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

细胞内染色 FACS 分选细胞的 TempO-Seq 基因表达谱

基本信息

批准号：
9410000
负责人：
BRUCE E. SELIGMANN
金额：
$ 192.39万
依托单位：
BIOSPYDER TECHNOLOGIES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-02-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9410000
关键词：
APEX1 gene Address Alzheimer&apos s Disease Antigens Area Autoimmune Process B-Lymphocytes Biological Assay Blast Cell CD4 Positive T Lymphocytes CD86 gene CD8B1 gene CXCR4 gene Cell Compartmentation Cell Cycle Cell Line Cells Centrocyte Cessation of life Client Collaborations Communicable Diseases Contracts DNA Data Dementia Development Disease Evaluation Flow Cytometry Gene Expression Gene Expression Profiling Genes Germinal Center B-Lymphocyte Government HIV HIV-1 Human Immune Immune Response Genes Immune response Immunity Immunization Immunology Infectious Diseases Research Inflammation Inflammatory Malignant Neoplasms Measurement Measures Messenger RNA Methods Modeling Molecular Biology Monitor Mus Mutation Names National Human Genome Research Institute Normal Cell Oligonucleotides Patients Performance Persons Phase Phenotype Protocols documentation Proviruses RNA Research Research Personnel Residual state Reverse Transcription Sampling Signal Transduction Sorting - Cell Movement Staining method Stains Stroke Structure of germinal center of lymph node Surface Surface Antigens T-Lymphocyte T-Lymphocyte Subsets Testing Text Therapeutic Time Transcriptional Regulation Validation Viral antiretroviral therapy base cell type cost diagnostic assay experimental study fluorescence activated cell sorter device frontier gene product genomic tools human DNA innovation instrument interest memory CD4 T lymphocyte mouse model novel novel diagnostics research and development response sample fixation single cell analysis tool transcriptome transcriptome sequencing vaccine development viral DNA viral RNA virtual

项目摘要

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.

项目摘要我们建议开发和验证细胞内染色的荧光激活的细胞分散器模板寡核苷酸测序基因分析测定法，ICSFACS/TEMPO-SEQ，以满足未满足的需求实现固定，透化和细胞内染色的低成本，定量基因表达分析，分类的单元格（一种用于净化许多不同功能上重要和稀有细胞子集的方法），包括单细胞在FACSARIA和BANCHTOP BD FACSMELODY，SONY SH800和BIORAD S3E SORETER上。我们成功证明了在第一阶段的可行性，开发了两个用于分析单个FACS的协议细胞。此外，我们能够证明有关随机表达的新发现由于测定的性能和虚拟背景信号的实际情况，单细胞水平的基因可以测量在固定时在单个细胞中关闭基因的真实“ 0”表达水平。在第二阶段我们将优化这些协议并验证人和鼠标的测定菜单 - 整个转录组测定法，替代测定法，由替代测定和免疫反应组成的面板小鼠和人类的基因，以及由宿主反应基因，HIV-1病毒RNA和DNA组成的面板。我们将展示实用程序，以生成正常T细胞激活的子集激活的子集的时间课程数据这些子集中的单元单元。最后，我们将在展示实用程序的过程中生成新的数据与专家顾问合作进行的鼠标模型，并通过HIV-1患者获得的数据在与艾滋病毒研究专家的测试客户安排中。在鼠标模型中，我们将介绍GC B细胞进行编程的超松和选择（增殖）的回合，并监视子集纯度臭名昭著地结合表面染色表型。在人类模型中，测试客户将介绍FACS 从HIV-1感染三重抗逆转录病毒疗法（ART）的样本中，以表征宿主RNA响应和HIV-1病毒DNA和病毒mRNA在CD4+ T记忆细胞中备有隔室和列举残留的HIV+ CD4+ T细胞。所使用的测定将包括病毒的测量 RNA和DNA与宿主基因同时。开发了ICSFACS/TEMPO-SEQ协议和测定经过验证将解决流式细胞仪和免疫学的未满足需求包括传染病，感染，癌症，中风和阿尔茨海默氏病，包括几种疾病。 Facsaria和Benchtop分选项的验证将满足核心实验室及其客户的需求，并选择使用或需要专门的单细胞性能的调查人员，这些台式分配器。它还为在这些台式分类器平台上开发诊断测定的基础奠定了基础 ICSFACS/TEMPO-SEQ。