USE OF NONRADIOACTIVE METHODS FOR THE DETERMINATION OF THE EXPRESSION, THE SEQUENCE AND THE COPY-NUMBER OF TRANSGENE IN MICE

Marshall's observation that ''toxicology goes molecular'', is turning out to be more true than ever; namely as it is observed that toxicolog ical 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 genet ically engineered animals such as p53 deficient mice, or mice carrying the v-Ha-ras oncogene. The latter were used in our laboratory for suc h toxicological assessments of chemicals. In order to verify that the transgene was expressed in normal, as well as in tumor cells, the tran sgene was detected in different tissues fixed with various solutions u sing in situ hybridization. It was also specifically retrotranscribed from paraffin-embedded tissues and consequently sequenced using a Tag polymerase reaction. We found that the transgene was expressed in vari ous organs. It carries a specific mutation of codon 12 leading to the activation of its encoded product (transducin: p21(v-Ha-ras)). Moreove r using a laser scanning densitometer, it has been demonstrated that 2 to 3 copies of the transgene were present per genome-equivalent in so me tissues. All experiments were realized using non-radioactive labell ing and detection (chemiluminescent or colorigenic) methods. Indeed, t he screening of such animals was realized in a easier and a safer mann er using the methods described in this paper than the usual methods ba sed on the use of radiolabelled precursors.