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. © 2004 Wiley Periodicals, Inc.

[1]  Paolo Sassone-Corsi,et al.  A Web of Circadian Pacemakers , 2002, Cell.

[2]  F. Gage,et al.  In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector , 1996, Science.

[3]  F. Fleury-Olela,et al.  Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. , 2000, Genes & development.

[4]  J E Bailey,et al.  Autoregulated Multicistronic Expression Vectors Provide One‐Step Cloning of Regulated Product Gene Expression in Mammalian Cells , 1997, Biotechnology progress.

[5]  Martin Fussenegger,et al.  SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus α-amylase , 2002 .

[6]  U. Schibler,et al.  A Serum Shock Induces Circadian Gene Expression in Mammalian Tissue Culture Cells , 1998, Cell.

[7]  S. Reppert,et al.  Molecular analysis of mammalian circadian rhythms. , 2001, Annual review of physiology.

[8]  S. Yamaguchi,et al.  Molecular machinery of the circadian clock in mammals , 2002, Cell and Tissue Research.

[9]  Peng Huang,et al.  The Circadian Gene Period2 Plays an Important Role in Tumor Suppression and DNA Damage Response In Vivo , 2002, Cell.

[10]  D. V. Leenen,et al.  Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms , 1999, Nature.

[11]  S. Honma,et al.  Characterization of three splice variants and genomic organization of the mouse BMAL1 gene. , 1999, Biochemical and biophysical research communications.

[12]  J. Hartikainen,et al.  Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty. , 2000, Human gene therapy.

[13]  I. Edery,et al.  Circadian rhythms in a nutshell. , 2000, Physiological genomics.

[14]  Steven M Reppert,et al.  mCRY1 and mCRY2 Are Essential Components of the Negative Limb of the Circadian Clock Feedback Loop , 1999, Cell.

[15]  Satchidananda Panda,et al.  Circadian rhythms from flies to human , 2002, Nature.

[16]  Mark J. Zylka,et al.  A Molecular Mechanism Regulating Rhythmic Output from the Suprachiasmatic Circadian Clock , 1999, Cell.

[17]  M. Fussenegger,et al.  New‐generation multicistronic expression platform: pTRIDENT vectors containing size‐optimized IRES elements enable homing endonuclease‐based cistron swapping into lentiviral expression vectors , 2004, Biotechnology and bioengineering.

[18]  A. Sehgal,et al.  Regulation of CLOCK and MOP4 by Nuclear Hormone Receptors in the Vasculature A Humoral Mechanism to Reset a Peripheral Clock , 2001, Cell.

[19]  Ueli Schibler,et al.  The Orphan Nuclear Receptor REV-ERBα Controls Circadian Transcription within the Positive Limb of the Mammalian Circadian Oscillator , 2002, Cell.

[20]  G M Edelman,et al.  A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. Karlsson,et al.  Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Fussenegger,et al.  Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction. , 2002, Nucleic acids research.

[23]  Hideki Mochizuki,et al.  High-Titer Human Immunodeficiency Virus Type 1-Based Vector Systems for Gene Delivery into Nondividing Cells , 1998, Journal of Virology.

[24]  Martin Fussenegger,et al.  Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells , 1998, Nature Biotechnology.

[25]  K. Yagita,et al.  Forskolin induces circadian gene expression of rPer1, rPer2 and dbp in mammalian rat‐1 fibroblasts , 2000, FEBS letters.

[26]  S. Reppert,et al.  The Circadian Clocks of Mice and Men , 2001, Neuron.

[27]  B. H. Miller,et al.  Coordinated Transcription of Key Pathways in the Mouse by the Circadian Clock , 2002, Cell.

[28]  M. Kozak.  Alternative ways to think about mRNA sequences and proteins that appear to promote internal initiation of translation. , 2003, Gene.

[29]  M. Elowitz,et al.  A synthetic oscillatory network of transcriptional regulators , 2000, Nature.

[30]  E. Nishida,et al.  Involvement of the MAP kinase cascade in resetting of the mammalian circadian clock. , 2000, Genes & development.

[31]  Y Sakaki,et al.  Entrainment of the circadian clock in the liver by feeding. , 2001, Science.

[32]  M. Fussenegger,et al.  The Impact of Mammalian Gene Regulation Concepts on Functional Genomic Research, Metabolic Engineering, and Advanced Gene Therapies , 2001, Biotechnology progress.

[33]  Steven A. Brown,et al.  Resetting of circadian time in peripheral tissues by glucocorticoid signaling. , 2000, Science.

[34]  M. Fussenegger,et al.  pTRIDENT, a novel vector family for tricistronic gene expression in mammalian cells. , 1998, Biotechnology and bioengineering.

[35]  U. Albrecht.  Functional Genomics of Sleep and Circadian Rhythm Invited Review: Regulation of mammalian circadian clock genes , 2002 .

[36]  M. Fussenegger,et al.  An Update of pTRIDENT Multicistronic Expression Vectors: pTRIDENTS Containing Novel Streptogramin‐Responsive Promoters , 2000, Biotechnology progress.