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A yeast-based rapid prototype platform for gene control elements in mammalian cells

Authors

  • Kathy Y. Wei,

    1. Department of Bioengineering, Stanford University, 473 Via Ortega, MC 4201, Stanford, CA 94305; telephone: 650-721-6371, fax: 650-721-6602
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  • Yvonne Y. Chen,

    1. Harvard Society of Fellows, Harvard University, Cambridge, MA
    2. Department of Systems Biology, Harvard Medical School, Boston, MA 02115
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  • Christina D. Smolke

    Corresponding author
    1. Department of Bioengineering, Stanford University, 473 Via Ortega, MC 4201, Stanford, CA 94305; telephone: 650-721-6371, fax: 650-721-6602
    • Department of Bioengineering, Stanford University, 473 Via Ortega, MC 4201, Stanford, CA 94305; telephone: 650-721-6371, fax: 650-721-6602.
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  • Author Contributions: K.Y.W. and Y.Y.C. designed research, constructed and characterized ribozyme-based devices in HEK cells, and wrote the paper; K.Y.W. characterized devices in HeLa and U2OS cells and performed quantitative comparisons against yeast data; Y.Y.C. generated HEK cell lines stably expressing ribozyme devices; C.D.S. designed research and wrote the paper.

Abstract

Programming genetic circuits in mammalian cells requires flexible, tunable, and user-tailored gene-control systems. However, most existing control systems are either mechanistically specific for microbial organisms or must be laboriously re-engineered to function in mammalian cells. Here, we demonstrate a ribozyme-based device platform that can be directly transported from yeast to mammalian cells in a “plug-and-play” manner. Ribozyme switches previously prototyped in yeast are shown to regulate gene expression in a predictable, ligand-responsive manner in human HEK 293, HeLa, and U2OS cell lines without any change to device sequence nor further optimization. The ribozyme-based devices, which exhibit activation ratios comparable to the best RNA-based regulatory devices demonstrated in mammalian cells to-date, retain their prescribed functions (ON or OFF switch), tunability of regulatory stringency, and responsiveness to different small-molecule inputs in mammalian hosts. Furthermore, we observe strong correlations of device performance between yeast and all mammalian cell lines tested (R2 = 0.63–0.97). Our unique device architecture can therefore act as a rapid prototyping platform (RPP) based on a yeast chassis, providing a well-developed and genetically tractable system that supports rapid and high-throughput screens for generating gene-controllers with a broad range of functions in mammalian cells. This platform will accelerate development of mammalian gene-controllers for diverse applications, including cell-based therapeutics and cell-fate reprogramming. Biotechnol. Bioeng. 2013; 110: 1201–1210. © 2012 Wiley Periodicals, Inc.

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