Besaratinia Ahmad, Tommasi Stella
Department of Preventive Medicine, USC Keck School of Medicine, University of Southern California;
Department of Preventive Medicine, USC Keck School of Medicine, University of Southern California.
J Vis Exp. 2018 Apr 26(134):57510. doi: 10.3791/57510.
A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (λ) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.
已经开发了许多转基因动物模型和突变检测系统，用于哺乳动物细胞中致癌物的致突变性测试。其中，转基因小鼠和λ选择cII突变检测系统已被全球许多研究小组用于致突变性实验。在此，我们描述了λ选择cII突变测定的详细方案，该方案可应用于转基因小鼠/大鼠的培养细胞或用感兴趣的化学/物理剂处理的相应动物。该方案包括以下步骤：(1) 分别从体外或体内用测试化合物处理的转基因动物的细胞或器官/组织中分离基因组DNA；(2) 从基因组DNA中回收携带突变报告基因（即cII转基因）的λ穿梭载体；(3) 将拯救的载体包装成感染性噬菌体；(4) 感染宿主细菌并在选择性条件下培养，以允许诱导的cII突变繁殖；(5) 对cII突变体进行评分并进行DNA序列分析，以分别确定cII突变频率和突变谱。
