Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene
Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene
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DOI：
10.3791/51742
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发表时间：
2014-12-01
影响因子：
1.2
通讯作者：
Diaz, Elva
中科院分区：
文献类型：
--
作者：
Alvarez, Angel;Barisone, Gustavo A.;Diaz, Elva
细胞的恶性转化通常与实验动物中增殖增加、接触抑制丧失、获得锚定非依赖性生长潜力和形成肿瘤的能力相关（1）。在NIH 3T3细胞中，已知Ras信号转导途径触发许多这些事件，即所谓的Ras转化。在NIH 3T3细胞中引入过表达的基因可以促进形态转化和接触抑制的丧失，这可以帮助确定感兴趣的基因的致癌潜力。病灶形成试验（FFA）（2）提供了一种简单的方法来评估癌基因转化潜力的一个方面。当NIH 3T3细胞在培养物中正常分裂时，它们会这样做，直到达到融合的单层。然而，在存在过表达的癌基因的情况下，这些细胞可以开始在致密的多层病灶中生长（1），其可以通过结晶紫或Hema 3染色来可视化和定量。在这篇文章中，我们描述了FFA协议与逆转录病毒转导的目的基因到NIH 3T3细胞，以及如何量化灶的数量，通过染色。逆转录病毒转导提供了一种比转染更有效的基因递送方法，并且当与潜在的人类致癌基因一起工作时，使用亲嗜性鼠逆转录病毒提供了生物安全控制。
Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals(1). In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)(2). When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci(1) that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.