Abstract
Primary mouse embryonic fibroblasts (PMEFs) have a number of properties that make them an attractive cell culture model. Relative to other primary explant cultures they are easy to establish and maintain, proliferate rapidly and, as a result, large numbers of cells can be produced from a single embryo within several days following explantation. This allows, for instance, for ready comparison of wild-type and knockout cells derived from the same litter of animals. PMEFs can be expanded through several passages before they reach crisis and can be used to establish cell lines following spontaneous transformation or following derivation from strains carrying mutations, such as in the gene encoding the tumour suppressor Trp53. They have been widely used as feeders to support other cultured cell types, notably embryonic stem cells, as well as for the study of a diverse range of cellular phenomena using microscopic, biochemical and molecular biological techniques. Here, we describe a simple and reliable method for the derivation and maintenance of PMEFs.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Keshishian, H. (2004) Ross Harrison’s “The outgrowth of the nerve fiber as a model of protoplasmic movement”. J. Exp. Zool. 301A, 201–203.
Van Gansen, P., and Van Lerberghe, N. (1988) Potential and limitations of cultivated fibroblasts in the study of senescence in animals. A review of the murine skin fibroblast system. Arch. Gerontol. Geriatr. 7, 31–74.
Hoki, Y., Araki, R., Fujimori, A., Ohhata, T., Koseki, H., Fukumura, R., Nakamura, M., Takahashi, H., Noda, Y., Kito, S., and Abe, M. (2003) Growth retardation and skin abnormalities of the Recql4-deficient mouse. Hum. Mol. Genet. 12, 2293–2299.
Harvey, M., Sands, A. T., Weiss, R. S., Hegi, M. E., Wiseman, R. W., Pantazis, P., Giovanella, B. C., Tainsky, M. A., Bradley, A., and Donehower, L. A. (1993) In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice. Oncogene 8, 2257–2267.
Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Butel, J. S., and Bradley, A. (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215–221.
Puck, T. T., and Marcus, P. I. (1955) A rapid method for viable cell titration and clone production with HeLa cells in tissue culture: the use of X-irradiated cells to supply conditioning factors. Proc. Natl. Acad. Sci. (USA) 41, 432–437.
Pantoja, C., de los Rios, L., Matheu, A., Antequera, F., and Serrano, M. (2005) Inactivation of imprinted genes induced by cellular stress and tumourigenesis. Cancer Res. 65, 26–33.
Green, A. E., Athreya, B., Lehr, H. B., and Coriell, L. L. (1967) Viability of cell cultures following extended preservation in liquid nitrogen. Proc. Soc. Exp. Biol. Med. 124, 1302–1307.
Lovelock, J. E., and Bishop, M. W. H. (1983) Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature 183, 1394–1395.
Karrow, A. M., Jr. (1969) Cryoprotectants – a new class of drugs. J. Pharm. Pharmacol. 21, 209–223.
Leibo, S. P., and Mazur, P. (1971) The role of cooling rates in low-temperature preservation. Cryobiology 8, 447–452.
Acknowledgements
I would like to thank Dr. A Ward, in whose lab these techniques have been utilised and for his assistance on the production of this chapter. Special thanks must also go to both Dr. Robert Kelsh and Dr. Ian Jones for their valued assistance with the microscopy and to Dr. David Tosh for the gift of the α-tubulin antibody.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Garfield, A.S. (2010). Derivation of Primary Mouse Embryonic Fibroblast (PMEF) Cultures. In: Ward, A., Tosh, D. (eds) Mouse Cell Culture. Methods in Molecular Biology, vol 633. Humana Press. https://doi.org/10.1007/978-1-59745-019-5_2
Download citation
DOI: https://doi.org/10.1007/978-1-59745-019-5_2
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-772-3
Online ISBN: 978-1-59745-019-5
eBook Packages: Springer Protocols