Department of Biochemistry, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118, USA.
Methods. 2011 Mar;53(3):214-9. doi: 10.1016/j.ymeth.2010.11.005. Epub 2010 Nov 27.
Although a great deal of progress has been made in elucidating the molecular identity of the infectious agent in prion diseases, the mechanisms by which prions kill neurons, and the role of the cellular prion protein (PrP(C)) in this process, remain enigmatic. A window into the normal function of PrP(C), and how it can be corrupted to produce neurotoxic effects, is provided by a PrP deletion mutant called ΔCR, which produces a lethal phenotype when expressed in transgenic mice. In a previous study, we described the unusual observation that cells expressing ΔCR PrP are hyper-sensitive to the toxic effects of two cationic antibiotics (G418 and Zeocin) that are typically used for selection of transfected cell lines. We have used this drug-sensitizing effect to develop a simple Drug-Based Cell Assay (DBCA) that reproduces several features of mutant PrP toxicity observed in vivo, including the rescuing activity of wild-type PrP. In this paper, we present a detailed guide for executing the DBCA in several, different experimental settings, including a new slot blot-based format. This assay provides a unique tool for studying PrP cytotoxic and cytoprotective activities in cell culture.
尽管在阐明朊病毒疾病感染因子的分子特征、朊病毒杀死神经元的机制以及细胞朊蛋白（PrP(C)）在这一过程中的作用方面已经取得了很大进展，但仍有许多未解之谜。一种名为ΔCR 的 PrP 缺失突变体为研究 PrP(C) 的正常功能以及它如何被破坏产生神经毒性效应提供了一个窗口，当在转基因小鼠中表达时，该突变体会产生致命表型。在之前的一项研究中，我们描述了一个不寻常的观察结果，即表达 ΔCR PrP 的细胞对两种阳离子抗生素（G418 和 Zeocin）的毒性作用非常敏感，这两种抗生素通常用于转染细胞系的选择。我们利用这种药物敏感效应开发了一种简单的基于药物的细胞检测法（DBCA），该方法复制了体内观察到的突变型 PrP 毒性的几个特征，包括野生型 PrP 的拯救活性。在本文中，我们提供了在几种不同实验条件下执行 DBCA 的详细指南，包括一种新的基于斑点印迹的格式。该检测法为在细胞培养中研究 PrP 细胞毒性和细胞保护活性提供了独特的工具。
