Patterning of Transcription Factor Activity in T cells During Influenza Infection

流感感染期间 T 细胞转录因子活性的模式

• 批准号: 7732677
• 负责人: Rajat Varma
• 金额: $ 77.81万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 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/7732677
• 关键词: Acids; Affect; Affinity; Affinity Chromatography; Antibodies; Antigens; Arts; Binding; Biophysics; CD80 gene; Calcium; Carbon Dioxide; Cell Communication; Cell Differentiation process; Cell membrane; Cell surface; Cells; Computer software; Confocal Microscopy; Cyclophosphamide/Fluorouracil/Prednisone; Dialysis procedure; Diffuse; Dose; Electroporation; Event; FOS gene; Family; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Future; Gel Chromatography; Glass; Goals; Histidine; Histocompatibility Antigens Class II; Hybridomas; Incubators; Individual; Infection; Influenza; Insecta; Intercellular adhesion molecule 1; JUN gene; Kinetics; Lasers; Ligand Binding; Ligands; Lipid Bilayers; Lipids; Liposomes; MHC Class II Genes; Mammalian Cell; Measurement; Methods; Microscope; Molecular Biology; Mus; NF-Kappa B p65; NF-kappa B; Pattern; Peptides; Phospholipids; Physical Dialysis; Proteins; Receptor Signaling; Recruitment Activity; Relative (related person); Resolution; Signal Transduction; Signaling Molecule; Solutions; Spectrum Analysis; Staging; Surface; System; T-Cell Receptor; T-Lymphocyte; Techniques; Testing; Time; Transcription Factor AP-1; Transgenic Mice; Vacuum; Work; cellular imaging; density; fast protein liquid chromatography; fluorescence microscope; human IL27RA protein; interest; mouse Il27ra protein; optical imaging; receptor; red fluorescent protein; research study; response; tissue culture; transcription factor

This is the first year of the lab setup. Lab renovations were completed on the February 1st 2008. As mentioned in the goals and objectives of the project we are interested in understanding the temporal dynamics of transcription factors in response to TCR stimulation. We will be performing these experiments in primary mouse T cells from TCR transgenic mice. We will be setting up a glass supported lipid bilayer system to perform these stimulation experiments. Purified proteins are incorporated in such bilayers such that they are free to diffuse. Such substrates are ideal for quantitatively controlling the ligand densities as well as ligand types. We will be incorporating proteins such as MHC class II, ICAM-1, CD80 and CD48. Such bilayers are ideal for optical imaging and studying molecular interactions that take place between cell-cell contacts. These proteins are either expressed in mammalian cells as GPI-anchored proteins or in insect cells as histidine tagged proteins. GPI-anchored proteins are incorporated in phospholipid liposomes by method of dialysis. These liposomes can fuse with a acid cleaned glass surface to form a glass supported bilayer. Ni-NTA lipids are also incorporated in liposomes which can be used to form bilayers. Histidine tagged proteins are made to interact with Ni-NTA lipid bilayers. The proteins will be purified using affinity chromatography. To this end we have setup tissue culture to grow large amounts of mammalian cells. We purchased two carbon-dioxide incubators to this end. We have also purified several antibodies from hybridomas. We have grown several grams of cells for purifying each of these proteins. We have also setup a system to grow insect cells. To this end we have purchased to stacked shaking incubators. Soluble MHC class II molecules purified from insect cells will be loaded with specific peptides in solution and the separation of free peptide with loaded protein will be done by subjecting the protein to gel filtration using FPLC. To this end we have purchased a FPLC machine. To make liposomes out of bilayers we need to treat the lipids under high vacuum for which we have purchased a lyophilizer. To visualize the transcription factors we will be genetically tagging them to different fluorescent proteins. NFATc1 will be tagged to CFP, c-Fos and c-Jun will be tagged to YFP and p65 subunit of NF-kB will be tagged to tag-RFP, a monomeric version of red fluorescent proteins. Molecular biology work is underway to produce these constructs. These constructs will be introduced in primary T cells using electroporation using kits developed by a company called Amaxa. We have purchased the electroporator developed by this company. We have setup a state of the art fluorescence microscope capable of wide field fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), spinning disc confocal microscopy and fluorescence correlation spectroscopy. Wide field fluorescence microscopy will allow us to perform calcium measurements to test the functionality of the T cell interaction with the bilayers. TIRFM will allow us to visualize single peptide loaded MHC class II molecules and also cytoplasmic signaling molecules that get recruited to activated receptors. Spinning disc confocal microscopy will allow us to follow the spatial and temporal dynamics of transcription factors in single cells. And lastly, fluorescence correlation spectroscopy will allow us to study the dynamics of individual subunits in multi-subunit receptor systems. This microscope is also equipped with 5 lasers (wavelengths 405, 440, 488, 514, 561, 640 nms), an automated XYZ stage and incubator systems to keep cells at 37 degrees. All components of the microscope are controlled via software allowing precise control of the experiments. Individual components of this microscope were purchased, assembled and tested. Using a combination of these different techniques we will start understanding the spatial and temporal dynamics of transcription factors.

这是实验室成立的第一年。2008年2月1日，实验室装修完成。正如在本项目的目标和目的中所提到的，我们有兴趣了解转录因子对TCR刺激的反应的时间动力学。我们将在来自TCR转基因小鼠的原代小鼠T细胞中进行这些实验。我们将建立一个玻璃支持的脂质双层系统来进行这些刺激实验。将纯化的蛋白质掺入这样的双层中，使得它们自由扩散。这种底物对于定量控制配体密度以及配体类型是理想的。我们将整合蛋白质，如MHC II类，ICAM-1，CD 80和CD 48。这种双层是光学成像和研究细胞与细胞接触之间发生的分子相互作用的理想选择。这些蛋白质在哺乳动物细胞中表达为GPI锚定蛋白或在昆虫细胞中表达为组氨酸标记蛋白。通过透析方法将GPI锚定蛋白掺入磷脂脂质体中。这些脂质体可以与酸清洁的玻璃表面融合以形成玻璃支撑的双层。Ni-NTA脂质也掺入脂质体中，其可用于形成双层。组氨酸标记的蛋白质与Ni-NTA脂质双层相互作用。将使用亲和色谱法纯化蛋白质。为此，我们建立了组织培养来培养大量的哺乳动物细胞。为此，我们购买了两个二氧化碳培养箱。我们还从杂交瘤中纯化了几种抗体。我们已经培养了几克细胞来纯化这些蛋白质。我们还建立了一个培养昆虫细胞的系统。为此，我们购买了堆叠式振荡培养箱。从昆虫细胞纯化的可溶性MHC II类分子将在溶液中负载特异性肽，并且通过使用FPLC使蛋白质经受凝胶过滤来进行游离肽与负载的蛋白质的分离。为此，我们购买了一台FPLC机器。为了从双层中制备脂质体，我们需要在高真空下处理脂质，为此我们购买了冻干机。为了使转录因子可视化，我们将在不同的荧光蛋白上对它们进行遗传标记。NFATc 1将被标记到CFP上，c-Fos和c-Jun将被标记到YFP上，NF-kB的p65亚基将被标记到tag-RFP上，tag-RFP是红色荧光蛋白的单体形式。分子生物学工作正在进行中，以产生这些结构。这些构建体将使用由一家名为Amaxa的公司开发的试剂盒通过电穿孔引入原代T细胞。我们购买了这家公司开发的电转化仪。我们已经建立了一个国家的最先进的荧光显微镜能够宽视场荧光显微镜，全内反射荧光显微镜（TIRFM），旋转光盘共聚焦显微镜和荧光相关光谱。宽视野荧光显微镜将使我们能够进行钙测量，以测试T细胞与双层相互作用的功能。TIRFM将使我们能够可视化单个肽加载的MHC II类分子，以及被激活受体招募的细胞质信号分子。旋转圆盘共聚焦显微镜将使我们能够跟踪单细胞中转录因子的时空动态。最后，荧光相关光谱将使我们能够研究多亚基受体系统中单个亚基的动力学。该显微镜还配备了5个激光器（波长405，440，488，514，561，640 nm），自动XYZ载物台和培养箱系统，以保持细胞在37度。显微镜的所有组件都通过软件进行控制，从而可以精确控制实验。购买、组装和测试了该显微镜的各个组件。使用这些不同技术的组合，我们将开始了解转录因子的空间和时间动态。