Autoregulated, bidirectional and multicistronic gas-inducible mammalian as well as lentiviral expression vectors.
We present a novel set of autoregulated, bidirectional and multicistronic mammalian as well as lentiviral expression vectors which enable transgene expression fine-tuning by gaseous acetaldehyde. The acetaldehyde-inducible regulation (AIR) technology capitalizes on Aspergillus nidulans components evolved to convert ethanol into metabolic energy. AIR is based on functional interaction of the fungal transactivator AlcR and AlcR-specific chimeric promoters (P(AIR)) which drive desired transgene expression in mammalian cells only in the presence of gaseous acetaldehyde. We have engineered AIR technology into a variety of different mammalian and lentiviral expression vector systems including (i) a most compact autoregulated expression format harboring alcR and the transgene in a single P(AIR)-driven transcription unit, (ii) a bidirectional P(AIR) derivative supporting expression of two transgenes with strict 1:1 transcription stoichiometry and (iii) a multicistronic expression arrangement providing simultaneous translation of three independent transgenes from a single P(AIR)-controlled transcript. All expression vectors have been validated in Chinese hamster ovary (CHO-K1), baby hamster kidney (BHK-21) and human HeLa cells for gas-inducible (co-)expression of the reporter transgenes such as Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY), human vascular endothelial growth factor 121 (VEGF121), human placental-secreted alkaline phosphatase (SEAP) and Escherichia coli-derived chloramphenicol acetyl-transferase (CAT). The panoply of mammalian/lentiviral vectors presented here provides a robust and versatile expression platform for the first gas-inducible transgene control system which we expect to foster future advances in gene therapy, tissue engineering as well as biopharmaceutical manufacturing.
Hartenbach
Shizuka
S
Institute for Chemical and Bio-Engineering, Swiss Federal Institute of Technology, ETH Hoenggerberg, HCI F115, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland.
Fussenegger
Martin
M
D000079:Acetaldehyde / Q000008:administration & dosage*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005246:Feedback / Q000502:physiology; D005740:Gases / Q000008:administration & dosage; D015967:Gene Expression Regulation, Viral / Q000187:drug effects / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000254:growth & development; D008322:Mammals; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection / Q000379:methods*
D005740:Gases; D011994:Recombinant Proteins; D000079:Acetaldehyde