Quantification of gene expression in targeted rare cells in vivo

体内靶稀有细胞基因表达的定量

• 批准号: 7945366
• 负责人: TIMOTHY R BRAZELTON
• 金额: $ 49.98万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7945366
• 关键词: Address; Aging; American; Animals; Area; Award; Biological Assay; Biological Process; Biotechnology; Budgets; Cells; Cities; Complex; Data; Employment; Environment; Evaluation; Facilities and Administrative Costs; Failure; Fibroblasts; Fostering; Functional disorder; Funding; Gene Expression; Gene Expression Profile; Genes; Genomics; Hospitals; Human; Hybridization Array; In Vitro; Individual; Malignant Neoplasms; Mediator of activation protein; Messenger RNA; Metabolism; Methods; Molecular; Monitor; Muscle; Muscle satellite cell; Muscular Dystrophies; Myoblasts; Nature; Occupations; Organ; Pattern; Pediatric Hospitals; Pennsylvania; Pericytes; Play; Positioning Attribute; Process; Proteomics; Protocols documentation; Recreation; Regulatory Pathway; Research; Research Support; Role; Sampling; Signal Transduction; Skeletal Muscle; Skeletal Muscle Satellite Cells; Solid; Species Specificity; Specific qualifier value; Stem cells; Students; Techniques; Technology; Testing; Time; Tissue Sample; Tissues; Transcript; United States; United States National Institutes of Health; Universities; Work; Xenograft Model; Xenograft procedure; aged; base; cancer cell; cell behavior; cell type; density; economic impact; genome-wide; in vivo; member; metabolomics; muscle regeneration; novel; operation; response; satellite cell; scale up; self-renewal; stem cell biology; stem cell population; technique development; tissue regeneration; tumor progression

DESCRIPTION (provided by applicant): This application is intended for the (06) "Enabling Technologies" challenge area and (06- HG-102*) "Technologies for obtaining genomic, proteomic, and metabolomic data from individual viable cells in complex tissue" challenge topic. The specialized microenvironment surrounding stem cells or cancer cells is thought to be a major regulator of critical functions such as stem cell quiescence, self-renewal, proliferation and differentiation, as well as cancer progression. The amount of speculation implicating microenvironments in these processes currently exceeds the ability to generate supporting experimental data. This is, in part, due to the nature of micro-environments: they must be studied in vivo as in vitro recreations have not been convincing; the roles of individual cell types are often unclear; and there is a lack of techniques to systematically evaluate micro-environmental regulatory pathways in an unbiased manner. One means of evaluating cellular responses is to monitor changes in gene expression. We have established new methods that increase sensitivity and species-selectivity of TaqMan-based probes when combined with a PCR-based pre-amplification protocol. This allows homologous transcripts from two closely-related species to be distinguished, even when the mRNA from one species is up to 100,000,000-fold higher than in the other species. Thus, allows the quantification of gene expression in as few as 1-10 cells of a specified type (e.g., muscle stem cells) as they reside in cellular heterogeneous in vivo microenvironments in a distinct species. Similarly, this Q-PCR- based approach can be used to quantify the total contribution of a stem cell population to a specific tissue, allowing repopulating assays to be efficiently completed on solid organs. Finally, the utility of this approach to prospectively isolate satellite cells from skeletal muscle at the single cell level is explored. While these advances in Q-PCR will have a large impact on the study up to 200 genes per sample, the current techniques are not scalable to genome wide discovery. Several alternative approaches to scale up species-specific gene quantification are explored. While specific studies are proposed here, we believe that this powerful approach is broadly applicable to the study of a wide variety of biological processes such as tissue regeneration and cancer. While our primary rational for creating this technology is to identify the molecular mechanisms of regulating skeletal muscle stem cells, this technique has broad applicability to study the reciprocal interactions between stem cells and their niche as well as malignant cells within the supporting stroma. CHOP contributes substantially to the local economy. In 2008, CHOP's operations created and supported over 16,882 jobs in the region, and CHOP's total economic impact was over $2.01 billion. Moreover, through a combination of private donations, NIH funding, and allocations from its hospital operations, CHOP receives more total research support than any other children's hospital in the United States -- $180 million in fiscal year 2007-2008. The direct funding in this proposal will create or retain 4 full-time staff positions in academic research plus 2 trainee/work study positions for undergraduate students at the University of Pennsylvania. In addition, $390,000 in indirect costs will directly enable an additional 4 CHOP staff members to retain their employment. Furthermore, approximately, over 95% of the material and supplies will be purchased from American, biotechnology companies. Approximately 50% of the supply budget will go to Applied Biosystems in Foster City, CA where it will create an estimated 1.3 jobs. Thus, this award will create or maintain approximately 11.3 U.S. jobs. The specialized microenvironments surround stem cells or cancer cells and are thought to be major regulators of cell behavior. We present here a technique to quantify expression of up to 100 genes in 1-10 cells residing in intact tissue in an animal. This powerful approach is broadly applicable to the study of a wide variety of biological processes. Specifically, we propose that these techniques can distinguish gene expression patterns in a selected cell type in gross tissue samples and allow the recognition of regulatory pathways and metabolic processes that are fundamental to stem cell and cancer cell behaviors.

描述（由申请人提供）：此应用程序旨在（06）“启用技术”挑战区域和（06-HG-102*）“从复杂组织中获得基因组，蛋白质组学和代谢细胞的基因组，蛋白质组学和代谢组数据的技术”。围绕干细胞或癌细胞周围的专门微环境被认为是关键功能的主要调节剂，例如干细胞静止，自我更新，增殖和分化以及癌症的进展。目前，在这些过程中暗示微环境的投机量超出了生成支持实验数据的能力。这在某种程度上是由于微环境的性质：必须在体内研究它们，因为体外娱乐并不令人信服。单个细胞类型的作用通常不清楚。而且缺乏系统地评估微环境调节途径的技术。评估细胞反应的一种方法是监测基因表达的变化。我们已经建立了新方法，以与基于PCR的基于PCR的预扩增方案结合使用，从而提高了基于Taqman的探针的敏感性和物种选择性。这允许从两个密切相关物种的同源转录本被区分，即使来自一个物种的mRNA比其他物种高至100,000,000倍。因此，允许将基因表达定量在少于指定类型的1-10个细胞（例如肌肉干细胞）中，因为它们驻留在不同物种的体内微环境中的细胞异质性微环境中。同样，这种基于Q-PCR的方法可用于量化干细胞群体对特定组织的总贡献，从而可以在固体器官上有效地完成重量测定。最后，探索了这种方法对前瞻性分离卫星细胞与单细胞水平上的骨骼肌的实用性。尽管Q-PCR中的这些进展将对每个样品的研究最多200个基因产生很大的影响，但当前技术不可扩展到基因组广泛的发现。探索了几种扩展物种特异性基因定量的替代方法。尽管这里提出了特定的研究，但我们认为这种强大的方法广泛适用于对组织再生和癌症等各种生物学过程的研究。尽管我们创建这项技术的主要理性是确定调节骨骼肌干细胞的分子机制，但该技术在研究干细胞及其利基市场之间以及支持基质中的恶性细胞之间具有广泛的适用性。 CHOP对当地经济做出了重大贡献。 2008年，CHOP的运营创造并为该地区的16,882个工作岗位提供了支持，而CHOP的总经济影响超过了20.1亿美元。此外，通过将私人捐款，NIH资金和医院运营分配的结合，与美国其他任何儿童医院相比，CHOP获得了更多的研究支持 - 在2007-2008财政年度为1.8亿美元。该提案的直接资金将为宾夕法尼亚大学的本科生创建或保留4个全职员工职位，以及2个学术研究人员/工作研究职位。此外，390,000美元的间接费用将直接使额外的4个砍伐人员能够保留其工作。此外，大约95％的材料和耗材将从美国生物技术公司购买。供应预算的大约50％将用于加利福尼亚州福斯特市的应用生物系统，在那里它将创造大约1.3个就业机会。因此，该奖项将创造或维持约11.3个美国工作岗位。专门的微环境围绕干细胞或癌细胞，被认为是细胞行为的主要调节剂。我们在这里提出了一种量化在动物完整组织中的1-10个细胞中多达100个基因表达的技术。这种强大的方法广泛适用于对各种生物学过程的研究。具体而言，我们建议这些技术可以区分总体组织样品中选定的细胞类型中的基因表达模式，并允许识别对干细胞和癌细胞行为至关重要的调节途径和代谢过程。