SFVS FOR STUDYING HORMONE RESPONSIVE SIGNALING

信号转导途径最终汇聚在

Signal transduction pathways converge ultimately at the level of transcriptional activation to produce specific patterns of gene expression in response to environmental stimuli. The initiation of transcription mediated by these signalling pathways is regulated by the coordinate expression and/or activation of specific transcription factors that bind to the control regions of genes. Specific insights into the mechanisms underlying transcriptional activation have recently arisen from studies of the structure and functions of these transcription factors and signalling molecules. A key component missing in the study of the functions of these molecules is a system in which negative effectors can be studied using gene transfer. This is especially apparent in cases where the negative effector will have a deleterious effect on cell growth. In this paradigm, the cells expressing the negative effector cannot be isolated and studied since a low percentage of the cells are actually transfected by conventional technology, and negative growth phenotypes are selected against. In this application, we will describe the second phase of developing and optimizing a system for the isolation of transiently transfected cells (or cells from transgenic mice) away from total populations in a non-destructive manner, which will allow subsequent culturing and/or biochemical analyses. The simplest system utilizes single chain fragments of immunoglobulin variable domains (sFv's) displayed on the surface of transfected cells in combination with magnetic beads coated with the antigen as a "Hook". PROPOSED COMMERCIAL APPLICATION: The commercialization of the Capture- Technology has allowed researchers to isolate transiently transfected cells from total populations in a non-destructive manner, allowing subsequent culturing and/or biochemical analyses. The studies described in this second phase of the development of this technology will yield multiple new products to be added to the Capture-Tec(TM) product line. These include, detachable beads and fluorophores to use with the system, multiple different "Hook" molecules with differing specificities, an inducible vector system for displaying the "Hook" only when desired, and a line of cDNA libraries that are developmentally staged, and cell-type specific. In addition, the transgenic mouse lines could be commercialized through an animal vendor, and we will offer the cell-type specific isolation beads.