tailieunhanh - A novel vector platform for vitamin H-inducible transgene expression in mammalian cells

A novel vector platform for vitamin H-inducible transgene expression in mammalian cells have therefore designed a strategy to convert antibiotic-responsive transcription factors into gene regulation systems responsive to non-toxic biotin, also known as vitamin H. Constitutive ligation of biotin to the Avitag-containing VP16 transactivation domain by the Escherichia coli biotin ligase BirA enables heterodimerization with tetracycline- (TetR), streptogramin- (Pip), and macrolide- (E) dependent repressors fused to streptavidin, which creates synthetic transactivators able to activate specific promoters (PhCMV*−1, PPIR , PETR).  | ELSEVIER Available online at ScienceDirect Journal of Biotechnology 131 2007 150-158 Journal of BIOTECHNOLOGY locate jbiotec A novel vector platform for vitamin H-inducible transgene expression in mammalian cells Wilfried Weber William Bacchus Franziska Gruber Mattia Hamberger Martin Fussenegger Institute for Chemical and Bioengineering ETH Zurich HCIF115 Wolfgang-Pauli-Strasse 10 CH-8093 Zurich Switzerland Received 22 February 2007 received in revised form 30 May 2007 accepted 14 June 2007 Abstract Inducible transgene control systems have been instrumental to gene therapy biopharmaceutical manufacturing drug discovery synthetic biology and functional genomic research. The most widely used heterologous gene regulation systems are responsive to antibiotics of the tetracycline streptogramin and macrolide classes. Although these antibiotics are clinically licensed concerns about the emergence of resistant bacteria sideeffects in animal studies and economic considerations associated with clearance of antibiotics in biopharmaceutical manufacturing have limited the use of heterologous transgene control modalities to basic research activities. We have therefore designed a strategy to convert antibiotic-responsive transcription factors into gene regulation systems responsive to non-toxic biotin also known as vitamin H. Constitutive ligation of biotin to the Avitag-containing VP16 transactivation domain by the Escherichia coli biotin ligase BirA enables heterodimerization with tetracycline- TetR streptogramin- Pip and macrolide- E dependent repressors fused to streptavidin which creates synthetic transactivators able to activate specific promoters PhCMV -1 PPIR PETR . We have demonstrated i that exogenous biotin 40 nM can induce heterologous transgene expression in a biotin- serum- free culture environment biotin-dependent heterodimerization of transactivator ii that excess biotin above 200 M gradually represses transgene expression in