Transcriptional target-based expression cloning of immunoregulatory molecules
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Transcriptional target-based expression cloning of immunoregulatory molecules Rebecca L. Lamason • Stefanie M. Lew • Joel L. Pomerantz
Published online: 13 January 2010 Ó Springer Science+Business Media, LLC 2010
Abstract Immunologic research has benefited tremendously from expression-cloning strategies designed to isolate genes responsible for a wide variety of immunomodulatory activities, including cytokines, receptors, signaling proteins, and transcription factors. Here, we discuss the use of expression-cloning strategies that have been modified to detect cDNAs that influence gene expression as assayed by a transcriptional reporter. We summarize our experience with these screens, review important parameters, and discuss potential modifications. Keywords Expression-cloning Gene discovery NF-jB HIV-1 cDNA library
Introduction Our expansive understanding of the development and function of the mammalian immune system depends upon the appreciation of the molecular machinery that governs cellular and tissue responses. Despite the wealth of understanding of innate and adaptive immunity, gaps in our collective knowledge continue to suggest that many important players are yet to be discovered. A wide variety of approaches have been described for the identification of novel relevant molecules. These include, for example, biochemical purification of an activity followed by mass spectrometry, interaction cloning by yeast two-hybrid, RNA interference screens, and screening in silico for molecules with interesting homology to known regulators. Another broad approach has involved expression-cloning, in which gene libraries are expressed in a heterologous cell or tissue, an activity is assayed, and then the gene responsible for the desired activity is isolated and identified. R. L. Lamason S. M. Lew J. L. Pomerantz (&) Department of Biological Chemistry, Institute for Cell Engineering, and Graduate Program in Immunology, The Johns Hopkins University School of Medicine, 733 N. Broadway, BRB 607, Baltimore, MD 21205, USA e-mail: [email protected]
Immunol Res (2010) 47:172–178
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Our laboratory has modified the expression-cloning approach to isolate cDNAs that influence gene regulation by incorporating into the screen the quantitative assay of expression of a particular reporter gene. This strategy has allowed us to survey a wide variety of biological activities, including the components of signaling pathways that influence gene activity as well as transcription factors that act directly upon the reporter gene target. The methodology is broadly applicable, unbiased, and easily modified to enrich for genes that satisfy a particular biological interest. Here, we describe our experience with this methodology and discuss its critical parameters and potential.
Cloning molecules that signal to NF-jB We established this approach [1] with the aim of isolating novel components of signaling pathways that activated NF-jB, a pleiotropic transcription factor that is critical for both innate and adaptive immunity [2]. P
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