Imaging Airway Myocyte Ca2+ Signaling in Living Animals

活体动物气道肌细胞 Ca2 信号传导成像

• 批准号: 7155830
• 负责人: LISA R ABSTON-LEFTRIDGE
• 金额: $ 4.88万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7155830
• 关键词: adenosine triphosphate; animal tissue; bioimaging /biomedical imaging; biological signal transduction; biotechnology; bronchoconstrictors; calcium flux; fluorescent dye /probe; genetic promoter element; genetically modified animals; intravital microscopy; laboratory mouse; luciferin monooxygenase; plasmids; postdoctoral investigator; reporter genes; respiratory epithelium; respiratory imaging /visualization; smooth muscle; temperature; tissue /cell culture; trachea; transfection /expression vector

DESCRIPTION (provided by applicant): Intracellular free calcium ion concentration ([Ca2+]i) controls a number of intracellular signaling systems, and as such is of interest to researchers who study those systems or their downstream effector systems. Other studies have demonstrated that protein-protein approximation can be detected by reconstitution of luciferase function when N- and C-terminal portions of either firefly or Renilla luciferase are each fused to one of another pair of interacting proteins. Based on this background information, we conceived, designed, implemented, and tested a novel split synthetic Renilla luciferase [Ca2+] reporter system, currently realized as a mammalian expression plasmid with CMV promoter that drives expression of a chimeric cDNA encoding a single fusion protein. We found that the luciferase activity of our probe falls substantially with increasing free [Ca2+] in a physiologically relevant range. Additional experiments are planned to more fully evaluate the probe's sensitivity to other ions; to ATP concentration; and to ambient temperature in cell lysates. In addition, we plan to evaluate whether the probe can detect intracellular calcium concentration in living cells in culture, tissue origins, and eventually in cells in living animals.

描述（由申请人提供）：细胞内游离钙离子浓度（[[Ca2+] i）控制许多细胞内信号传导系统，因此研究这些系统或其下游效应器系统的研究人员感兴趣。其他研究表明，当萤火虫或氟氟荧光素酶的N-和C末端部分分别融合到另一对相互作用的蛋白质中时，可以通过重新建立荧光素酶功能来检测蛋白质蛋白近似。基于这些背景信息，我们构思，设计，实施和测试了一种新型的分裂合成肾荧光素酶[Ca2+]报告基因系统，该系统目前用CMV启动子构成是一种哺乳动物表达质粒，该质粒具有CMV启动子，该质粒驱动了编码单个融合蛋白的嵌合cDNA的表达。我们发现，我们的探针的荧光素酶活性大大降低，随着生理相关范围内的游离[Ca2+]增加。计划进行其他实验，以更全面地评估探针对其他离子的敏感性；到ATP浓度；并在细胞裂解物中的环境温度。此外，我们计划评估探针是否可以检测培养细胞中活细胞的细胞内钙浓度，组织起源以及最终在活动物中的细胞中。