Rogers B J, Provost G S, Young R R, Putman D L, Short J M
Stratagene, La Jolla, CA 92037.
Mutat Res. 1995 Mar;327(1-2):57-66. doi: 10.1016/0027-5107(94)00081-f.
A lambda/lacI shuttle vector transgenic mouse mutagenesis assay has been optimized and standardized for reproducible mutant detection. The mutagenic endpoints are blue lacI- phage plaques on a bacterial lawn resulting from the de-repression of beta-galactosidase activity acting on the chromogenic substrate X-gal. Non-mutant lacI phage plaques remain colorless. Factors demonstrated to affect mutant detection include X-gal concentration per assay tray, plaque density per assay tray, pH of plating agar, incubation time at 37 degrees C and the use of a red translucent screening filter over a light source to enhance mutant plaque visibility. In vivo mutant frequencies for liver in untreated animals using standard protocols and internal controls were repeatable in separate experiments using lambda/lacI B6C3F1 mice (4.3 +/- 1.2 x 10(-5) and 4.1 +/- 0.8 x 10(-5)). These studies analyze the use of internal controls to monitor the level of mutant phage plaque detection in a given experiment and evaluate the repeatability of observed mutant frequencies obtained when using standardized procedures.
已对λ/lacI穿梭载体转基因小鼠诱变试验进行了优化和标准化，以实现可重复的突变体检测。诱变终点是在细菌菌苔上出现的蓝色lacI-噬菌体噬菌斑，这是由于作用于显色底物X-gal的β-半乳糖苷酶活性去阻遏所致。非突变型lacI噬菌体噬菌斑仍为无色。已证明影响突变体检测的因素包括每个试验平板的X-gal浓度、每个试验平板的噬菌斑密度、平板琼脂的pH值、37℃下的孵育时间以及在光源上方使用红色半透明筛选滤光片以提高突变体噬菌斑的可见度。使用标准方案和内部对照，未处理动物肝脏的体内突变频率在使用λ/lacI B6C3F1小鼠的单独实验中是可重复的(4.3±1.2×10⁻⁵和4.1±0.8×10⁻⁵)。这些研究分析了在给定实验中使用内部对照来监测突变体噬菌体噬菌斑检测水平，并评估了使用标准化程序时观察到的突变频率的可重复性。
