Abstract
Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.
Highlights
Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks
Using a set of mouse serine-threonine kinase (STK) genes as an example, we describe the development of protocols and vectors contained in the Alliance for Cellular Signaling (AfCS) plasmid database
We queried the simple modular architecture research tool (SMART) database [21, 22] to generate a comprehensive, yet unique, list of ORFs and identified 217 distinct murine STK ORFs based on the information publicly available in 2001
Summary
Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gatewayா entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. At the outset of the Alliance for Cellular Signaling (AfCS) project [1], reagent availability was a central issue, and in the case of cDNA clones, there was no publicly accessible repository of validated mouse sequences. As the described ORFeome cloning projects have concentrated on model organisms other than mouse, the AfCS-generated plasmids remain, to our knowledge, the largest collection of mouse ORFonly clones available through a non-commercial source
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