Microfluidic-microtiter interface for rapid large-scale screenings of C. elegans

描述（由申请人提供）：损伤后，与再生过程差或不存在的中枢神经系统的轴突不同，周围神经系统的轴突能够再生，尽管效率低。揭示轴突再生背后的分子机制将对创伤性神经损伤的医疗处理具有重要价值，也将为许多神经退行性疾病提供有价值的见解。使用大型脊椎动物模型进行基因鉴定需要复杂的测定和仪器。另一方面，由于它们的简单性和与哺乳动物的巨大相似性，使用小型无脊椎动物对快速全基因组筛选具有极大的兴趣。在这些动物中，线虫秀丽隐杆线虫（C. elegans）是提供广泛的遗传工具的最强大的模式生物之一。无论是研究给定基因的表型，还是研究特定表型中涉及的基因，正向和反向遗传学都特别通用。然而，直到最近，C. elegans用于神经再生研究是有限的，由于缺乏精确的手术技术，轴突切断。 我们最近的两项技术创新，超快激光纳米手术和微流体固定，使研究这种遗传上易于处理的模型生物体的轴突再生成为可能。虽然超快激光纳米手术最终使我们能够以高精度切断这种小蠕虫的轴突，但微流体固定芯片能够在不使用任何麻醉剂的情况下快速捕获蠕虫进行手术，将执行手术所需的时间减少了100倍（从几十分钟到几秒），并消除了麻醉剂可能的副作用。这种微流控芯片最终提供了进行高通量筛选的可能性，前提是可以自动加载大量样品而不是手动处理。 本研究的目标有三个：（1）制作一个微流控装置，可以自动将蠕虫从多孔板输送到微流控轴切芯片，（2）开发其计算机辅助自动化，（3）展示集成系统的性能，用于快速筛选影响C轴突再生的候选基因。RNA干扰（RNAi）。这种新的装置将有助于操纵大人口样本的轴突切断术芯片和他们的存储后，轴突切断术的再生结果的进一步研究。这种高通量筛选平台的开发需要整合用于RNAi喂养、纳米外科手术、恢复和成像的不同模块，以及通过计算机控制的自动化进行同步。一个能够从可单独寻址的威尔斯孔中处理1000个蠕虫的平台将有助于任何自动筛选研究，从而大大减少时间和成本。 公共卫生相关性：加速大规模C。elegans，我们提出设计一种新的微流体多路复用器，以与标准孔板（微量滴定仪）接口，用于将蠕虫从各个威尔斯自动且精确地转移到不同的成像和手术微流体模块中。这种微流体多路复用器的成功自动化将使我们能够使用RNAi干扰和飞秒激光纳米轴突切断术对影响神经再生的基因进行高通量筛选。由于人与C. elegans，更好地了解蠕虫神经再生的分子机制，最终将有助于开发人类退行性疾病的治疗和预防。

DESCRIPTION (provided by applicant): After injury, unlike the axons of the central nervous system where the regeneration process is poor or absent, axons of the peripheral nervous system are able to regenerate, though inefficiently. Uncovering the molecular mechanisms behind axon regeneration will be of great value for the medical handling of traumatic nerve injuries and will also offer valuable insights regarding many neurodegenerative diseases. The use of large vertebrate animal models for gene identification requires complex assays and instrumentations. On the other hand, thanks to their simplicity and large similarities to mammals, the use of small invertebrate animals is of great interest for rapid genome-wide screenings. Among these animals, the nematode Caenorhabditis elegans (C. elegans) is one of the most powerful model organisms providing a wide-range of genetic tools. It is especially versatile to forward and reverse genetics, whether phenotypes of a given gene are investigated or genes involved in a specific phenotype are. However, until recently, the use of C. elegans for nerve regeneration studies was limited due to a lack of precise surgical techniques for axotomy. Two of our recent technological innovations, ultrafast laser nanosurgery and microfluidics immobilization have made it feasible to study axonal regeneration in this genetically tractable model organism. While the ultrafast laser nanosurgery has finally enabled us to severe axons of this small worm with high precision, the microfluidic immobilization chip has enabled rapid trapping of worms for surgery without using any anesthetics, reducing the time required to perform the surgery by a factor of 100 (from tens of minutes to several seconds) and eliminating the possible side effects of the anesthetics. Such microfluidic chip finally offers the possibility to perform high-throughput screening, provided that large sample populations can be automatically loaded instead of manual handling. The goal of this research project is threefold: (1) fabricate a microfluidic device that can automatically deliver worms from multi-well plates to the microfluidic axotomy chip, (2) develop its computer assisted automation, and (3) demonstrate the performance of the integrated system for rapid screening of candidate genes affecting axonal regeneration in C. elegans by RNA interference (RNAi). This novel device will facilitate the manipulation of large population samples for delivery to the axotomy chip and for their storage after the axotomy for further study of the regeneration results. Development of such a high-throughput screening platform requires integration of different modules for RNAi feeding, nanosurgery, recovery, and imaging and their synchronization through computer controlled automation. A platform capable of handling 1000's of worms from individually addressable wells will facilitate any automated screening studies, thus greatly reducing time and cost. PUBLIC HEALTH RELEVANCE: To accelerate large-scale screening of C. elegans, we propose to engineer a novel microfluidic multiplexer to interface with standard well-plates (microtiters) for transferring worms automatically and precisely from individual wells into different imaging and surgery microfluidic modules. The successful automation of this microfluidic multiplexer will allow us to perform high throughput screening of genes affecting nerve regeneration using RNAi interference and femtosecond laser nano-axotomy. Owing to the genetic similarity between human and C. elegans, a better understanding of the molecular mechanisms underlying nerve regeneration in the worms will eventually enable the development of treatments and preventions of human degenerative diseases.