TempO-Seq Gene Expression Profiling of Intracellular Stained FACS Sorted Cells

项目摘要 我们建议开发和验证一种细胞内染色的荧光激活细胞分选仪模板 寡核苷酸测序基因分析检测，icsFACS/TempO-Seq，通过以下方式解决未满足的需求 能够对固定的、透化的和细胞内染色的低成本、定量基因表达分析， 分选细胞（用于纯化许多不同功能重要和罕见细胞亚群的方法），包括 单细胞，在FACSAria和台式BD FACSMelody、Sony SH 800和BioRad S3 e分选仪上。我们 在第一阶段成功地证明了可行性，开发了两种用于分析单个FACS分选的方案， 细胞此外，我们能够证明新的观察有关的随机表达， 由于测定的性能和实际上不存在背景信号，因此基因处于单细胞水平， 可以测量在固定时在单细胞中关闭的基因的真实“0”表达水平。在 在第二阶段，我们将优化这些方案，并验证一系列针对人类和小鼠的检测方法--一个完整的 转录组测定、替代测定、由替代测定加上免疫应答组成的组 小鼠和人类的基因，以及由宿主反应基因、HIV-1病毒RNA和DNA组成的一组基因。 我们将证明产生正常T细胞的时间过程数据的实用性， 在这些子集中的单个细胞。最后，我们将在演示实用程序的过程中生成新数据， 与专家顾问合作进行的小鼠模型，以及从HIV-1患者获得的数据 与一位艾滋病研究专家进行了一次测试性客户安排。在小鼠模型中，我们将分析GC B细胞 进行程序化的多轮超突变和选择（增殖），并在 臭名昭著的表面染色表型在人体模型中，受试者将分析FACS 从接受三联抗逆转录病毒治疗（ART）的HIV-1感染患者中分选样本， 宿主RNA应答和HIV-1前病毒DNA和病毒mRNA在CD 4 + T记忆细胞中的存在 隔室并计数残留的HIV+ CD 4 + T细胞。所用试验将包括测量病毒 RNA和DNA同时作为宿主基因。开发的icsFACS/TempO-Seq方案和测定法 经过验证，将解决流式细胞术和免疫学在越来越多的领域中未满足的需求。 疾病，包括传染病、炎症、癌症、中风和阿尔茨海默氏症，仅举几例。 FACSAria和台式分拣机的验证将满足核心实验室及其客户的需求， 选择使用这些台式分选机或需要这些台式分选机的专门单细胞性能的研究人员。 它还为在这些台式分选机平台上开发诊断测定奠定了基础， icsFACS/TempO-Seq.

PROJECT SUMMARY We propose to develop and validate an intracellular stained Fluorescence Activated Cell Sorter Templated Oligonucleotide Sequencing gene profiling assay, icsFACS/TempO-Seq, addressing an unmet need by enabling low-cost, quantitative gene expression analysis of fixed, permeabilized, and intracellular stained and sorted cells (a method used to purify many different functionally important and rare subsets of cells), including single cells, on both the FACSAria and benchtop BD FACSMelody, Sony SH800, and BioRad S3e sorters. We successfully demonstrated feasibility in Phase I, developing two protocols for profiling single FACS sorted cells. In addition, we were able to demonstrate novel observations regarding the stochastic expression of genes at the single cell level due to the performance of the assay and virtual absence of background signal so that true ”0” expression levels of genes that were off in single cells at the time of fixation could be measured. In Phase II we will optimize these protocols and validate a menu of assays for human and mouse – a whole transcriptome assay, a surrogate assay, a panel comprised of the surrogate assay plus immune response genes for both mouse and human, and a panel comprised of host response genes, HIV-1 viral RNA and DNA. We will demonstrate utility to generate time course data of normal T-cells activated ex vivo for subsets and single cells within those subsets. Finally, we will generate novel data in the course of demonstrating utility in a mouse model carried out in collaboration with an expert consultant, and by data obtained from HIV-1 patients in a -test client arrangement with an expert in HIV research. In the mouse model we will profile GC B cells undergoing programmed rounds of hypermutation and selection (proliferation) and monitor subset purity in a notoriously coalescing surface-staining phenotype. In the human model, the -test client will profile FACS sorted samples from HIV-1 infected patients undergoing triple antiretroviral therapy (ART) to characterize the host RNA response and HIV-1 provirus DNA and viral mRNA harbored within the CD4+ T memory cell compartments and enumerate residual HIV+ CD4+ T cells. The assay used will include measurement of viral RNA and DNA at the same time as host genes. The icsFACS/TempO-Seq protocols and assays developed and validated will address unmet needs in flow cytometry and immunology across a growing range of diseases, including infectious diseases, inflammation, cancer, stroke, and Alzheimer’s to name a few. Validation for both the FACSAria and benchtop sorters will address the needs of core labs and their clients and investigators who choose to use, or require the specialized single cell performance, of these benchtop sorters. It also lays the groundwork for the development of diagnostic assays on these benchtop sorter platforms using icsFACS/TempO-Seq.