HIGH-THROUGHPUT IN VIVO SUBCELLULAR-RESOLUTION VERTEBRATE SCREENING PLATFORM

描述（由申请人提供）：研究整个生物体的能力使研究体外无法复制的复杂体内过程成为可能，如器官发育、肝脏、胰腺、心脏和神经元再生、癌症转移、神经变性、感染性疾病进展、发病机制、心血管、免疫、内分泌和神经系统功能。细胞不被转化，并且处于细胞-细胞、细胞外基质和其他相互作用的正常生理环境中。微阵列研究和体外筛选使用细胞系和数以百万计的组合合成的化合物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、效力、毒性和生物分布以及整个器官（如肾、肝、心脏和脑）上数千个基因的功能的鉴定不能在体外进行，并且需要使用体内动物模型。目前，在脊椎动物的体外和体内测定的通量和能力之间存在显著的差距。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。在动物的后期发展阶段进行的试验失败不仅会付出高昂的代价，而且会大大减缓进展。然而，迄今为止，由于缺乏关键技术，使用体内脊椎动物模型对基因功能和化合物进行高通量测试受到显著限制。在这里，我们提出了一种高度变革性的技术，该技术将首次允许在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）上以细胞分辨率进行大规模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。所提出的高速整体动物操作、定向、固定、成像、显微手术和注射平台将使得可以进行体内测定的通量和复杂性显著增加（取决于观察到的表型和操作复杂性，每个动物约5-10秒，而不是目前需要的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无偏见的全基因组脊椎动物研究，以显着加快基因功能鉴定以及药物先导发现的基础和转化研究。为了证明系统的能力，我们将进行第一次大规模的体内化学筛选，用于再生显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.