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  • Title: Conditional cell transformation by doxycycline-controlled expression of the ASV17 v-jun allele.
    Author: Bader AG, Hartl M, Bister K.
    Journal: Virology; 2000 Apr 25; 270(1):98-110. PubMed ID: 10772983.
    Abstract:
    To investigate the molecular basis of oncogenesis induced by the v-jun oncogene of avian sarcoma virus 17 (ASV17), we developed a conditional cell transformation system in which transcription of the ASV17 v-jun allele is controlled by a doxycycline-sensitive transactivator (tTA) or a reverse (doxycycline-dependent) transactivator (rtTA), respectively. Permanent cell lines of quail embryo fibroblasts conditionally transformed by a doxycycline-controlled v-jun allele revert to the normal phenotype within 3 days and lose their ability to grow in soft agar, strictly dependent on the addition or removal of the drug, respectively. The reverted cells are rapidly retransformed on conditional activation of v-jun. While full-level synthesis of v-jun mRNA and v-Jun protein in these cells is established within 2 and 14 h, respectively, after switching to the permissive conditions, the first morphological alterations are observed after 24 h, and as early as 2 days later the morphology has changed entirely from flat cells resembling normal fibroblasts to spindle-shaped fusiform cells showing a typical jun-transformed phenotype. Kinetic expression analysis revealed that transcriptional activation of the direct jun target gene BKJ precisely coincides with the establishment of full-level v-Jun protein synthesis. Furthermore, we have analyzed the expression of a novel candidate v-jun target gene, termed JAC, which shows no sequence homology to known genes. Similar to BKJ, JAC is specifically activated in jun-transformed fibroblasts, and induction of JAC is tightly linked to the conditional expression of oncogenic v-Jun. These results demonstrate the high stringency of the doxycycline-controlled v-jun expression system, and they also indicate that expression of v-jun in these cells is indispensable for enhanced proliferation, cell transformation, and the induction of specific expression patterns of downstream target genes.
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