SFVS FOR STUDYING HORMONE RESPONSIVE SIGNALING

用于研究激素反应信号传导的 SFVS

• 批准号: 2654540
• 负责人: JAMES P HOEFFLER
• 金额: $ 37.14万
• 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/2654540
• 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.

信号转导途径最终会聚在 转录激活产生特定的基因表达模式 对环境刺激的反应。 转录起始 由这些信号传导途径介导的是由协调调节 特异性转录因子的表达和/或激活，所述转录因子结合 基因的控制区域。 对机制的具体见解 潜在的转录激活最近出现的研究， 这些转录因子和信号传导的结构和功能 分子。 在对这些功能的研究中缺少一个关键组成部分， 分子是一个系统，其中可以使用 基因转移。 这一点在以下情况下尤其明显： 效应物将对细胞生长产生有害影响。 在这个范例中， 表达负效应子的细胞不能被分离和研究 因为低百分比的细胞实际上被 常规技术，选择负生长表型， 反对。 在本申请中，我们将描述 开发和优化一个系统的隔离瞬态 转染的细胞（或来自转基因小鼠的细胞）远离总的 以非破坏性的方式，这将允许随后的 培养和/或生化分析。 最简单的系统利用 免疫球蛋白可变结构域的单链片段（sFv） 显示在转染细胞的表面上， 用抗原包被的珠作为"钩"。 建议的商业应用：捕获的商业化- 技术使研究人员能够分离出瞬时转染的 以非破坏性的方式从总群体中提取细胞， 随后的培养和/或生化分析。 描述的研究 在这项技术发展的第二阶段， Capture-Tec（TM）产品线中将添加多个新产品。 这些包括与系统一起使用的可分离珠和荧光团， 具有不同特异性的多种不同"钩"分子， 用于仅在需要时显示"钩"的诱导型载体系统，和 一系列发育阶段的cDNA文库和细胞类型 特定. 此外，转基因小鼠品系可以商业化 通过一个动物供应商，我们将提供特定的细胞类型， 隔离珠。