Preparation and Immortalization of Primary Murine Cells
The generation of immortal cell lines from genetically defined mice has proven useful in the understanding of molecular pathways in mammalian biology. This article describes the preparation and immortalization of murine embryo fibroblasts (MEFs), the workhorse cell type of genetically defined mice. These cells are used most frequently because of their ease of preparation, relative homogeneity, and high frequency of immortalization with serial passage in culture. These techniques can be used, with modification, to immortalize rat embryo fibroblasts as well. Furthermore, variations of these techniques can be applied to other murine cell types (e.g., astrocytes or keratinocytes) for the study of cell-type specific pathways (see Section V).
The genetics of immortalization in murine cells are now largely understood [Fig. 1, reviewed in Sharpless and DePinho (1999)]. As opposed to human cells, the spontaneous immortalization of cultured rodent cells occurs with high frequency resulting from a stochastic genetic event. This difference in frequency is attributable to the additional requirement in human cells for telomerase expression as detailed in the previous article. In murine cells, the act of cell culture, with a few exceptions, potently induces both antiproliferative products (p16INK4a and p19ARF) of the Ink4a/Arf locus, which regulate the Rb and p53 pathways, respectively (Fig. 1). Immortalization of murine cells requires circumventing at least p19ARF-p53 or, in some cell types, both of the pathways regulated by the products of the Ink4a/Arf locus (for examples, see Bachoo et al., 2002; Kamijo et al., 1997; Randle et al., 2001). This can be done in one of three ways: spontaneous, stochastic genetic deletion through serial culture; the use of immortalizing oncogenes; or the use of genetically defined mice. This article deals predominantly with the former method, the latter two are described in Section V.
-Sterile 10-cm dishes (Falcon Cat. No. 353003)
-Sterile 6-cm dishes (Falcon Cat. No. 353002)
-Sterile phosphate-buffered saline (PBS, GIBCO Cat. No. 14190-144)
-100 and 70% ethanol
-0.25% trypsin-EDTA (GIBCO Cat. No. 25200-056).
-DMEM (with glucose and L-glutamine, GIBCO Cat. No. 11995-065)
-Heat-inactivated fetal calf serum (FCS, Sigma Cat. No. F-2442; to heat inactivate, thaw and then place in water bath at 56°C for 30min)
-100× penicillin/streptomycin (P/S 10,000 units/ml, GIBCO Cat. No. 15140-122)
-Optional: β-mercapoethanol (β-ME, Sigma Cat. No. M-7522)
-Liquid nitrogen-safe cryotybes (Corning Cat. No. 2028)
-Dimethyl Sulfoxide (DMSO, Mallinkrodt AR Cat. No. 4948)
III. INSTRUMENTATION (ALL STERILE)
- 6 ¾in. Mayo scissors (Fisher Cat. No. 13-804-8)
- Large forceps (Fisher Cat. No. 13-812-36)
- Curved fine scissors (Fisher Cat. No. 08-951-10)
- Two curved small forceps (Fisher Cat. No. 08-953D)
- Two 5 ½ in. Kelly clamps (Fisher Cat. No. 08-907)
- 9-in. cotton-plugged Pasteur pipettes (Fisher Cat. No. 13-678-8B).
- Razor blades (place in 100% ethanol in 15-m dish prior to use)
A. Murine Embryo Fibroblast Production
B. Thawing MEFs
C. Immortalizing MEFs
This can be done using a rigorously defined passaging protocol (e.g., 3T3 or 3T9 assay) or, more simply, by serial replating of cells. The advantage of the passaging protocol is that one learns about the growth kinetics and immortalization frequency of the cell (see Section V). The immortalization of wild-type MEFs requires a stochastic genetic event: generally p53 loss or, less frequently, loss of p19ARF (Kamijo et al., 1997). Other immortalizing events, such as mdm2 amplification (Olson et al., 1993), have also been noted, but these are considerably rarer. In our experience, this genetic event is most likely to be loss of p53 (~60-70% of immortalized lines) o r p19ARF (~20-30%) regardless of growth conditions (passage at high or low density). Therefore, serial passaging without counting cells at every passage is an appropriate way to derive immortalized cells. In fact, the "3T3 cell" has become a nondescript term referring to immortal murine embryo fibroblasts obtained through serial passaging, but few laboratories except those interested in growth and senescence do rigorous 3T3 assays to obtain them.
D. Immortalization by Serial Passage
E. 3T9 Assay
This assay was originally described by Todaro and Green (1963). The "3" refers to passaging every 3 days, and the "9" refers to 9 x 105 cells plated per passage. 3T3 (3 x 105 cells) and 3T12 (1.2 x 10 6 cells) assays can also be done to study growth and immortalization at varying densities. In our experience, immortalization frequency is highest for the 3T3 assay (and therefore this assay has lent its name to all immortalized lines of murine fibroblasts), whereas 3T9 is more useful for measuring immortalization frequency (Sharpless et al., 2001). As stated earlier, serial replating of murine fibroblasts at almost any density will eventually yield immortalized lines as long as the cells are not seeded too sparsely.
A. Immortalization through the Use of Oncogenes or Genetic Background
A problem with immortalization by serial passage is that the nature of the immortalizing genetic event is not known, although predominantly p53 or p19ARF is inactivated as MEFs escape senescence. As the behavior of p19ARF-deficient cells may be quite different from p53-deficient cells, however, the stochastic inactivation of these pathways may produce significant line-to-line variability and therefore be undesirable. For example, p53 null MEFs are more aneuploid and more resistant to DNA damage than p19ARF null MEFs (Kamijo et al., 1997; Pomerantz et al., 1998; Serrano et al., 1996; Stott et al., 1998). To assure that all lines will evade senescence via a similar mechanism, cells can either be immortalized through the use of an immortalizing oncogene or by using cells derived from animals of a genetic background that resists senescence.
Classically, the most commonly used oncoprotein to immortalize cells is the SV40 large T antigen (Colby and Shenk, 1982; Jat and Sharp, 1986; Todaro and Green, 1966). This molecule inactivates the p53 and Rb pathway, and the majority of murine cell types can be immortalized by transfection or retroviral transduction of TAg. A disadvantage of TAg, however, is that cells expressing this molecule are unstable and are prone to clonal in vitro evolution. Furthermore, as Rb pocket proteins are required for the differentiation of many cells types (Dannenberg et al., 2000), TAg generally impairs or precludes the study of differentiation. Alternatively, MEFs and several other murine cell types can be immortalized with a dominant-negative form of p53 [e.g., p53-DD (Bowman et al., 1996)], which preserves the Rb pathway, although these cells are still more prone to aneuploidy than p19ARF-deficient lines.
The most elegant method of obtaining immortal cell lines is by deriving cells from animals resistant to senescence. The most commonly used strains for this purpose are Ink4a/Arf-deficient (Serrano et al., 1996), p19ARF-deficient (Kamijo et al., 1997), or p53-deficient (Donehower et al., 1992) mice. These strains are widely available and can be obtained from the mouse models of the human cancer consortium (MMHCC http: / / web. ncifcrf, gov / researchresources / mmhcc/). For studies of genetically defined animals, the genetic background of interest can be crossed two generations to mice of these backgrounds and then MEFs (or other cell types) derived as described earlier, which will be immortal in most cases if derived from p53- or p19ARF-deficient embryos. This method can be employed to derive immortal cells from difficult genetic backgrounds that undergo premature senescence in culture (Frank et al., 2000; Jacobs et al., 1999). This strategy can also be employed to obtain cells of nonfibroblast lineages. For example, immortal melanocytes (Chin et al., 1997), skin keratinocytes (unpublished observations), glia (Bachoo et al., 2002), lymphocytes (unpublished observations; Randle et al., 2001), and macrophages (Randle et al., 2001) have been derived successfully from p19ARF- or Ink4a/Arf-deficient mice using standard culture methods for these cell types. To immortalize with high efficiency, cells must be homozygous null for p53, p19ARF, or Ink4a/Arf; therefore, the principal disadvantage of this approach is the extra time needed to backcross to these defined genetic backgrounds.
B. Data Analysis of 3T9 or 3T3 Assay
These data can be plotted as cell number per passage or population doublings (PDs) per passage (Fig. 2). PDs for any given passage = log2(Nf/No) = 1.44* In(Nf/No), where Nf = number of cells counted at the end of the passage and No= number of cells seeded at the beginning of the passage (i.e., 9 × 105 for a 3T9 assay). For the purpose of immortalization frequency, "senescence" occurs if less than 9 × 105 cells are recovered for two consecutive passages. The immortalization frequency = 1-number of senescent lines/total number of lines analyzed. Measured in this way, the immortalization frequency of wild-type MEFs can vary significantly depending on culture conditions, method of embryo preparation, and so on and therefore is only meaningful when compared to proper littermate control embryos analyzed concurrently.
Bachoo, R. M., Maher, E. A., Ligon, K. L., Sharpless, N. E., Chan, S. S., You, M. J., Tang, Y., DeFrances, J., Stover, E., Weissleder, R., et al. (2002). Epidermal growth factor receptor and Ink4a/Arf: Convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. Cancer Cell 1, 269-277.
Bowman, T., Symonds, H., Gu, L., Yin, C., Oren, M., and Van Dyke, T. (1996). Tissue-specific inactivation of p53 tumor suppression in the mouse. Genes Dev. 10, 826-835.
Chin, L., Pomerantz, J., Polsky, D., Jacobson, M., Cohen, C., Cordon- Cardo, C., Horner, J. W., II, and DePinho, R. A. (1997). Cooperative effects of INK4a and ras in melanoma susceptibility in vivo. Genes Dev. 11, 2822-2834.
Colby, W. W., and Shenk, T. (1982). Fragments of the simian virus 40 transforming gene facilitate transformation of rat embryo cells. Proc. Natl. Acad. Sci. USA 79, 5189-5193.
Dannenberg, J. H., van Rossum, A., Schuijff, L., and te Riele, H. (2000). Ablation of the retinoblastoma gene family deregulates G(1) control causing immortalization and increased cell turnover under growth- restricting conditions. Genes Dev. 14, 3051- 3064.
Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Jr., Butel, J. S., and Bradley, A. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215-221.
Frank, K. M., Sharpless, N. E., Gao, Y., Sekiguchi, J. M., Ferguson, D. O., Zhu, C., Manis, J. P., Horner, J., DePinho, R. A., and Alt, E W. (2000). DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway. Mol. Cell 5, 993-1002.
Jacobs, J. J., Kieboom, K., Marino, S., DePinho, R. A., and van Lohuizen, M. (1999). The oncogene and Polycomb-group genebmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature 397, 164-168.
Jat, P. S., and Sharp, P. A. (1986). Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts. J. Virol. 59, 746-750.
Kamijo, T., van de Kamp, E., Chong, M. J., Zindy, E, Diehl, J. A., Sherr, C. J., and McKinnon, P. J. (1999). Loss of the ARF tumor suppressor reverses premature replicative arrest but not radiation hypersensitivity arising from disabled atm function. Cancer Res. 59, 2464-2469.
Kamijo, T., Zindy, E, Roussel, M. E, Quelle, D. E., Downing, J. R., Ashmun, R. A., Grosveld, G., and Sherr, C. J. (1997). Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARE Cell 91, 649-659.
Olson, D. C., Marechal, V., Momand, J., Chen, J., Romocki, C., and Levine, A. J. (1993). Identification and characterization of multiple mdm-2 proteins and mdm-2-p53 protein complexes. Oncogene 8, 2353-2360.
Pomerantz, J., Schreiber-Agus, N., Liegeois, N. J., Silverman, A., Alland, L., Chin, L., Potes, J., Chen, K., Orlow, I., Lee, H. W., et al. (1998). The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell 92, 713-723.
Randle, D. H., Zindy, E, Sherr, C. J., and Roussel, M. E (2001). Differential effects of p19(Arf) and p16(Ink4a) loss on senescence of murine bone marrow-derived preB cells and macrophages. Proc. Natl. Acad. Sci. USA 98, 9654-9659.
Serrano, M., Lee, H., Chin, L., Cordon-Cardo, C., Beach, D., and DePinho, R. A. (1996). Role of the INK4a locus in tumor suppression and cell mortality. Cell 85, 27-37.
Sharpless, N. E., Bardeesy, N., Lee, K. H., Carrasco, D., Castrillon, D. H., Aguirre, A. J., Wu, E. A., Horner, J. W., and DePinho, R. A. (2001). Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 413, 86-91.
Sharpless, N. E., and DePinho, R. A. (1999). The INK4A/ARF locus and its two gene products. Curr. Opin. Genet. Dev. 9, 22-30.
Sherr, C. J., and DePinho, R. A. (2000). Cellular senescence: Mitotic clock or culture shock? Cell 102, 407-410.
Stott, E J., Bates, S., James, M. C., McConnell, B. B., Starborg, M., Brookes, S., Palmero, I., Ryan, K., Hara, E., Vousden, K. H., and Peters, G. (1998). The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2. EMBO J. 17, 5001-5014.
Todaro, G. J., and Green, H. (1963). Quantitative Studies of mouse embyo cells in cutture and their development into established linos. J. Cell Biol. 17, 299-313.
Todaro, G. J., and Green, H. (1966). High frequency of SV40 transformation of mouse cell line 3T3. Virology 28, 756-759.
© 2018 Biocyclopedia | All rights reserved.