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

项目总结 我们建议开发和验证细胞内染色荧光激活细胞分选器模板 寡核苷酸测序基因图谱分析，icsFACS/TEMPO-SEQ，通过以下方式解决未满足的需求 实现固定、渗透和细胞内染色的低成本、定量的基因表达分析 分类细胞(一种用于纯化许多不同的功能重要和稀有的细胞子集的方法)，包括 单电池，在FACSAria和台式BD FACSMelody、Sony SH800和BioRad S3e分选器上都是如此。我们 在第一阶段成功论证了可行性，开发了两种方案来分析分类的单个FAC 细胞。此外，我们还展示了关于随机表达的新的观察结果 单细胞水平的基因，这是由于分析的性能和几乎没有背景信号 可以测量在固定时在单个细胞中失去的基因的真实“0”表达水平。在……里面 第二阶段，我们将优化这些方案，并验证用于人类和小鼠的检测菜单--整体 转录组分析，一种替代试验，由替代试验和免疫反应组成的小组 老鼠和人类的基因，以及由宿主反应基因、HIV-1病毒RNA和DNA组成的小组。 我们将演示如何生成正常T细胞体外激活的时间进程数据 这些子集内的单个细胞。最后，我们将在演示实用程序的过程中生成新的数据 与专家顾问合作建立的小鼠模型，以及从HIV-1患者那里获得的数据 在与艾滋病毒研究专家的检测客户安排中。在小鼠模型中，我们将分析GC B细胞 接受程序化轮次的高度突变和选择(增殖)并监测子集纯度 臭名昭著的粘连表面染色表型。在人体模型中，-TEST客户端将分析FAC 从接受三重抗逆转录病毒治疗(ART)的HIV-1感染患者中分选样本以表征 宿主RNA反应与CD4T记忆细胞内HIV-1前病毒DNA和病毒mRNA 并计数残留的HIV-CD4T细胞。使用的化验将包括病毒的测量 RNA和DNA同时作为宿主基因。制定了ICSFACS/TEMPO-SEQ方案和检测方法 和经验证的将解决在流式细胞术和免疫学方面未得到满足的需求，涉及范围越来越广的 疾病，包括传染病、炎症、癌症、中风和阿尔茨海默氏症等等。 FACSAria和台式分选机的验证将满足核心实验室及其客户的需求 选择使用或要求这些台式分选器具有专门的单电池性能的研究人员。 它还为在这些台式分拣平台上开发诊断分析奠定了基础 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.