HIGH-THROUGHPUT IN VIVO SUBCELLULAR-RESOLUTION VERTEBRATE SCREENING PLATFORM

点击翻译按钮获取中文摘要

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.