HIGH-THROUGHPUT IN VIVO SUBCELLULAR-RESOLUTION VERTEBRATE SCREENING PLATFORM

描述(申请人提供)：研究整个生物体的能力使研究复杂的体内过程成为可能，这些过程在体外无法复制，如器官发育、肝脏、胰腺、心脏和神经元再生、癌症转移、神经退化、传染病进展、发病机制、心血管、免疫、内分泌和神经系统功能。细胞不转化，处于细胞与细胞、细胞外基质等相互作用的正常生理环境中。微阵列研究和使用细胞系和数百万种组合合成的化合物进行的体外筛选已经产生了数千种可能的遗传靶点和候选药物。药物的特异性、有效性、毒性和生物分布以及肾脏、肝脏、心脏和大脑等整个器官上数千个基因的功能不能在体外完成，需要使用体内动物模型。目前，脊椎动物的体外和体内检测的吞吐量和能力之间存在着显著的差距。因此，在药物筛选和开发的早期阶段，药物不能在体内进行测试。在动物发育的后期阶段进行失败的试验不仅代价高昂，而且还会显著减缓进展。然而，由于缺乏关键技术，使用活体脊椎动物模型对基因功能和化合物的高通量测试迄今受到极大限制。在这里，我们提出了一项具有高度变革性的技术，该技术将首次允许在细胞分辨率下对脊椎动物的复杂器官，如心脏、肝脏、肾脏、胰腺、视觉、免疫系统和中枢神经系统进行大规模的体内遗传和化学筛选。这项技术可以影响从神经生物学到再生生物学和癌症生物学的广泛领域。拟议的高速全动物操作、定位、固定、成像、显微手术和注射平台将使体内分析的吞吐量和复杂性大幅增加(根据观察到的表型和操作复杂性，每只动物大约5-10秒，而不是目前需要10-30分钟)。我们的建议与NIH的路线图目标高度相关，因为它将允许系统和公正的全基因组脊椎动物研究大大加快在识别基因功能以及发现药物先导方面的基础和翻译研究。为了展示系统的能力，我们将进行第一次大规模的体内化学筛查，以再生微手术损伤的脊髓纤维。

DESCRIPTION (provided by applicant): The ability to study whole organisms makes it possible to study complex in vivo processes that cannot be replicated in vitro such as organ development, liver, pancreas, heart, and neuronal regeneration, cancer metastasis, neural degeneration, infectious disease progression, pathogenesis, cardiovascular, immune, endocrine, and nervous system functions. Cells are not transformed and are in their normal physiological environment of cell-cell, extracellular matrix, and other interactions. Microarray studies and in vitro screens using cell lines and millions of combinatorially synthesized compounds have generated thousands of possible genetic targets and drug candidates. Identification of specificity, potency, toxicity, and biodistribution of pharmaceuticals as well as functions of thousands genes on entire organs like kidney, liver, heart, and brain cannot be done in vitro, and require use of in vivo animal models. Currently, there is significant gap between the throughput and capabilities of in vitro and in vivo assays on vertebrates. As a result, during early stages of drug screening and development, pharmaceuticals cannot be tested in vivo. Failure of tests on animals at later stages of development not only costs dearly, but also slows progress significantly. Yet, high-throughput testing of gene functions and compounds using in vivo vertebrate animal models has so far been significantly limited due to the absence of key technologies. Here, we propose a highly transformative technology that will allow, for the first time, large- scale in vivo genetic and chemical screens at cellular resolution on complex organs of vertebrates such as heart, liver, kidney, pancreas, vision, immune system, and central nervous system. This technology can impact a broad spectrum of fields ranging from neurobiology to regenerative biology, and cancer biology. The proposed high-speed whole-animal manipulation, orientation, immobilization, imaging, microsurgery, and injection platform will enable a dramatic increase in the throughput and complexity with which in vivo assays can be performed (~5-10 seconds per animal depending on the observed phenotype and manipulation complexity instead of the 10-30 minutes it currently takes). Our proposal is highly relevant to NIH's roadmap goals as it will allow systematic and unbiased genome-wide vertebrate studies to dramatically accelerate both fundamental and translational research in identification of gene functions as well as in discovery of drug leads. To demonstrate system capabilities, we will perform the first large-scale in vivo chemical screen for regenerating micro-surgically injured spinal-cord fibers.