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

 描述(申请人提供)：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。使用荧光指示剂记录这些钙信号的能力提供了进入分子机制的途径和跟踪亚细胞信号动力学的手段。基因编码钙指示剂(GECI)的最新进展现在提供了检测体内单个基因靶标细胞内微小、瞬时和快速波动的钙信号的能力。钙信号转导 调节淋巴细胞激活所必需的细胞-细胞接触的运动模式；其他方面包括与T细胞受体激活的紧密耦合，对Orai1的依赖，钙依赖的转录激活，以及调节性T细胞对钙信号的抑制。需要利用内源性GECI的转基因表达来同时跟踪和读出钙浓度，以(1)揭示调节性T细胞抑制的机制，(2)在疾病模型中可视化内源性T细胞激活的治疗调节(3)首次识别淋巴结中的内源性多克隆激活事件。此外，寻求为总统的大脑倡议、大脑活动图和类似项目测量、修改大脑回路功能并对其进行建模的神经科学家也有同样的需求。我们通过将绿色荧光指示剂GCaMP6f与红色荧光蛋白tdTomato融合，创造了一种新型的比例计壁虎，我们称之为“Salsa6f”，并已成功地在体外进行了测试。该探头可以定量读出钙离子浓度和不依赖于钙离子的跟踪。该建议旨在(1)通过定点靶向普遍表达的ROSA-26基因座来获得依赖Cre的Geci转基因小鼠品系Salsa6f，以及(2)将该品系与Cre重组酶驱动系杂交，并验证Salsa6f在免疫细胞、神经元和神经胶质细胞中的功能。在交叉驱动CD4+T细胞和Vav+B细胞的表达后，纯化的细胞将被测试Salsa6f指示物的功能、表达以及在免疫细胞归巢、定位、运动、增殖、表面标记表达和细胞因子产生方面的缺乏干扰，这在定量分析中进行了测量。类似的杂交将推动CaMKII+神经元和GFAP+星形胶质细胞的表达。转基因小鼠的大脑切片将被评估是否有适当的表达以及神经元和胶质细胞的形态。将培养神经元和神经胶质细胞，并在场强刺激和自发活动时测量Salsa6f的速度和灵敏度。Salsa6f转基因小鼠的新能力将使识别、映射和关联共同定义免疫和神经系统功能的复杂分子机制和细胞行为的新方法成为可能。

 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.