COPAS BIOSORT Worm Sorter

描述（申请人提供）：秀丽隐杆线虫（Caenorhabditis elegans）是一种由大约1000个成年动物分化细胞组成的多细胞蛔虫，它具有许多优点，即它的世代时间极短，并且它的基因组可以很容易地诱变，因此，与现成的遗传和基因组工具一起，克隆突变基因是高度可行的，极大地便利了。另一个优点是生物体的光学透明性和许多基因特异性启动子的可用性，这些启动子可将转基因（如荧光蛋白）引导到特定的细胞，包括神经元。荧光指示器可以简单地通过用荧光修饰细胞来报告细胞的存在，或者作为生物报告者发挥作用，例如基因编码的钙指示器，用于记录区隔（如细胞质）钙的变化。为荧光细胞分选而开发的分选技术已应用于适当大小的多细胞生物的分选，如秀丽隐杆线虫。在这里，我们提出了一个秀丽隐杆线虫的自动分选器，将位于杜克大学，达勒姆，北卡罗来纳州，供杜克大学和邻近的北卡罗来纳大学教堂山分校的一组研究人员使用。三个主要用户将使用该仪器调查与R01拨款相关的具体问题，所有这些都将分离单个活蠕虫，其中荧光转基因将通过特定启动子定向到特定细胞。通过利用蠕虫分选的力量进行高通量分析，再结合向前遗传学，分选机将极大地促进向前遗传学，他们各自的研究议程将被一个量子飞跃放大，在这个水平上没有仪器是不可实现的。Liedtke博士，PI，将使用哺乳动物化蠕虫模型来研究哺乳动物TRPV4阳离子通道的传导机制，该通道指向ASH伤害性头神经元，相当于脊椎动物的三叉神经节，以研究有害渗透刺激反应的感觉传导的分子机制。这些研究将有助于阐明TRPV4在三叉神经痛中的作用。同样来自杜克大学的Aballay博士将研究秀丽隐杆线虫对细菌入侵的免疫反应的分子机制。他的研究将有助于阐明炎症性肠病、败血症和多重耐药细菌感染等疾病的免疫发病机制。北卡罗来纳大学教堂山分校的戈尔茨坦博士将研究原肠胚形成的分子机制，这是所有多细胞生物的早期发育事件，特别是在他的实验室发现的一组新的候选基因。他的研究将增加我们对与人类疾病状态（如神经管闭合缺陷、癌症和先天性心脏病）相关的早期形态发生的细胞和分子机制的理解。一个强大而多样化的小用户项目团体也将做出贡献并从中受益。

DESCRIPTION (provided by applicant): Caenorhabditis elegans, a multicellular roundworm of approximately 1000 differentiated cells in adult animals, offers numerous advantages, namely it has an extremely short generation time, and its genome can be readily mutagenized, so that together with readily available genetic and genomic tools, cloning of mutated genes is highly feasible and greatly facilitated. Another advantage is optic transparency of the organism and availability of numerous gene-specific promoters that direct transgenes such as fluorescent proteins to specific cells, including neurons. Fluorescent indicators can be simply reporting the presence of a cell by decorating it with fluorescence or by functioning as biological reporters, such as genetically-encoded calcium indicators for changes in compartmental (such as cytoplasmic) calcium. Sorting technology developed for fluorescent cell sorting has been applied for sorting of multicellular organisms of appropriate size, such as C. elegans. Here we propose an automated sorter for C. elegans, to be located at Duke University, Durham, NC, for use by a group of investigators at Duke University and neighboring UNC at Chapel Hill. Three major users will be investigating specific questions pertinent to their R01 grants with the proposed instrument, all will be separating individual live worms, in which a fluorescent transgene will be directed to a specific cell by a specific promoter. By harnessing the power of worm sorting to conduct high-throughput assaying, combined with forward genetics, which in turn will be greatly facilitated by the sorter, their respective research agendas will be amplified by a quantum leap, at that level not feasible without the instrument. Dr. Liedtke, the PI, will be using a mammalianized worm model to study transduction mechanisms of the mammalian TRPV4 cation channel directed to the ASH nociceptive head neuron, the cellular equivalent of the trigeminal ganglion of vertebrates, in order to study molecular mechanisms of sensory transduction in response to noxious osmotic stimulation. These studies will help elucidate the role of TRPV4 in trigeminal pain. Dr. Aballay, also of Duke, will be investigating molecular mechanisms of C. elegans' immune response to bacterial invasion. His studies will help elucidate immun-pathogenetic mechanisms operative in diseases such as inflammatory bowel disease, sepsis, and multi-drug resistant bacterial infections. Dr. Goldstein, of UNC Chapel Hill, will be studying molecular mechanisms of gastrulation, an early developmental event of all multicellular organisms, in particular a set of novel candidate genes discovered in his lab. His studies will increase our understanding of cellular and molecular mechanisms of early morphogenesis relating to human disease states such as neural tube closure defects, cancer, and congenital heart disease. A strong and diverse group of minor users' projects will be contributing and benefitting as well.