or hypothetical biochemical links exist between these genes and the pathways or processes to be analysed in the respective experiment.
We describe an easy and reproducible method of establishing permanent keratinocyte lines from spontaneously immortalized primary murine keratinocytes. This method employs co-cultivation of keratinocytes with 3T3-J2 fibroblast feeder cells for several passages during which immortalization occurs. The resulting keratinocyte lines do not only grow infinitely but, in many cases, individual lines from the same genetic background also exhibit similar growth characteristics, hence they are especially valuable for comparative studies.
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References
Woodworth, C.D., Gaiotti, D., Michael, E., Hansen, L., and Nees, M. (2000) Targeted disruption of the epidermal growth factor receptor inhibits development of papillomas and carcinomas from human papillomavirus-immortalized keratinocytes. Cancer Res 60, 4397–402.
Jat, P.S., Noble, M.D., Ataliotis, P., Tanaka, Y., Yannoutsos, N., Larsen, L., and Kioussis, D. (1991) Direct derivation of conditionally immortal cell lines from an H-2 Kb-tsA58 transgenic mouse. Proc Natl Acad Sci USA 88, 5096–100.
DiPersio, C.M., Shao, M., Di Costanzo, L., Kreidberg, J.A., and Hynes, R.O. (2000) Mouse keratinocytes immortalized with large T antigen acquire alpha3beta1 integrin-dependent secretion of MMP-9/gelatinase B. J Cell Sci 113, 2909–21.
Lamar, J.M., Iyer, V., and DiPersio, C.M. (2008) Integrin alpha3beta1 potentiates TGFbeta-mediated induction of MMP-9 in immortalized keratinocytes. J Invest Dermatol 128, 575–86.
Reichelt, J., Breiden, B., Sandhoff, K., and Magin, T.M. (2004) Loss of keratin 10 is accompanied by increased sebocyte proliferation and differentiation. Eur J Cell Biol 83, 747–59.
Reichelt, J., Bussow, H., Grund, C., and Magin, T.M. (2001) Formation of a normal epidermis supported by increased stability of keratins 5 and 14 in keratin 10 null mice. Mol Biol Cell 12, 1557–68.
Reichelt, J., Furstenberger, G., and Magin, T.M. (2004) Loss of keratin 10 leads to mitogen-activated protein kinase (MAPK) activation, increased keratinocyte turnover, and decreased tumor formation in mice. J Invest Dermatol 123, 973–81.
Reichelt, J., and Magin, T.M. (2002) Hyperproliferation, induction of c-Myc and 14-3-3sigma, but no cell fragility in keratin-10-null mice. J Cell Sci 115, 2639–50.
Pasparakis, M., Courtois, G., Hafner, M., Schmidt-Supprian, M., Nenci, A., Toksoy, A., Krampert, M., Goebeler, M., Gillitzer, R., Israel, A., Krieg, T., Rajewsky, K., and Haase, I. (2002) TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2. Nature 417, 861–6.
Tscharntke, M., Pofahl, R., Chrostek-Grashoff, A., Smyth, N., Niessen, C., Niemann, C., Hartwig, B., Herzog, V., Klein, H.W., Krieg, T., Brakebusch, C., and Haase, I. (2007) Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1. J Cell Sci 120, 1480–90.
Watt, F.M. (1998) Cultivation of human epidermal keratinocytes with a 3T3 feeder layer. In: Celis, J.E., ed. Cell Biology: A Laboratory Handbook, Vol. 1. New York, Academic Press, pp. 113–8.
Todaro, G.J., and Green, H. (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17, 299–313.
Rheinwald, J.G. (1989) Methods for clonal growth and serial cultivation of normal human epidermal keratinocytes and mesothelial cells. In: Baserga, R., ed. Cell Growth and Division. A Practical Approach. IRL Press, Oxford, pp. 81–94.
Sugimachi, K., Sosef, M.N., Baust, J.M., Fowler, A., Tompkins, R.G., and Toner, M. (2004) Long-term function of cryopreserved rat hepatocytes in a coculture system. Cell Transplant 13, 187–95.
Kobayashi, C., Kagami, H., Kito, K., Ishikawa, K., Ebisawa, K., Ueda, M., and Terasaki, H. (2005) Selective and efficient culturing of retinal pigment epithelial cells using a feeder layer. Cytotherapy 7, 427–37.
Honda, M.J., Shimodaira, T., Ogaeri, T., Shinohara, Y., Hata, K., and Ueda, M. (2006) A novel culture system for porcine odontogenic epithelial cells using a feeder layer. Arch Oral Biol 51, 282–90.
Acknowledgements
We thank Semra Frimpong for excellent technical assistance and Dr. Penny Lovat and Dr. Anne Vollmers for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG).
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Reichelt, J., Haase, I. (2010). Establishment of Spontaneously Immortalized Keratinocyte Lines from Wild-Type and Mutant Mice. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 585. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-380-0_5
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DOI: https://doi.org/10.1007/978-1-60761-380-0_5
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