A comparison of strategies for immortalizing mouse embryonic fibroblasts
The genetically amenable mouse model has led to a large collection of genetically defined lines from which mouse embryonic fibroblasts (MEFs) have been derived. Despite their widespread use, MEFs are time consuming to generate and have a limited lifespan. Immortalizing primary MEFs with the desired genetic manipulations greatly reduces culture maintenance time, enables the generation of near limitless amounts of protein lysate, and facilitates biological replicates during experimentation. In this work, we have evaluated several approaches for MEF immortalization. When cultivated at 3% O2, some primary MEF lines could be proliferated for > 40 passages with a median doubling rate of 45 ± 55 h (n = 8). However, serial passaging at 3% O2 achieved spontaneous immortalization with varying success. If cultures seemed to be reaching their Hayflick limit when cultivated at 3% O2, supplementing the culture media with 5 µM ROCK inhibitor Y-27632 helped to extend proliferation and achieve spontaneous immortalization. MEFs immortalized via SV40 Ta infection reliably produced cell lines with a median doubling rate of 25 ± 9 h (n = 9) and viability greater than 90%. In addition to a discussion of the characteristics of cell lines generated with various immortalization strategies, pros and cons of each strategy are included as are recommendations for generating immortalized MEFs.
Hayflick L (1965) Limited in vitro lifetime of human diploid cell strains. Experimental Cell Research 37(3):614-636.
2. Xu J (2005) Preparation, culture, and immortalization of mouse embryonic fibroblasts. Current protocols in molecular biology / edited by Frederick M Ausubel [et al], Chapter 28:Unit 28.21.
3. Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J (2003) Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nature Cell Biology 5(8):741-747.
4. Di Micco R, Cicalese A, Fumagalli M, Dobreva M, Verrecchia A, et. al. (2008) DNA damage response activation in mouse embryonic fibroblasts undergoing replicative senescence and following spontaneous immortalization. Cell Cycle 7(22):3601-3606.
5. Chapman S, Liu XF, Meyers C, Schlegel R, McBride AA (2010) Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. Journal of Clinical Investigation 120(7):2619-2626.
6. Liu XF, Ory V, Chapman S, Yuan H, Albanese C, et al. (2012) ROCK Inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. American Journal of Pathology 180(2):599-607.
7. Palechor-Ceron N, Suprynowicz FA, Upadhyay G, Dakic A, Minas T, Simic V, et. al. (2013) Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK inhibitor to conditionally reprogram and immortalize epithelial cells. American Journal of Pathology 183(6):1862-1870.
8. Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, et al. (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology 25(6):681-686.
9. Ali SH, DeCaprio JA (2001) Cellular transformation by SV40 large T antigen: interaction with host proteins. Seminars in Cancer Biology 11(1):15-22.
10. Singh SV, Srivastava SK, Choi S, Lew KL, Antosiewicz J, et al. (2005) Sulforaphane-induced cell death in human prostate cancer cells is initiated by reactive oxygen species. Journal of Biological Chemistry 280(20):19911-19924.
11. Wagoner KL, Bader RA (2012) Evaluation of SV40-transformed synovial fibroblasts in the study of rheumatoid arthritis pathogenesis. Rheumatology International 32(7):1885-1891.
12. Oh HY, Jin X, Kim JG, Oh MJ, Pian X, et al. (2007) Characteristics of primary and immortalized fibroblast cells derived from the miniature and domestic pigs. Bmc Cell Biology 8:8.
13. Kohno S, Murata T, Koide N, Hikita K, Kaneda N (2011) Establishment and characterization of a noradrenergic adrenal chromaffin cell line, tsAM5NE, immortalized with the temperature-sensitive SV40 T-antigen. Cell Biology International 35(4):325-334.
14. Liu SH, Hatton MP, Khandelwal P, Sullivan DA (2010) Culture, immortalization, and characterization of human meibomian gland epithelial cells. Investigative Ophthalmology and Visual Science 51(8):3993-4005.
15. Lundberg AS, Randell SH, Stewart SA, Elenbaas B, Hartwell KA, et al. (2002) Immortalization and transformation of primary human airway epithelial cells by gene transfer. Oncogene 21(29):4577-4586.
16. Jeong YK, Kang JS, Kim JW, Suh SK, Lee M, et al. (2009) Differences in gene expression profiles reflecting differences in drug sensitivity to acetaminophen in normal and transformed hepatic cell lines in vitro. Molecular and Cellular Toxicology 5(1):32-43.
17. Benchimol S, Pim D, Crawford L (1982) Radioimmunoassay of the cellular protein P 53 in mouse and human cell-lines. Embo Journal 1(9):1055-1062.
18. Cawthon RM (2002) Telomere measurement by quantitative PCR. Nucleic Acids Research 30(10):e47.