Abstract
High-efficiency gene transfer into mammalian fibroblasts is a routine procedure that can be performed by a variety of methods (1), but transfection of epithelial cells has been more difficult to achieve. Each of the standard techniques has associated problems; special equipment is required for electroporation (2) and also for direct microinjection (3). Protoplast fusion (4) is suitable only for cloned genes. Retroviral transduction of genes (5) is a method of high efficiency for animal cells, but again is applicable only to cloned genes of restricted size, and safety considerations are likely to restrict greatly the use of retroviral vectors suitable for human cells. Calcium phosphate coprecipitation has been used with success in several epithelial lines (6–8) and, more recently, strontium phosphate coprecipitation (9). These techniques are very simple to perform, require no special equipment, and can be applied to genomic DNA transfection.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pollard, J. W., Luqmani, Y., Bateson, A., and Chotai, K. (1984) DNA transformation of mammalian cells, in Methods in Molecular Biology, vol. 2 (Walker, J. M., ed.), Humana Press, Clifton NJ, pp. 321–332.
Tur-Kaspa, R., Teicher, L., Levine, B. J., Skoultchi, A. I., and Shafritz, D. A. (1986) Use of electroporation to introduce biologically active genes into primary rat hepatocytes. Mol. Cell Biol. 6, 716–718.
Garcia, I., Sordat, B., Rauccio-Farinon, E., Dunand, M., Kraehenbuhl, J.-P., and Diggelmann, H. (1986) Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes. Mol. Cell Biol. 6, 1974–1982.
Yoakum, G. H., Lechner, J. F., Gabrielson, E. W., Korba, B. E., Malan-Shibley, L., Willey, J. C., Valerio, M. G., Shamsuddin, A. M., Trump, B. F., and Harris, C. C. (1985) Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene. Science 22, 1174–1179.
Wolff, J. A., Yee, J.-K., Skelly, H. F., Moores, J. C., Respess, J. G., Friedmann, T., and Leffert, H. (1987) Expression of retrovirally transduced genes in primary cultures of adult rat hepatocytes. Proc. Natl. Acad. Sci. USA 84, 3344–3348.
Hynes, N. E., Jaggi, R., Kozma, S. C., Ball, R., Muellener, D., Wetherall, N. T., Davis, B. W., and Groner, B. (1985) New acceptor cell for transfected genomic DNA oncogene transfer into a mouse mammary epithelial cell line. Mol. Cell Biol. 5, 268–272.
Storer, R. D., Stein, R. B., Sina, J. F., DeLuca, J. G., Allen, H. L., and Bradley, M. O. (1986) Malignant transformation of a preneoplastic hamster epidermal cell line by the EJ c-Ha-ras oncogene. Cancer Res. 46, 1458–1464.
Summerhayes, I. C., Malone, P., and Visvanathan, K. (1986) Altered growth properties and cell surface changes in ras transformed mouse bladder epithelium. Int. J. Cancer 37, 233–240.
Brash, D. E., Reddel, R. R., Quanrud, M., Yang, K., Farrell, M. P., and Harris, C. C. (1987) Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells. Mol. Cell Biol. 7, 2031–2034.
Lemoine, N. R. and Wynford-Thomas, D. (1987) The thyroid follicular cell as a recipient for DNA transfection. Br. J. Cancer 55, 342.
Boffey, S. A. (1984) Plasmid DNA isolation by the cleared lysate method, in Methods in Molecular Biology, vol. 2 (Walker, J. M., ed.), Humana Press, Clifton, NJ, pp 177–183.
Kaiser, K. and Murray, N. E. (1985) The use of phage lambda replacement vectors in the construction of representative genomic DNA libraries, in DNA Cloning, vol. 1 (Glover, D. M., ed.), IRL, pp. 1–74.
Gluzman, Y., Sambrook, J. R., and Frisque, R. J. (1980) Expression of early genes of origin-defective mutants of SV40. Proc. Natl. Acad. Sci. USA 77, 3898–3902.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1997 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Lemoine, N.R., Wynford-Thomas, D. (1997). Transfection and Transformation of Human Thyroid Epithelial Cells. In: Pollard, J.W., Walker, J.M. (eds) Basic Cell Culture Protocols. Methods in Molecular Biology™, vol 75. Humana Press, Totowa, NJ. https://doi.org/10.1385/0-89603-441-0:441
Download citation
DOI: https://doi.org/10.1385/0-89603-441-0:441
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-441-9
Online ISBN: 978-1-59259-561-7
eBook Packages: Springer Protocols