Exogenous sequences are frequently silenced following random integration into the genomes of many organisms. """"""""Trans-sensing"""""""", first described in plants and more recently in Drosophila, describes a class of phenomena that share the common feature of one gene sensing the presence of its homologue in trans. Perhaps as a result of the lack of appropriate genetic assays, Trans-sensing has yet to be described in mammals. We propose to develop a FACS-based dual reporter gene/selectable marker system, using the Green Fluorescent Protein and the cell surface marker CD8, allowing for the independent analysis of expression from two transgenes within the same cell. We will introduce retroviral vectors with identical regulatory elements encoding each of the reporter genes into MEL cells,and apply this dual reporter system to test the hypothesis that one transgene can silence an unlinked homologous transgene. Deletion analysis using the Cre-lox system will reveal if the continuous presence of homologous elements is necessary for trans-sensing. Furthermore, the histone deacetylase inhibitors trichostatin A and trapoxin will be used to determine whether disruption of histone acetylation status influences trans-sensing in mammalian cells. Demonstration of trans-sensing could have important implications. For example, the introduction of multiple copies of a transgene for the purposes of gene therapy could potentially be counterproductive. If so, then current efforts to maximize transduction efficiencies may require steps to minimize the introduction of supernumary copies of the relevant transgene.
| Lorincz, Matthew C; Schubeler, Dirk; Hutchinson, Shauna R et al. (2002) DNA methylation density influences the stability of an epigenetic imprint and Dnmt3a/b-independent de novo methylation. Mol Cell Biol 22:7572-80 |
