Use of non-radioactive 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 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.
Rihn
B
B
Institut National de Recherche et de Sécurité, Vandoeuvre, France.
Bottin
M C
MC
Coulais
C
C
Zissu
D
D
Edorh
A
A
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D015870:Gene Expression; D011905:Genes, ras; D005821:Genetic Techniques; D017403:In Situ Hybridization; D008297:Male; D008325:Mammary Neoplasms, Experimental / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011868:Radioisotopes
D017931:DNA Primers; D004273:DNA, Neoplasm; D011868:Radioisotopes