Patterning of Transcription Factor Activity in T cells During Influenza Infection

这是实验室建立的第一年。实验室装修于2008年2月1日完成。正如在项目的目标和目的中提到的，我们感兴趣的是了解转录因子响应TCR刺激的时间动态。我们将在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撑的脂质双分子层系统来进行这些刺激实验。纯化的蛋白质被掺入这样的双层中，使它们可以自由扩散。这种底物对于定量控制配体密度和配体类型是理想的。我们将纳入MHC II类，ICAM-1， CD80和CD48等蛋白质。这种双层结构是理想的光学成像和研究发生在细胞-细胞接触之间的分子相互作用。这些蛋白要么在哺乳动物细胞中作为gpi锚定蛋白表达，要么在昆虫细胞中作为组氨酸标记蛋白表达。通过透析方法将gpi锚定蛋白纳入磷脂脂质体中。这些脂质体可以与酸清洗过的玻璃表面融合，形成玻璃支撑的双层结构。Ni-NTA脂质也被纳入脂质体中，脂质体可用于形成双层。组氨酸标记蛋白与Ni-NTA脂质双分子层相互作用。蛋白质将使用亲和层析纯化。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。我们还建立了一个系统来培养昆虫细胞。为此，我们购买了两台堆叠式摇箱。从昆虫细胞中纯化的可溶性MHC II类分子将在溶液中装载特定的肽，并将蛋白质通过FPLC凝胶过滤进行游离肽与负载蛋白的分离。为此，我们购买了一台FPLC机器。为了从双分子层中提取脂质体，我们需要在高真空下处理脂质体，为此我们购买了冷冻干燥机。为了使转录因子可视化，我们将对它们进行不同荧光蛋白的遗传标记。NFATc1将被标记为CFP， c-Fos和c-Jun将被标记为YFP， NF-kB的p65亚基将被标记为标签- rfp，这是一种红色荧光蛋白的单体版本。分子生物学的工作正在进行中，以产生这些结构。使用Amaxa公司开发的电穿孔试剂盒，这些结构将被引入原代T细胞。我们购买了这家公司开发的电穿孔器。我们已经建立了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），旋转圆盘共聚焦显微镜和荧光相关光谱。宽视场荧光显微镜将允许我们进行钙测量，以测试T细胞与双层相互作用的功能。TIRFM将使我们能够看到装载MHC II类分子的单个肽，以及被募集到激活受体的细胞质信号分子。旋转圆盘共聚焦显微镜将使我们能够跟踪转录因子在单个细胞中的时空动态。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），一个自动化的XYZ台和培养系统，以保持细胞在37度。显微镜的所有部件都通过软件控制，从而可以精确控制实验。这台显微镜的各个部件都是购买、组装和测试的。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。

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.