CAREER: Automated MEMS-based Drosophila Embryo Injection Technologies for High-Throughput Functional Genomics Screens

果蝇是人类生物学中一种重要的模式生物。除了黑腹果蝇的基因组序列外，在过去两年中，其他11种果蝇的基因组序列也已公开。这些基因组序列为研究基因功能及其在发育和疾病中的意义带来了前所未有的机会。基因功能通常是从对生物体基因组成的特定扰动和随后对生物体影响的分析中推断出来的。过去已经建立了两种强大的方法来分析果蝇：用转座遗传元件进行永久遗传转化和通过RNA干扰（RNAi）进行瞬时特异性基因沉默。这两种方法都需要在胚胎发育的早期阶段分别可靠和快速地注射DNA和双链RNA （dsRNA）。在这个NSF CAREER项目的框架内，将开发两个自动化的、基于mems的果蝇胚胎注射系统，称为“搜索和注射”和“喂养和注射”，以实现果蝇胚胎基因功能的高通量筛选。“搜索和注射”将支持RNAi实验；“饲料和注射”将支持基因转化实验。与最先进的人工注射程序相比，自动化注射技术将使实验吞吐量增加约20倍。超越原理证明的强大技术的发展将使它们能够在果蝇研究界传播和广泛使用。根据美国国家科学基金会CAREER奖开发的注射系统将与果蝇胚胎高通量共聚焦成像系统以及可靠的图像分析软件相辅相成，自动识别基因沉默导致的表型。这些新工具的应用将有助于更好地了解人类发育和疾病的分子机制，预计将对新疗法和改善公共卫生状况产生重大影响。提出的研究将推进有关MEMS器件的设计，制造，封装和应用的基础工程知识。所产生的知识可以帮助创建自动化处理DNA、RNA、其他生化试剂、细胞、卵母细胞、胚胎以及微粒子和纳米粒子的系统，广泛应用于生物研究、生物技术、药物发现、高通量筛选和医学诊断。教育和推广活动的目的是吸引初中生、高中生和本科生在科学事业的早期，并培养能够创造性地识别和解决生物学和医学技术需求的新一代工程师。拓展活动包括针对少数民族学生的为期10周的本科生暑期研究项目，针对高中生的为期6周的暑期研究项目，以及针对中学生的周末研讨会。研究也直接整合到两个新的跨学科课程中：“生物医学和生物医学纳米技术”和“干细胞工程”。

0748062ZappeThe fruit fly Drosophila serves as an important model organism for human biology. In addition to the genome sequence of D. melanogaster, the genome sequences of 11 other Drosophila species have been made publicly available over the past two years. These genome sequences have brought unprecedented opportunities to study functions of genes and their implications in development and disease. Gene functions are often inferred from specific perturbation of the genetic makeup of an organism and subsequent analysis of the effects on the organism. Two powerful methods have been established in the past for such analysis in Drosophila: permanent genetic transformation with transposable genetic elements and transient specific gene silencing through RNA interference (RNAi). Both methods require reliable and rapid injection of DNA and doublestranded RNA (dsRNA), respectively, at the earliest stages of embryonic development.Within the frame of this NSF CAREER project, two automated, MEMS-based Drosophila embryo injection systems termed 'Search and Inject' and 'Feed and Inject' will be developed to enable high-throughput screens for gene functions in Drosophila embryos. 'Search and Inject' will support RNAi experiments; 'Feed and Inject' will support genetic transformation experiments. The automated injection technologies will lead to an approximately 20-fold increase in experimental throughput compared to state-of-the-art manual injection procedures. Development of robust technologies beyond proof of principle will enable their dissemination and widespread use within the Drosophila research community. The injection systems developed under the NSF CAREER award will be complemented with a system for high-throughput, confocal imaging of Drosophila embryos as well as image analysis software for reliable, automated recognition of phenotypes due to gene silencing. Application of these new tools will lead to a better understanding of molecular mechanisms of development and disease in humans, with expected significant impact on new therapies and improvement of the state of public health. The proposed research will advance fundamental engineering knowledge regarding design, fabrication, packaging, and application of MEMS devices. The generated knowledge can help create systems for automated handling of DNA, RNA, other biochemical reagents, cells, oocytes, embryos, as well as micro- and nanoparticles, with widespread applications in biological research, biotechnology, drug discovery, high-throughput screening, and medical diagnostics.Educational and outreach activities are designed to interest middle school, high school, and undergraduate students early on in a career in science and to educate a new breed of engineers who can creatively identify and address technological needs in biology and medicine. Outreach activities include a ten-week-long undergraduate summer research program for minority students, a six-week-long summer research program for high school students, and a weekend workshop for middle school students. Research is also directly integrated in two new, interdisciplinary classes: 'BioMEMS and Biomedical Nanotechnology' and 'Stem Cell Engineering'.