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Nucleic Acids Research 2005 33(4):e39; doi:10.1093/nar/gni039
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Published online 24 February 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

Screening of retroviral cDNA libraries for factors involved in protein phosphorylation in signaling cascades

J. Stitz1,2, P. O. Krutzik1,2 and G. P. Nolan1,2,*

1 Department of Microbiology and Immunology, School of Medicine, Stanford University Stanford, CA 94305, USA 2 Baxter Laboratory in Genetic Pharmacology, School of Medicine, Stanford University Stanford, CA 94305, USA

*To whom correspondence should be addressed at Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, CCSR 3230, School of Medicine, 269 Campus Drive, Stanford, CA 94305, USA. Tel: +1 723 723 7002; Fax: +1 650 723 2383; Email: gnolan{at}stanford.edu

Received December 20, 2004. Revised January 25, 2005. Accepted February 4, 2005.

We report a novel approach that allows for the rapid identification of proteins mediating phosphorylation in signaling cascades after specific stimulation. As a proof of concept, we used the interferon- {gamma} (IFN-{gamma})-induced phosphorylation of signal transducer and activator of transcription-1 (Stat1) in a human promonocytic cell line, which was previously shown to be deficient in this signaling pathway. By using retroviral cDNA expression libraries, transduced selector cells expressing single cDNAs were stimulated with IFN-{gamma}, then fixed, permeabilized and stained intracellularly for phospho-Stat1 levels. Cells responding to the stimulation, which showed increased levels of phosphorylated Stat1, were enriched using fluorescence activated cell sorting (FACS). Genomic DNA was isolated from the enriched cell population and served as a template for cDNA amplification using PCR. After only one round of selection, a cDNA encoding the ß-chain of the IFN-{gamma} receptor (IFNGR2) was obtained and demonstrated to restore the selected phenotype. The approach now allows one to use phospho-events as reporters, alone or in tandem, for screening of signaling network states, overcoming a prior need to rely on the reporter genes that are often only indirect measures of phenotypes desired in a screen.


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C. A. Merten, J. Stitz, G. Braun, J. Medvedovska, K. Cichutek, and C. J. Buchholz
Fusoselect: cell-cell fusion activity engineered by directed evolution of a retroviral glycoprotein
Nucleic Acids Res., March 15, 2006; 34(5): e41 - e41.
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