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Codon improved reverse tetracycline repressor fused to virion protein 16 activation domain (rtTA); rtTA-ligand binding domain transactivator fusions; Insulators
Abstract:
Several strategies for regulated stable transgene expression in mammalian cells have been described. These strategies have different strengths and weaknesses, however they all share a common problem, namely predictability in application. Here we address this problem using the leading strategy for ligand inducible transgene expression, the tetracycline repressor system. Initially, we found the best stable clone out of 48 examined showed only 6−fold inducibility. Hence we looked for additions and modifications that improve the chances of a successful outcome. We document three important aspects; first, use of a mammalian codon−optimized tetracycline repressor gene; second, addition of a steroid hormone receptor ligand binding domain to the tetracycline repressor−virion protein 16 fusion protein activator; third, flanking the tet−operator/transgene cassette with insulator elements from the chicken beta−globin locus. By inclusion of these three design features, 18/18 clones showed low basal and highly inducible (>50 x) expression
