QSB: Charting the Regulatory Space of Hematopoiesis by Microfluidic Digital PCR

QSB：通过微流控数字 PCR 绘制造血调控空间

• 批准号: 0535870
• 负责人: Stephen Quake
• 金额: --
• 依托单位: Stanford University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-04 至 2008-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0535870&HistoricalAwards=false
• 关键词: QSB; Charting; Regulatory; Space; Hematopoiesis

All the cells of a developing organism carry the same instructions for making proteins, yet the execution of the genetic program is systematically varied to produce the specialized tissues that comprise the fully-formed adult. While the character of each cell reflects the expression of many thousands of genes, this expression pattern is itself determined by a smaller network of regulatory genes, or 'transcription factors.' The causal basis for cellular differentiation can be traced back to the activity of these regulatory networks. For example, in response to diffusive signals called cytokines, self-renewing hematopoietic stem cells give rise to daughter cells that commit to progressively more specialized fates, ultimately becoming terminally differentiated cells, such as erythrocytes, macrophages, and the B and T cells of immunity. This project will develop and apply a new technology - a digital RT-PCR assay using silicone elastomer microfluidic chips - to take a focused look at the behavior of genetic regulatory networks in individual developing blood cells. This research will profile the activity of networks of genes implicated as hematopoietic regulators with single-cell resolution. A database of such profiles will reveal how network activity varies within ostensibly uniform populations of progenitor cells and support comparisons between distinct, developmentally-staged populations. RT-PCR readings will be collated with flow cytometry data to facilitate population analysis that encompasses both surface marker phenotype and internal regulatory network state. Sibling assay studies will be conducted to explore the possibility that metastable regulatory network states bias lineage choice responses to instructive signaling from cytokines.Broader Impact: This project will further creation of new, interdisciplinary approaches to biological investigations by integrating mainstream developmental genetics, advanced 'lab-on-a-chip' technology, and a quantitative, engineering-oriented approach to the analysis of complex systems. Software developed for the project, including a powerful new program for designing multiplexed PCR primers, will be made available to the research community on an open source basis. In addition, this project will provide opportunity for cross-disciplinary education and training of graduate students; undergraduate students will be involved through a CalTech- sponsored summer research fellowship program (SURF). Valuable research experience will also be available to underrepresented groups through investigator involvement with the Pasadena City College Biotech Program that sponsors research science internships by providing opportunity for interns to work on this cross-disciplinary project.

发育中的有机体的所有细胞都携带着相同的蛋白质制造指令，但遗传程序的执行是系统性变化的，以产生构成完全形成的成体的专门组织。虽然每个细胞的特征反映了数千个基因的表达，但这种表达模式本身是由较小的调节基因或转录因子网络决定的。细胞分化的因果基础可以追溯到这些调节网络的活动。例如，响应于被称为细胞因子的扩散信号，自我更新的造血干细胞产生子细胞，子细胞致力于逐渐更特化的命运，最终成为终末分化的细胞，如红细胞、巨噬细胞以及免疫的B和T细胞。该项目将开发和应用一种新技术-使用硅橡胶微流控芯片的数字RT-PCR分析-重点关注个体发育中血细胞的遗传调控网络的行为。这项研究将描绘作为造血调节因子的基因网络的活性，具有单细胞分辨率。这样的数据库将揭示网络活动如何在表面上均匀的祖细胞群体中变化，并支持不同的发育阶段群体之间的比较。将RT-PCR读数与流式细胞术数据进行比较，以促进包括表面标志物表型和内部调控网络状态的群体分析。同胞分析研究将进行探索的可能性，亚稳态调节网络状态的偏见谱系选择反应的指导性信号从cytokines.Broader影响：该项目将进一步创建新的，跨学科的方法，生物学研究整合主流发育遗传学，先进的“芯片实验室”技术，和定量，工程导向的方法来分析复杂的系统。为该项目开发的软件，包括用于设计多重PCR引物的功能强大的新程序，将以开放源代码的方式提供给研究界。 此外，该项目将为研究生提供跨学科教育和培训的机会;本科生将通过加州理工学院赞助的夏季研究奖学金计划（SURF）参与。 宝贵的研究经验也将提供给代表性不足的群体，通过研究人员参与帕萨迪纳城市学院生物技术计划，赞助研究科学实习，为实习生提供机会，在这个跨学科的项目工作。