Adenovirus vector systems permitting regulated protein expression and their use for in vivo splicing studies

摘要: We have constructed two adenovirus-based gene expression vector systems permitting regulated protein expression. They are based on the tetracycline-regulated Tet-ON- and progesterone antagonist RU 486-regulated systems, which were rescued into E1-deficient adenovirus vectors. The vectors function in a number of cell types representing broad species-variety regulation was shown to be tightly controlled cells not permissive for virus replication. Furthermore, adenovirus-Tet-ON system perform mice after intramuscular administration.The novel adenovirus-vector then used study effects overexpression selected proteins replication during lytic infection, with focus alternative splicing. Expression transcription units is large extent temporally at level pre-mRNA splicing, where viral splice site usage shifts from proximal distal selection as infection proceeds. This makes an appropriate model mechanistic studies show that essential host splicing factor ASF/SF2 inhibits this shift by promoting sites. As consequence, displayed markedly inhibited growth. Interestingly, mRNA major late promoter almost completely lost consequence overexpression. Collectively, cellular prevents entering phase infection. strongly argues need block enhancer activity establishment Further, analysis strict inhibition region 1 we propose temporal merely fitness rather than profoundly deleterious unregulated During our noted 293 cells, growth Ad vectors, unwanted background reporter evident systems. therefore introduced additional regulatory element, functioning transcriptional road-block, showed methodological innovation represents way overcome potentially modified should make it possible reconstruct recombinant viruses expressing highly toxic proteins.In conclusion, work presents new vivo involved RNA other mechanisms.