Sun Hong, Taneja Reshma
Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York, NY, USA.
Methods Mol Biol. 2007;383:303-10. doi: 10.1007/978-1-59745-335-6_19.
An important step in cellular transformation and tumorigenesis is immortalization, in which cells gain the ability to grow indefinitely by bypassing cellular senescence that imposes a finite number of divisions in culture. Primary mouse embryonic fibroblast (MEF) cells have a limited growth capacity and on prolonged passaging spontaneously immortalize at a low frequency. In contrast to transformation of primary MEF cells that requires the presence of two cooperating oncogenes, immortalized MEF cells can be transformed by a single oncogene (Ras) resulting in a loss of contact inhibition, anchorage-independent growth, and tumor formation in nude mice. Studies of MEF cells have played an important role in the elucidation of the molecular mechanisms underlying cellular immortalization, transformation, and tumorigenesis. Additionally, utilization of MEF cells disrupted for specific genes has provided a powerful tool to analyze the genetic regulation of these cellular processes. In this chapter, methods for analysis of cellular immortalization using the 3T3 protocol, as well as transformation of MEF cells using oncogenic retroviruses are provided. This is followed by protocols for analysis of transformed cell characteristics such as foci formation, anchorage independent growth, and tumor formation in nude mice.
细胞转化和肿瘤发生过程中的一个重要步骤是永生化，即细胞通过绕过细胞衰老获得无限生长的能力，细胞衰老会在培养中限制细胞分裂的次数。原代小鼠胚胎成纤维细胞（MEF）生长能力有限，长时间传代后会以低频率自发永生化。与需要两个协同癌基因才能转化原代MEF细胞不同，永生化的MEF细胞可被单个癌基因（Ras）转化，导致接触抑制丧失、不依赖贴壁生长，并在裸鼠体内形成肿瘤。对MEF细胞的研究在阐明细胞永生化、转化和肿瘤发生的分子机制方面发挥了重要作用。此外，利用特定基因缺失的MEF细胞提供了一个强大的工具来分析这些细胞过程的基因调控。在本章中，提供了使用3T3方案分析细胞永生化以及使用致癌逆转录病毒转化MEF细胞的方法。随后是分析转化细胞特征的方案，如集落形成、不依赖贴壁生长和在裸鼠体内形成肿瘤。
