SFVS FOR STUDYING HORMONE RESPONSIVE SIGNALING

用于研究激素反应信号传导的 SFVS

• 批准号: 2016974
• 负责人: JAMES P HOEFFLER
• 金额: $ 36.95万
• 依托单位: INVITROGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2016974
• 关键词: antibody; antibody specificity; biological signal transduction; cell type; chimeric proteins; fluorescent dye /probe; gene targeting; genetically modified animals; haptens; hormone regulation /control mechanism; immunoglobulin genes; laboratory mouse; magnetism; method development; microcapsule; reporter genes; tissue /cell culture; transcription factor; transfection; transfection /expression vector

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.

信号转导通路最终汇聚在 转录激活以产生特定的基因表达模式 对环境刺激的反应。转录的启动 通过这些信号通路的调节受坐标的调节 结合特定转录因子的表达和/或激活 到基因的控制区。对机制的具体见解 潜在的转录激活最近出现在对 这些转录因子和信号转导的结构和功能 分子。在对这些功能的研究中缺少的一个关键组成部分 分子是一种可以用来研究负效应的系统 基因转移。这一点在以下情况下尤为明显 效应器会对细胞生长产生有害影响。在这个范例中， 表达负效应的细胞不能被分离和研究 由于很低比例的细胞实际上是通过 选择了常规技术和负增长表型 反对。在本应用程序中，我们将描述 暂态隔离系统的开发与优化 转基因细胞(或转基因小鼠的细胞) 以非破坏性的方式传播，这将使后续 培养和/或生化分析。最简单的系统利用 免疫球蛋白可变区单链片段(SFV‘s) 结合磁性材料在转基因细胞表面展示 涂有抗原的珠子就成了“钩子”。 拟议的商业应用：捕获物的商业化- 技术使研究人员能够分离出瞬时转基因的 以非破坏性的方式从总种群中提取细胞，从而 随后的培养和/或生化分析。这些研究描述了 在这项技术发展的第二阶段将产生 多款新产品将加入Capture-Tec(TM)产品线。 这些包括与系统一起使用的可拆卸的珠子和荧光团， 具有不同特性的多个不同的“钩”分子，以及 仅在需要时才显示“钩子”的诱导式载体系统，以及 一系列发育阶段的、细胞类型的cDNA库 具体的。此外，转基因小鼠品系还可以商业化。 通过动物供应商，我们将提供特定细胞类型的 隔离珠子。