Toward construction of a self-sustained clock-like expression system based on the mammalian circadian clock.
Despite recent advances in circadian biology, detailed understanding of how a biological pacemaker system is assembled, maintained, and regulated continues to be a significant challenge. We have assembled and characterized a first-generation, regulatable, self-sustained clock-like expression system based on key components of the mammalian circadian clock. The molecular setup of the clock-like oscillator was reduced to the core set of positive and negative elements common to all known circadian pacemakers. Sophisticated tetracycline-responsive multi-cistronic expression integrated with forefront lentiviral transduction tools enabled autoregulated reporter transgene expression in a human cell line. We characterized transgene expression kinetics of an artificial oscillator and showed that its expression profiles could be modulated by a serum shock and administration of regulating tetracycline antibiotics. Design of a generic mammalian clock-like expression system will offer novel opportunities to study circadian biology and may provide a unique tool for rhythmic expression of desired transgenes fostering advances in biopharmaceutical manufacturing, gene therapy, and tissue engineering.
Chilov
Dmitri
D
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT D74, CH-8093 Zurich, Switzerland.
Fussenegger
Martin
M
D056930:ARNTL Transcription Factors; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins; D001683:Biological Clocks / Q000235:genetics* / Q000502:physiology; D015153:Blotting, Western; D056926:CLOCK Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D002940:Circadian Rhythm / Q000235:genetics* / Q000502:physiology; D056931:Cryptochromes; D025461:Feedback, Physiological / Q000235:genetics; D005420:Flavoproteins / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D005809:Genes, Regulator / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics; D056950:Period Circadian Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013752:Tetracycline / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection
D056930:ARNTL Transcription Factors; D000469:Alkaline Phosphatase; D001426:Bacterial Proteins; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D018797:Cell Cycle Proteins; D056931:Cryptochromes; D005420:Flavoproteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D056950:Period Circadian Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D013752:Tetracycline; D013937:Thymidine Kinase; D015534:Trans-Activators; D014157:Transcription Factors; C538960:BMAL1 protein, human; C538961:Bmal1 protein, mouse; C539022:PER1 protein, human; C539023:Per1 protein, mouse; C034811:tetracycline resistance-encoding transposon repressor protein; C054562:yellow fluorescent protein, Bacteria; C538943:CLOCK protein, human; C538944:Clock protein, mouse