Visualization of the Specific Gene Transcription in the Nucleus with a Novel In Situ RNase Protection Method.

Abstract

The ordinary in situ hybridization technique, using the labeled DNA or RNA probes, visualizes the mRNA species that have previously been transcribed and accumulated in the cytoplasm. In contrast, we aimed to visualize the transcription of particular genes itself by use of the hybridization between radioisotope−labeled newly transcribed cellular RNA and unlabeled RNA probes(riboprobes) followed by digestion of unhybridized RNA with RNase. In the present study, the cultured mouse 3T3−L1 cell line with or without the induction for the adipocyte was labeled with 3H-uridine, fixed and was hybridized in situ with the complementary(antisense) or homologous(sense) riboprobe for 28S rRNA or glycerol−3−phosphate dehydrogenase(GPDH) mRNA, an adipocyte marker. Following extensive digestion with RNase, the signal for remaining radioactivity in nuclei was quantitatively analyzed with autoradiography. In the uninduced cells hybridized with 28S rRNA probes, a significantly stronger signal was obtained with the complementary probe than with the homologous probe. With respect to GPDH probes, the adipocyte−induced cells showed a significantly stronger signal with the complementary probe than with the homologous probe, whereas the uninduced cells showed no significant difference in signal with both probes. These results demonstrated the possibility of visualizing the transcription of particular genes in particular cells at particular time point by applying “in situ RNase protection” to histological specimens.