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

描述（由申请人提供）：损伤后，与中枢神经系统轴突的再生过程较差或缺失不同，周围神经系统的轴突能够再生，尽管效率较低。揭示轴突再生背后的分子机制将对创伤性神经损伤的医学处理具有重要价值，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分析和仪器。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非常感兴趣。在这些动物中，秀丽隐杆线虫（秀丽隐杆线虫）是最强大的模式生物之一，提供了广泛的遗传工具。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，向前和反向遗传学都是特别通用的。然而，直到最近，秀丽隐杆线虫在神经再生研究中的使用受到限制，因为缺乏精确的手术技术进行轴截。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化，使得研究这种遗传上可处理的模式生物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高精度地切断这种小蠕虫的轴突，但微流体固定芯片可以在不使用任何麻醉剂的情况下快速捕获蠕虫进行手术，将手术所需的时间减少了100倍（从几十分钟到几秒钟），并消除了麻醉药可能产生的副作用。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样本群，而不是人工处理。本研究项目的目标有三个：(1)制造一种微流控装置，可以自动将蠕虫从多孔板输送到微流控轴突切割芯片上；(2)开发其计算机辅助自动化；(3)通过RNA干扰（RNAi）快速筛选影响秀丽隐杆线虫轴突再生的候选基因的集成系统的性能。这种新颖的装置将便于操作大量的样本，以便将其运送到轴切开术芯片中，并在轴切开术后将其存储，以进一步研究再生结果。开发这样的高通量筛选平台需要集成RNAi馈送、纳米手术、恢复和成像的不同模块，并通过计算机控制的自动化进行同步。一个能够处理来自单个可寻址井的数千个蠕虫的平台将有助于任何自动化筛选研究，从而大大减少时间和成本。

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.