Infectious Diseases Research Technologies Core - RML

传染病研究技术核心 - RML

基本信息

批准号：
8336696
负责人：
Robert Hohman
金额：
$ 289.29万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8336696
关键词：
3-Dimensional Address Alternative Splicing Antigens Area Bacteria Biological Biological Assay Biological Preservation Biotechnology Blood capillaries Cells Collaborations Communicable Diseases Computers Confocal Microscopy Containment Contrast Media Copy Number Polymorphism Custom DNA Microarray Chip DNA Sequence Data Detection Development Dimensions Discipline Discrimination Electron Microscope Electron Microscopy Electrons Environment Equipment Eukaryotic Cell Experimental Designs Explosion Fixatives Flow Cytometry Freeze Substitution Freezing Genes Genome Genomics Genotype Hour Human Hypersensitivity Ice Image Imagery Imaging technology Immunology Industry Infectious Diseases Research Institutes Internet Intervention Intramural Research Intramural Research Program Investigation Laboratories Lasers Life Light Link Macromolecular Complexes Maps Metabolic Methods Microarray Analysis Microscope Microscopy Mission Molecular Biology National Institute of Allergy and Infectious Disease Optics Organelles Organic solvent product Parasites Phenotype Play Preparation Process Proteomics Protocols documentation Reaction Research Research Personnel Resolution Rest Role SNP genotyping Sampling Scanning Scientist Site Small RNA Solutions Sorting - Cell Movement Specimen Structure System Technology Testing Time Titania Titanium Training Transportation United States National Institutes of Health Vaccines Validation Variant Virus base biodefense capillary data management data mining design exome flexibility genome sequencing improved instrument instrumentation irradiation microwave electromagnetic radiation new technology next generation pathogen programs research and development research study technology development technology/technique tissue/cell culture transcriptomics transmission process

项目摘要

This project provides state-of-the-art research technologies for NIAID's intramural infectious diseases, allergy, and immunology research programs. The new technologies are developed and validated and then applied in support of NIAID research. Technologies developed outside the NIH are likewise tested, evaluated, validated and, if appropriate, incorporated into the technology portfolio of the NIAID intramural program. The technologies supported include flow cytometry, confocal microscopy, electron microscopy, DNA microarray, DNA sequencing, Next Generation sequencing, bacterial phenotyping and quantitative PCR. Many of these technologies are used in high containment laboratories critical to the Institute's infectious diseases and biodefense research agenda. In addition to technology development, the RTB provides advanced training in all aspects of the technologies in the Branch's portfolio.   Sequencing.  The RTB develops applications using capillary DNA sequencing technology. Applications are developed in close collaboration with DIR investigators. All data is uploaded to a server, which tracks and manages all of the sequencing data generated for the Institute.   Next Generation sequencing employs the Illumina GA II, the Illumina HiSeq 2000, and the 454 FLX Titanium sequencers towards small RNA discovery, ChIP-Seq, transcriptomics, exome sequencing, de novo and ref-map genome sequencing, and copy number variation studies. Phenotyping microarray.  The RTB develops applications using a phenotyping microarray technology. This technology has the capability to analyze >2,000 metabolic reactions for bacterial strains, clones, or tissue culture cells through the use of a 96-well plate format.   Microarrays . The RTB develops project-specific research applications on Affymetrix microarray platform including custom chip design, experimental design, sample processing and chip processing. In addition to developing new applications for microarray research, the RTB develops statistical analysis, data management, and data mining solutions for DIR research programs; focusing on interpreting data generated by highly parallel detection systems used in genomics. The RTB also performs QPCR for high throughput microarray data validation, sample optimization, and Next gen data validation    Human/Pathogen Genotyping . Several technologies are used for human and pathogen genotyping depending on the scope of the genotyping project and they range from capillary-based re-sequencing of entire human genes for de novo SNP or In/Del discovery, to high throughput targeted SNP genotyping via allelic discrimination assays using Taqman quantitative PCR, to SNPlex multiplexed assays (3730XL sequencer), to Affymetrix SNP chip-based arrays and custom pathogen SNP (MIP technology) arrays.   Next Generation sequencing technologies also are playing a larger and developing role in de novo SNP, InDel, copy number, alternative splice variant and new expressed region discovery for both human and pathogen genomes. Flow Cytometry.  Project-specific research applications are developed for flow cytometry analysis and sorting in BSL-2 and BSL-3 environments.   Electron Microscopy.  Project-specific research applications are developed in the areas of sample preparation and analysis ranging from basic structural studies to immuno-localization of selected antigens for a wide array of specimens. A variety of methods, protocols, and equipment are employed to accommodate different preparative and imaging needs. Recent technological advancements have focused on two principle areas. One is the introduction and optimization of sophisticated preparative technologies and techniques for improved retention and visualization of labile structures often lost during routine processing and improving structural preservation. The other area involves the introduction of advanced imaging technologies including high resolution transmission and scanning electron microscopes. Freeze substitution is the lengthy process of replacing vitreous ice in rapidly-frozen hydrated samples with an organic solvent containing fixatives and electron-dense contrasting agents. In order to determine whether controlled microwave irradiation could facilitate the process, the Electron Microscopy Unit developed and assessed methods for maintaining cryo conditions in a laboratory microwave processor. Further development resulted in the fabrication of a thermally controllable unit decreasing processing periods from several days to a few hours while achieving excellent structural and antigenic preservation. The 300 kV TEM microscope recently acquired by the EM Unit is the most advanced instrument for high resolution 3-D biological imaging configured to have optimal flexibility to respond to the needs of multiple investigators and provide them with the highest quality images available with extant technology. It is the platform upon which improvements in ultrastructural imaging will likely be made over the next decade resulting in the highest level resolution possible for the characterization of macromolecular complexes, cellular organelles, viruses, bacteria and other parasites as well as the ability to observe in three dimensions the host pathogen interactions occurring within eukaryotic cells. These technologies improve our ability to relate structure to function, providing information which may identify vaccine targets or other intervention strategies.

该项目为NIAID的壁内传染病，过敏和免疫学研究计划提供了最先进的研究技术。新技术经过开发和验证，然后应用于支持NIAID研究。同样对NIH之外开发的技术进行了测试，评估，验证，并在适当的情况下纳入NIAID壁内计划的技术组合中。支持的技术包括流式细胞仪，共聚焦显微镜，电子显微镜，DNA微阵列，DNA测序，下一代测序，细菌表型和定量PCR。这些技术中的许多技术用于对该研究所的传染病和生物形式研究议程至关重要的高遏制实验室。除技术开发外，RTB还提供了分支机构投资组合中技术各个方面的高级培训。   测序。  RTB使用毛细管DNA测序技术开发应用程序。应用程序是与DIR调查人员密切合作开发的。所有数据都将上传到服务器，该服务器跟踪和管理为研究所生成的所有测序数据。   下一代测序采用Illumina GA II，Illumina HISEQ 2000和454 FLX钛序列仪，用于小RNA发现，ChIP-Seq，Transcriptomics，Exomememics，Exome Squeencing，DE NOGO和REF-MAP基因组测序以及拷贝数变异研究。表型微阵列。  RTB使用表型微阵列技术开发应用程序。该技术具有通过使用96孔板格式分析细菌菌株，克隆或组织培养细胞的2,000个代谢反应的能力。   微阵列。 RTB在Affymetrix微阵列平台上开发了特定项目的研究应用程序，包括自定义芯片设计，实验设计，样品处理和芯片处理。除了为微阵列研究开发新的应用程序外，RTB还开发了DIR研究计划的统计分析，数据管理和数据挖掘解决方案；着重解释由基因组学中使用的高度平行检测系统产生的数据。 RTB还对高吞吐量微阵列数据验证，样本优化和下一个Gen数据验证执行QPCR    人/病原体基因分型。根据基因分型项目的范围，将几种技术用于人类和病原体的基因分型，从基于毛细管的重新序列对从头SNP或/del Discovery进行整个人类基因的重新序列，到使用Taqman定量PCR的高吞吐量SNP基因分型的高吞吐量SNP基因分型，到Sneple semement snepers snepers snple snepected snple（3730）基于芯片的阵列和自定义病原体SNP（MIP技术）阵列。   下一代测序技术还在从头SNP，INDEL，拷贝数，替代剪接变体以及人类和病原体基因组的新表达区域发现中发挥更大而发展的作用。流式细胞仪。  在BSL-2和BSL-3环境中开发了特定于项目的研究应用程序，用于流式细胞仪分析和分类。   电子显微镜。  在样本制备和分析领域开发了特定于项目的研究应用，从基本的结构研究到各种标本的选定抗原的免疫定位。采用多种方法，协议和设备来满足不同的制备和成像需求。最近的技术进步集中在两个主要领域。一种是对复杂的制备技术和技术的引入和优化，以改善在常规处理和改进结构保存过程中经常丢失的不稳定结构的保留和可视化。另一个领域涉及引入高级成像技术，包括高分辨率传输和扫描电子显微镜。冻结替代是用有机溶剂含有固定剂和电子致密对比剂的有机溶剂来代替快速冷冻水合样品中玻璃冰的漫长过程。为了确定受控的微波辐射是否可以促进该过程，电子显微镜单元开发和评估了用于维持实验室微波处理器中冷冻条件的方法。进一步的发展导致制造可热控制的单元从几天减少到几个小时，同时实现出色的结构和抗原保存。 EM单元最近获得的300 kV TEM显微镜是高分辨率3-D生物成像的最先进的仪器，该仪器配置为具有最佳的灵活性，可以响应多个研究者的需求，并为它们提供具有现存技术的最高质量图像。在接下来的十年中，可能会改善超微结构成像的平台，从而可以表征大分子，细胞器细胞器，病毒，细菌和其他寄生虫的最高水平分辨率，以及在三个维度中观察到的宿主病原体的能力。这些技术提高了我们将结构与功能联系起来的能力，提供了可以识别疫苗目标或其他干预策略的信息。