A Transgenic Mouse Line to Map Cell-Type Specific Calcium Signals In Vivo

用于绘制体内细胞类型特异性钙信号的转基因小鼠系

基本信息

批准号：
9001251
负责人：
MICHAEL D CAHALAN
金额：
$ 23.18万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-02-01 至 2018-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9001251
关键词：
Action Potentials Affinity Astrocytes Autoimmune Diseases B-Lymphocytes BRAIN initiative Behavior Biological Assay Brain CD4 Positive T Lymphocytes Calcium Calcium Signaling Calcium ion Cell Communication Cells Cellular Morphology Coupling Development Disease model Eukaryotic Cell Event Generations Glial Fibrillary Acidic Protein Health Homing Image Immune Immune response Immune system Immunologic Surveillance In Vitro Individual Infection Interneurons Investigation Letters Life Lymphocyte Activation Maps Measurement Measures Membrane Modeling Molecular Molecular Machines Molecular Profiling Morphology Mouse Strains Movement Mus Nervous System Physiology Nervous system structure Neuroglia Neurons Pattern Positioning Attribute Production Proteins Reading Receptor Activation Regulatory T-Lymphocyte Reporting Rosa Signal Transduction Site Speed Surface T-Cell Activation T-Cell Receptor Technology Testing Therapeutic Time Transcriptional Activation Transgenic Mice Transgenic Organisms Validation adaptive immunity base body system calcium indicator calmodulin-dependent protein kinase II cell behavior cell motility cell type cytokine immunoregulation in vivo innovation lymph nodes meetings mouse model nervous system disorder novel novel strategies novel therapeutic intervention ratiometric recombinase red fluorescent protein two-photon

项目摘要

DESCRIPTION (provided by applicant): In eukaryotic cells calcium ions are the primary means of controlling protein-based molecular machines. The ability to record these calcium signals using fluorescent indictors has provided an entry into molecular mechanism and a means to follow subcellular signaling dynamics. Recent advances in genetically encoded calcium indicators (GECIs) now provide the ability to detect even small, transient, and rapidly fluctuating calcium signals within individual genetically-targeted cells in vivo. Calcium signaling regulates movement patterns that govern cell-cell contact essential for lymphocyte activation; other facets include close coupling to T cell receptor activation, reliance on Orai1, calcium- dependent transcriptional activation, and suppression of calcium signaling by regulatory T cells. There is a need to simultaneously track and read out calcium concentrations using endogenous transgenic expression of GECIs to (1) uncover mechanisms of regulatory T cell suppression, (2) visualize therapeutic modulation of endogenous T cell activation in disease models (3) identify endogenous polyclonal activation events in the lymph node for the first time. Moreover, the need for these capabilities are shared by neuroscientists seeking to measure, modify, and model brain circuit function for the President's Brain Initiative, the Brain Activity Map, and similar projects. We have created a novel; ratiometric GECI we call "Salsa6f" by fusing the green fluorescent indicator GCaMP6f with the red fluorescent protein tdTomato and have successfully tested it in vitro. This probe permits quantitative readout of Ca2+ concentrations and Ca2+-independent tracking. This proposal seeks to (1) generate a Cre-dependent GECI transgenic mouse strain with Salsa6f through site directed targeting of the ubiquitously expressed Rosa-26 locus, and (2) cross this line to Cre recombinase driver lines and validate Salsa6f function in immune cells, neurons, and glia. After crossing to drive expression in CD4+ T cells and Vav+ B cells, purified cells will be tested for Salsa6f indicator function, expression, and lack of interference in immune cell homing, localization, motility, proliferation, surface marker expression, and cytokine production, as measured in quantitative assays. Similar crosses will drive expression in CaMKII+ neurons and GFAP+ astrocytes. Brains sections from transgenic mice will be assessed for appropriate expression and neuronal and glial morphology. Neurons and glia will be cultured and the speed and sensitivity of Salsa6f measured upon field stimulation and during spontaneous activity. The new capabilities of Salsa6f transgenic mice will enable new approaches to identify, map, and relate the intricate molecular mechanisms and cell behaviors that collectively define immune and nervous system function.

描述（由适用提供）：在真核细胞中，钙离子是控制基于蛋白质的分子机器的主要手段。使用荧光指示器记录这些钙信号的能力已提供了一种分子机制，并遵循亚细胞信号传导动力学的手段。现在，遗传编码的钙指标（GECIS）的最新进展现在提供了检测体内个体遗传靶向细胞内的小型，瞬态和快速波动的钙信号的能力。钙信号传导调节控制细胞 - 淋巴细胞激活必不可少的细胞接触的运动模式；其他方面包括与T细胞受体激活的密切偶联，ORAI1上的缓解，依赖钙的转录激活以及调节T细胞抑制钙信号传导。需要使用GECI的内源性转基因表达对（1）发现调节性T细胞抑制的机制同时跟踪和读取钙浓度，（2）可视化治疗。疾病模型中内源性T细胞激活的调节（3）首次鉴定淋巴结中的内源性多克隆活化事件。此外，神经科学家寻求为总统的大脑倡议，大脑活动图和类似项目衡量，修改和模型的大脑循环功能所共享的这些功能的需求。我们创作了一本小说；比例测量geci我们将绿色荧光指示剂GCAMP6F与红色荧光蛋白TDTOMATO融合并成功地在体外测试了它来称为“ salsa6f”。该探针允许对Ca2+浓度和CA2+非依赖性跟踪的定量读数。该提案试图通过定向靶向无处不在表达的ROSA-26基因座的位点，生成依赖CRE的GECI转基因小鼠菌株，（2）越过CRE重组酶驱动线并验证免疫细胞，神经元和Glia中的SALSA6F功能。在越过CD4+ T细胞和VAV+ B细胞中表达表达后，将测试纯化的细胞，以测试免疫球体的归因，定位，运动，增殖，表面标志物表达和细胞因子的产生，如定量分析所测量。类似的十字会在CAMKII+神经元和GFAP+星形胶质细胞中驱动表达。将评估来自转基因小鼠的大脑切片，以进行适当的表达以及神经元和神经胶质形态。神经元和神经胶质将被培养，并在野外刺激和赞助活动期间测得的SALSA6F的速度和灵敏度。 SALSA6F转基因小鼠的新能力将实现新的方法来识别，绘制和关联复杂的分子机制和细胞行为，这些机制和细胞行为共同定义了免疫和神经系统功能。