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.
A yeast-based rapid prototype platform for gene control elements in mammalian cells†
Article first published online: 17 JAN 2013
Copyright © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 110, Issue 4, pages 1201–1210, April 2013
How to Cite
Wei, K. Y., Chen, Y. Y. and Smolke, C. D. (2013), A yeast-based rapid prototype platform for gene control elements in mammalian cells. Biotechnol. Bioeng., 110: 1201–1210. doi: 10.1002/bit.24792
- Issue published online: 22 FEB 2013
- Article first published online: 17 JAN 2013
- Accepted manuscript online: 26 NOV 2012 09:09AM EST
- Manuscript Accepted: 12 NOV 2012
- Manuscript Revised: 23 OCT 2012
- Manuscript Received: 8 AUG 2012
- National Institutes of Health. Grant Number: RC1GM091298
- Defense Advanced Research Projects Agency. Grant Number: HR0011-11-2-0002
- National Science Foundation
- RNA controller;
- synthetic biology
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.