Analysis of Growth Properties and Cell Cycle Regulation Using Mouse Embryonic Fibroblast Cells

A balance between proliferation and apoptosis is crucial for cellular homeostasis, and its disruption leading to enhanced cellular proliferation and uncontrolled growth are hallmarks of cancer. Genetic manipulation in the mouse offers a powerful approach to delineate the roles of genes in carcinogenesis and determine the molecular and cellular basis of their function. Mouse embryonic fibroblast cells derived from mice that are disrupted for tumor suppressors or oncogenes have served as an invaluable tool to study altered growth properties of cells and identify regulatory molecules involved in neoplastic transformation. In this chapter, protocols for isolation of mouse embryonic fibroblast cells from midgestation mouse embryos and their applications to study altered growth properties by growth curves and colony formation assays are provided. Methods to analyze cell cycle profiles by flow cytometry using bromodeoxyuridine and propidium iodide staining were also provided, entry of cells in S-phase by [³ H] thymidine incorporation studies, and the analysis of cells in mitosis by staining with antiphospho-H3 antibodies are also provided.