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Preparation of primary cultures of embryo fibroblasts from genetically engineered mouse strains can provide a valuable resource for analyzing the consequences of genetic alterations at the cellular level. Mouse embryo fibroblasts (MEFs) have been particularly useful in cancer research, as they have facilitated the identification of the genetic changes that allow cells to overcome senescence and proliferate indefinitely in culture. The immortalized MEFs can then acquire additional mutations that lead to anchorage-independent growth and the ability to form tumors in mice. Recently we developed an MEF model system for analysis of the role of the tumor suppressor gene DLC1 in cellular transformation (Qian et al., 2012). In this communication we describe a protocol for the isolation of MEFs from day 13.5-day 14.5 mouse embryos. The MEFs obtained by this procedure are suitable for use in biochemical assays and for further genetic manipulations.
Note: Depending on the viability of the particular strain, 6–10 embryos can be expected from each pregnant female, and they should yield enough MEFs for several experiments.
Note: Scissors and forceps can be placed in inverted Petri dish lids when not in use, and they can be cleaned by wiping with gauze pads wetted with 70% ethanol.
Note: if some cell lysis has occurred, the cell suspension may be viscous, and it may not be possible to avoid transferring the larger fragments to the flasks. This is not a major problem, since some fibroblasts will migrate out from the tissue fragments and attach to the dish.
Note: Some protocols call for 20% fetal bovine serum in the freezing medium, but 10% has worked for us.
This protocol was based on the method of Todaro and Green (1963) and subsequent modifications of the technique, as described in publications such as Coats et al. (1999). This work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health.