Transferring a synthetic gene circuit from yeast to mammalian cells

The emerging field of synthetic biology builds gene circuits for scientific, industrial, and therapeutic needs. Adaptability of synthetic gene circuits across different organisms could enable a synthetic biology pipeline, where circuits are designed in silico, characterized in microbes and reimplemented in mammalian settings for practical usage. However, the processes affecting gene circuit adaptability have not been systematically investigated. Here we construct a mammalian version of a negative feedback-based “linearizer” gene circuit previously developed in yeast. The first naïve mammalian prototype was non-functional, but a computational model suggested that we could recover function by improving gene expression and protein localization. After rationally developing and combining new parts as the model suggested, we regained function and could tune target gene expression in human cells linearly and precisely as in yeast. The steps we have taken should be generally relevant for transferring any gene circuit from yeast into mammalian cells.

[1]  J. Collins,et al.  Synthetic Biology Moving into the Clinic , 2011, Science.

[2]  Robert Gentleman,et al.  flowCore: a Bioconductor package for high throughput flow cytometry , 2009, BMC Bioinformatics.

[3]  S. Smale,et al.  DNA sequence requirements for transcriptional initiator activity in mammalian cells. , 1994, Molecular and cellular biology.

[4]  Demin Zhou,et al.  A more efficient RNAi inducible system for tight regulation of gene expression in mammalian cells and xenograft animals. , 2007, RNA.

[5]  M. C. Hu,et al.  The inducible Iac operator-repressor system is functional in mammalian cells , 1987, Cell.

[6]  J. Debus,et al.  Role of PP2Cα in cell growth, in radio- and chemosensitivity, and in tumorigenicity , 2007, Molecular Cancer.

[7]  S Falkow,et al.  FACS-optimized mutants of the green fluorescent protein (GFP). , 1996, Gene.

[8]  G. Stephanopoulos.  Challenges in Engineering Microbes for Biofuels Production , 2007, Science.

[9]  A B Rose,et al.  Intron-mediated regulation of gene expression. , 2008, Current topics in microbiology and immunology.

[10]  Zhen Xie,et al.  Rationally-designed logic integration of regulatory signals in mammalian cells , 2010, Nature nanotechnology.

[11]  M. Bennett,et al.  A fast, robust, and tunable synthetic gene oscillator , 2008, Nature.

[12]  S. Gorfien,et al.  Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells. , 2005, Biotechnology and bioengineering.

[13]  Martin Fussenegger,et al.  Artificial regulatory networks and cascades for discrete multilevel transgene control in mammalian cells , 2003, Biotechnology and bioengineering.

[14]  A. Ørbo,et al.  MCF-7 cell apoptosis and cell cycle arrest: non-genomic effects of progesterone and mifepristone (RU-486). , 2010, Anticancer research.

[15]  Farren J. Isaacs,et al.  Phenotypic consequences of promoter-mediated transcriptional noise. , 2006, Molecular cell.

[16]  T. Dryja,et al.  Transgenic mice with a rhodopsin mutation (Pro23His): A mouse model of autosomal dominant retinitis pigmentosa , 1992, Neuron.

[17]  Lingchong You,et al.  Spatiotemporal modulation of biodiversity in a synthetic chemical-mediated ecosystem , 2009, Nature chemical biology.

[18]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Collins,et al.  Combinatorial promoter design for engineering noisy gene expression , 2007, Proceedings of the National Academy of Sciences.

[20]  M. Fussenegger,et al.  An engineered epigenetic transgene switch in mammalian cells , 2004, Nature Biotechnology.

[21]  C. Tyler-Smith,et al.  Attenuation of green fluorescent protein half-life in mammalian cells. , 1999, Protein engineering.

[22]  J. Collins,et al.  Construction of a genetic toggle switch in Escherichia coli , 2000, Nature.

[23]  C. Gatz,et al.  Control of expression of the Tn10-encoded tetracycline resistance genes. Equilibrium and kinetic investigation of the regulatory reactions. , 1983, Journal of molecular biology.

[24]  Christina D Smolke,et al.  Reprogramming Cellular Behavior with RNA Controllers Responsive to Endogenous Proteins , 2010, Science.

[25]  G. Balázsi,et al.  Negative autoregulation linearizes the dose–response and suppresses the heterogeneity of gene expression , 2009, Proceedings of the National Academy of Sciences.

[26]  M. Lewandoski.  Conditional control of gene expression in the mouse , 2001, Nature Reviews Genetics.

[27]  Y. Koide,et al.  Codon optimization effect on translational efficiency of DNA vaccine in mammalian cells: analysis of plasmid DNA encoding a CTL epitope derived from microorganisms. , 1999, Biochemical and biophysical research communications.

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

[29]  M. Fussenegger,et al.  Macrolide-based transgene control in mammalian cells and mice , 2002, Nature Biotechnology.

[30]  R. Jaenisch,et al.  A generic intron increases gene expression in transgenic mice , 1991, Molecular and cellular biology.

[31]  M. Win,et al.  Higher-Order Cellular Information Processing with Synthetic RNA Devices , 2008, Science.

[32]  J. Stelling,et al.  A tunable synthetic mammalian oscillator , 2009, Nature.

[33]  Pamela A Silver,et al.  Synthetic memory circuits for tracking human cell fate. , 2012, Genes & development.

[34]  M. Kozak.  An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. , 1987, Nucleic acids research.

[35]  W. Hillen,et al.  Structural requirements of tetracycline-Tet repressor interaction: determination of equilibrium binding constants for tetracycline analogs with the Tet repressor , 1991, Antimicrobial Agents and Chemotherapy.

[36]  E. Huang,et al.  Transfer and Expression of Plasmids Containing Human Cytomegalovirus Immediate‐Early Gene 1 Promoter‐Enhancer Sequences in Eukaryotic and Prokaryotic Cells , 1988, Biotechnology and applied biochemistry.

[37]  A. Long,et al.  A human cell line from a pleural effusion derived from a breast carcinoma. , 1973, Journal of the National Cancer Institute.

[38]  R. Weiss,et al.  A universal RNAi-based logic evaluator that operates in mammalian cells , 2007, Nature Biotechnology.

[39]  J. Collins,et al.  Tuning and controlling gene expression noise in synthetic gene networks , 2010, Nucleic acids research.

[40]  Ahmad S. Khalil,et al.  Synthetic biology: applications come of age , 2010, Nature Reviews Genetics.

[41]  C. Gorman,et al.  Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA. , 1990, Nucleic acids research.

[42]  Hermann Bujard,et al.  A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells , 1999, The journal of gene medicine.

[43]  A. V. van Ooyen,et al.  Comparison of total sequence of a cloned rabbit beta-globin gene and its flanking regions with a homologous mouse sequence. , 1979, Science.

[44]  Tobias Mourier,et al.  Eukaryotic Intron Loss , 2003, Science.

[45]  Priscilla E. M. Purnick,et al.  The second wave of synthetic biology: from modules to systems , 2009, Nature Reviews Molecular Cell Biology.

[46]  S. McKnight,et al.  Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. , 1988, Genes & development.

[47]  James T Kadonaga,et al.  Rational design of a super core promoter that enhances gene expression , 2006, Nature Methods.

[48]  S. Kain,et al.  Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. , 1996, Nucleic acids research.

[49]  M. T. Hasan,et al.  Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[50]  J. Mallet,et al.  Optimization of transgene expression at the posttranscriptional level in neural cells: implications for gene therapy. , 2003, Molecular therapy : the journal of the American Society of Gene Therapy.

[51]  Hermann Bujard,et al.  Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements , 1993 .

[52]  Timothy S. Ham,et al.  Production of the antimalarial drug precursor artemisinic acid in engineered yeast , 2006, Nature.

[53]  Sandya Liyanarachchi,et al.  Genome-wide analysis of core promoter elements from conserved human and mouse orthologous pairs , 2006, BMC Bioinformatics.

[54]  Hamid Bolouri,et al.  Dizzy: Stochastic Simulation of Large-scale Genetic Regulatory Networks (supplementary Material) , 2005, J. Bioinform. Comput. Biol..

[55]  M. Fussenegger,et al.  De novo design and construction of an inducible gene expression system in mammalian cells. , 2011, Methods in enzymology.

[56]  William D. Richardson,et al.  A short amino acid sequence able to specify nuclear location , 1984, Cell.

[57]  L. Banaszynski,et al.  A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules , 2006, Cell.

[58]  K. Thorn,et al.  Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae , 2004, Yeast.

[59]  A. Schambach,et al.  Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression , 2006, Gene Therapy.

[60]  D. Endy,et al.  Refinement and standardization of synthetic biological parts and devices , 2008, Nature Biotechnology.

[61]  James J. Collins,et al.  A Tunable Genetic Switch Based on RNAi and Repressor Proteins for Regulating Gene Expression in Mammalian Cells , 2007, Cell.

[62]  W. Hillen,et al.  Permeation of tetracyclines through membranes of liposomes and Escherichia coli. , 2000, European journal of biochemistry.

[63]  Michael Boshart,et al.  A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus , 1985, Cell.

[64]  P. Lowenstein,et al.  Turning the gene tap off; implications of regulating gene expression for cancer therapeutics , 2008, Molecular Cancer Therapeutics.

[65]  Zhen Xie,et al.  Molecular Systems Biology Peer Review Process File Synthetic Incoherent Feed-forward Circuits Show Adaptation to the Amount of Their Genetic Template. Transaction Report , 2022 .

[66]  S. Basu,et al.  A synthetic multicellular system for programmed pattern formation , 2005, Nature.

[67]  B. Fang,et al.  Gene transfer: the challenge of regulated gene expression. , 2008, Trends in molecular medicine.

[68]  Christopher A. Voigt,et al.  A Synthetic Genetic Edge Detection Program , 2009, Cell.

[69]  R. Palmiter,et al.  Introns increase transcriptional efficiency in transgenic mice. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[70]  Raphael Kopan,et al.  rtTA toxicity limits the usefulness of the SP-C-rtTA transgenic mouse. , 2009, Developmental biology.

[71]  F. Yao,et al.  Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. , 1998, Human gene therapy.