Living Cell Arrays for Real Time Functional Genomics

描述(由申请人提供)：新的工具正在给生物医学研究带来革命性的变化，使关于正常和患病状态下的基因、蛋白质及其结构和功能的数据的获取呈指数级增长。在这些进步中，大规模监测基因图谱的能力是值得注意的。然而，通常很难将正常和异常状态下观察到的趋势和关系与基因表达谱所产生的表型联系起来。对于涉及代谢紊乱的生理状态，基因表达谱不能完全解释所涉及的复杂分子机制。因此，为了全面了解代谢状态，既要了解基因表达事件，也要了解控制代谢物变化的细胞质事件。拟议的生物工程研究伙伴关系寻求开发一种新的功能基因组学方法来研究基因表达：使用完整的细胞以大规模平行、高通量的形式使用Aequorin型荧光蛋白(AFP)同时分析多个基因的时间表达谱。具体目标是：(1)建立和鉴定一组监测炎症细胞因子信号转导通路中主要事件的报告细胞系；(2)设计一个微流控系统，动态控制一组原代大鼠肝细胞和H35肝癌报告细胞的输入刺激和荧光反应；(3)表征细胞因子信号转导的动力学以及脂肪变性和不同代谢状态对这些细胞急性时相反应的影响。这个项目将由三个不同的研究小组进行，他们将进行广泛的互动。首席研究员Martin Yarmush(MGH)博士将监督行政和技术核心，并领导细胞生理学和成像小组，该小组将整合其他PL开发的技术，并进行大部分细胞生理学实验。MGH微尺度工程设施主任Mehmet Toner博士将领导微制造和微流体小组，专注于微制造设备的开发。分子生物学家和基因治疗专家杰弗里·摩根博士(布朗大学，普罗维登斯，RI)将领导专注于报告细胞系开发的分子生物学和细胞分析小组。

DESCRIPTION (provided by applicant): New tools are revolutionizing biomedical research, enabling an exponential growth in the acquisition of data regarding genes, proteins, and their structures and functions in normal and diseased states. Among these advances, the ability to monitor profiles of genes on a large scale is notable. However, it is often difficult to correlate the trends and relationships observed in normal and abnormal states to the phenotype resulting from the gene expression profile. For physiological states involving metabolic derangements, gene expression profiling does not fully explain the complex molecular mechanisms involved. Thus, in order to develop a comprehensive understanding of metabolic states, it is essential to understand both the gene expression events as well as the cytoplasmic events, which control changes in metabolites. The proposed Bioengineering Research Partnership seeks to develop a new functional genomics approach for studying gene expression: the use of intact cells for the simultaneous temporal expression profiling of multiple genes using aequorin-type fluorescent proteins (AFP) in a massively parallel, high throughput format. The specific aims are: (1) to generate and characterize a panel of reporter cell lines that monitors the major events in the inflammatory cytokine signaling cascades; (2) to design a microfluidic system to dynamically control the input stimulus as well as the fluorescent response of an array of primary rat hepatocytes and H35 hepatoma reporter cell lines; (3) to characterize the dynamics of cytokine signal transduction and the impact of steatosis and different metabolic states on the acute phase response of these cells. This project will be carried out by three distinct research groups, which will interact extensively. Dr. Martin Yarmush (MGH), the Principal Investigator, will oversee the Administrative and Technical Core, and lead the Cell Physiology and Imaging group, which will integrate the technologies developed by the other Pl's and carry out the bulk of the cell physiology experiments. Dr. Mehmet Toner, Director of the Microscale Engineering Facility at the MGH, will lead the Microfabrication and Microfluidics group focusing on the development of microfabricated devices. Dr. Jeffrey Morgan (Brown Univ., Providence, RI), a molecular biologist and expert in gene therapy, will lead the Molecular Biology and Cell Analysis group focusing on reporter cell line development.