Rihn B, Bottin M C, Coulais C, Zissu D, Edorh A
Institut National de Recherche et de Sécurité, Vandoeuvre, France.
Cell Mol Biol (Noisy-le-grand). 1995 Nov;41(7):907-15.
Marshall's observation that "toxicology goes molecular", is turning out to be more true than ever; namely as it is observed that toxicological endpoints are the point of interaction between proteins and genes following the administration of toxicants. Transgenic mice represent a valuable tool for studying the adverse effects of chemicals in genetically engineered animals such as p53 deficient mice, or mice carrying the v-Ha-ras oncogene. The latter were used in our laboratory for such toxicological assessments of chemicals. In order to verify that the transgene was expressed in normal, as well as in tumor cells, the transgene was detected in different tissues fixed with various solutions using in situ hybridization. It was also specifically retrotranscribed from paraffin-embedded tissues and consequently sequenced using a Taq polymerase reaction. We found that the transgene was expressed in various organs. It carries a specific mutation of codon 12 leading to the activation of its encoded product (transducin: p21v-Ha-ras). Moreover using a laser scanning densitometer, it has been demonstrated that 2 to 3 copies of the transgene were present per genome-equivalent in some tissues. All experiments were realized using non-radioactive labelling and detection (chemiluminescent or colorigenic) methods. Indeed, the screening of such animals was realized in a easier and a safer manner using the methods described in this paper than the usual methods based on the use of radiolabelled precursors.
马歇尔提出的“毒理学走向分子层面”这一观点，如今已比以往任何时候都更具真实性；也就是说，正如人们所观察到的，毒理学终点是毒物给药后蛋白质与基因之间的相互作用点。转基因小鼠是研究化学物质对基因工程动物（如p53缺陷小鼠或携带v-Ha-ras癌基因的小鼠）不良影响的宝贵工具。后者在我们实验室被用于此类化学物质的毒理学评估。为了验证转基因在正常细胞以及肿瘤细胞中是否表达，我们使用原位杂交技术在不同固定液固定的组织中检测转基因。还从石蜡包埋组织中特异性逆转录，并随后使用Taq聚合酶反应进行测序。我们发现转基因在各个器官中均有表达。它携带密码子12的特定突变，导致其编码产物（转导素：p21v-Ha-ras）被激活。此外，使用激光扫描密度计已证明，在某些组织中，每个基因组当量存在2至3个转基因拷贝。所有实验均使用非放射性标记和检测（化学发光或显色）方法进行。事实上，与基于使用放射性标记前体的常规方法相比，使用本文所述方法以更简便、更安全的方式对这类动物进行筛选。
