New Models for Molecular-Level Imaging of Cell Signaling in vivo.

描述（由申请人提供）：活体成像已成为研究天然环境中细胞的重要工具，但几乎没有适用于活组织中信号传导的实时空间分辨分析的方法或工具。基于光的荧光对于信号的亚细胞分辨率具有最佳分辨率，并且可以实时使用。许多研究小组已经开发出将多光子荧光成像应用于小鼠靶组织和器官的方案。这些协议目前仅限于观察细胞形态，定位和运动参数，而进展需要开发的探针系统，响应信号的开始。 在体外，使用GFP标记的生物标志物，基因表达和用于跟踪细胞内的分子行为已被证明是非常有用的跟踪细胞活化。然而，体内的情况被认为是困难的，由于考虑到探针强度，在没有基准的情况下难以解释，以及区域自发荧光。我们表明，新的改进GaAs检测在2-光子显微镜，激发和检测弱荧光蛋白的表达是不是主要的障碍分子成像，只要感兴趣的分子表达在或接近现在定义的水平。相反，限制是自发荧光减少和提供基准标记来表征生物传感融合蛋白的策略。 我们建议，多路复用的生物传感器阵列的集合将允许成像的核输入的转录因子，激活PI 3激酶和T细胞受体。编码这些的基因将作为基于P2 A的融合体整合到具有lox-stop- lox控制的组织非依赖性ROSA-26基因座中，使得它们可以在许多现有的组织特异性启动子Cre驱动菌株下有条件地表达。基于这种设计，它模仿了我们之前在体外和体内首次成功使用T细胞受体的设计，即使在具有不均匀自发荧光水平的器官中，这些受体也将发挥作用。我们的目标是开发这些工具，并随后将其应用于与新改进的自发肿瘤生长模型相互作用的T细胞。我们还将通过非限制性共享和/或将小鼠品系放入仓库，使表达这些生物传感器的小鼠可供社区使用。项目叙述： 虽然我们的科学在理解我们的细胞在孤立状态下的行为方面取得了巨大的成功，但研究细胞在其自然环境中的工作方式却更加困难。这项提议将创造新的小鼠品系，使我们能够使用显微镜观察组织，并观察细胞被激活以分裂和/或重塑它们所参与的组织。对我们来说，了解哪些细胞在人类许多疾病的背景下被激活和失活是很重要的，我们的研究结果可能会对其中许多疾病的性质提供很好的见解。

DESCRIPTION (provided by applicant): Intravital imaging has become a vital tool to study cells in their native context and yet there are almost no methods or tools suitable for real-time spatially-resolved analysis of signaling in living tissues. Light-based fluorescence has the best resolution for subcellular resolution of signaling and can be used in real-time. Numerous groups have developed protocols for applying multiphoton fluorescence imaging to target tissues and organs in the mouse. These protocols are currently limited to observation of cell morphology, positioning and motility parameters while progress requires development of probe systems that respond to signaling onset. In vitro, the use of GFP-tagged biomarkers, genetically expressed and used to track molecular behaviors within cells has proven to be extremely useful in tracking cellular activation. However, the situation in vivo has been considered difficult, as a result of considerations of probe intensity, difficulty with interpretation in the absence of fiducials, and regional autofluorescence. We show that, with new improved GaAs detection in 2-photon microscopy, excitation and detection of weak fluorescent protein expression is not the primary hindrance to molecular imaging so long as the molecule of interest is expressed at or near a now-defined level. Instead, the limitations are autofluorescence reduction and strategies that provide fiducial markers to characterize biosensing fusion-proteins. We propose that a collection of multiplexed biosensor arrays will permit the imaging of nuclear import of transcription factors, activation of PI3kinase enzymes and T cell receptors. The genes encoding these will be integrated as P2A-based fusion into the tissue-independent ROSA-26 locus with lox-stop- lox control so that they can be conditionally expressed under the many existing tissue-specific- promoter Cre driver-strains. Based on the design, which mimics our previous success with the T cell receptor first in vitro and now in vivo, these will be functional even in an organ with uneven levels of autofluorescence. Our goal is to develop these tools and subsequently apply them to a T cell interacting with a newly improved model for spontaneous tumor outgrowth. We will also make the mice expressing these biosensors available to the community through non-restrictive sharing and/or the placement of mouse strains into a repository.7. Project Narrative: While our science is achieving great success at understanding the behavior of our cells in isolation, it is more difficult to study the way cells work in their native context. This proposal will create new mouse strains that will permit us to look into tissues using microscopy and observe cells being activated to divide and/or remodel the tissue of which they are a part. It is important for us to understand which cells are being activated and deactivated in the context of many diseases of humans, and our results are likely to provide great insight into the nature of many of these.