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Creation of genome-wide protein expression libraries using random activation of gene expression

Nature Biotechnology volume 19, pages 440–445 (2001)Cite this article

Abstract

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 × 106 individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, ∼53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.

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Figure 1: Schematic diagram of gene expression using a RAGE vector.
Figure 2: Schematic diagrams of RAGE vectors.
Figure 3: Comparison of experimental and predicted transcript structure.

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  1. Athersys, Inc., 3201 Carnegie Ave., Cleveland, 44115, OH

    John J. Harrington, Bruce Sherf, Stephen Rundlett, P. David Jackson, Rob Perry, Scott Cain, Christina Leventhal, Mark Thornton, Rakesh Ramachandran, Jessica Whittington, Laura Lerner, Dana Costanzo, Karen McElligott, Sherry Boozer, Robert Mays, Emery Smith, Neil Veloso, Alison Klika, Jennifer Hess, Kevin Cothren, Kalok Lo, Jason Offenbacher, Joel Danzig & Matt Ducar

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Correspondence to John J. Harrington.

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Harrington, J., Sherf, B., Rundlett, S. et al. Creation of genome-wide protein expression libraries using random activation of gene expression. Nat Biotechnol 19, 440–445 (2001). https://doi.org/10.1038/88107

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