Analyzing Cytokine- and TCR-Mediated Lymphocyte Responses by RNAi

通过 RNAi 分析细胞因子和 TCR 介导的淋巴细胞反应

• 批准号: 7964549
• 负责人: William Paul
• 金额: $ 36.39万
• 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/7964549
• 关键词: Biological Assay; Biological Process; CD4 Positive T Lymphocytes; Candidate Disease Gene; Cell Differentiation process; Cell Separation; Cell physiology; Cells; Cytokine Gene; Development; Elements; Extinction (Psychology); Gene Proteins; Genes; Genetic; Genome; Goals; Immunoglobulins; In Vitro; Interleukin-2; Lentivirus Vector; Libraries; Lymphocyte; Mediating; Methods; Modeling; Mus; Participant; Pathway interactions; Peripheral; Phosphoric Monoester Hydrolases; Play; Population; Preparation; Process; Protein Tyrosine Kinase; Puromycin; RNA Interference; Regulation; Resistance; Rest; Role; Scientist; Screening procedure; Set protein; Signal Pathway; Signal Transduction; Staging; Subfamily lentivirinae; System; T-Cell Receptor; T-Lymphocyte; Technology; Testing; Th1 Cells; Th1/Th2 Differentiation Pathway; base; cytokine; insight; instrument; interest; member; protein expression; response; small hairpin RNA; tool

In order to gain a deeper insight into the genetic regulation of cytokine-determined and immunoglobulin/ T cell receptor based signaling in lymphocytes, efforts to use RNA interference (RNAi) technology as a screening tool have been undertaken. An shRNA library consisting of all the phosphatases has been prepared in a lentivirus vector with a puromycin resistance element. Unit scientists have chosen to analyze the importance of phosphatases in peripheral CD4 T cell differentiation to Th1 cells. The model utilized takes advantage of an indicator mouse prepared in the Unit in which GFP expression marks the presence of the master Th1 regulatory T-bet. When T cells differentiate in vitro ino Th1 cells, they express veery large amounts of GFP and are easily detectable and can be purified by cell sorting. The initial screen carried out was aimed at determining what phosphatases were important for the expression or the extinction of T-bet during the Th differentiation process. Nave CD4 T cells were exposed to a library of all shRNAs complementary to phosphatases (over 1000 members), The shRNAs were in lentiviruses so that they could be introduced into nave CD4 T cells. The cels were then differentiated under Th1 conditions for 4 days, rested in IL-2 and exposed to puromycin, to eliminate cells that had not incorporated and expressed a member of the library. At this stage the great majority of the cells were already GFP+. The cells were then shifted to a Th2 culture. At the end of four additional days of culture under Th2 conditions, 1/2 of the cells were T-bet negative. The T-bet negative and positive cells were purified and the incorporated shRENAs were PCR amplified, utilizing a method to avoid the difficulties resulting from the hairpin. The resulting large set of amplified shRNAs were subjected to deep sequencing using an ABI instrument. More than 20 shRNAs were found to be uniquely expressed in the T-bet negative cells and a similar number in the T-bet positives. The technical aspects were were shown to be reproducible using PSR amplification with one extewrnal and one internal primer. Currently those that scored in both assays, are being tested individually to determine whether they will block T-bet expression or extinction. This screen promises to reveal much exciting new information regarding the role of phosphatases in Th1 and Th2 differentiation and to outline new pathways.

为了更深入地了解淋巴细胞中由精氨酸决定的和基于免疫球蛋白/ T细胞受体的信号传导的遗传调控，已经进行了使用RNA干扰（RNAi）技术作为筛选工具的努力。已在具有嘌呤霉素抗性元件的慢病毒载体中制备了由所有磷酸酶组成的shRNA文库。 单位科学家选择分析磷酸酶在外周CD 4 T细胞分化为Th 1细胞中的重要性。 利用的模型利用了在该单位中制备的指示小鼠，其中GFP表达标志着主Th 1调节T-bet的存在。 当T细胞在体外分化为Th 1细胞时，它们表达非常大量的GFP，并且很容易检测到，并且可以通过细胞分选纯化。 进行的初始筛选旨在确定在Th分化过程中哪些磷酸酶对于T-bet的表达或消退是重要的。 将幼稚CD 4 T细胞暴露于与磷酸酶互补的所有shRNA（超过1000个成员）的文库。shRNA在慢病毒中，使得它们可以被引入幼稚CD 4 T细胞中。 然后在Th 1条件下使细胞分化4天，在IL-2中静止并暴露于嘌呤霉素，以消除未掺入和表达文库成员的细胞。 在这个阶段，绝大多数细胞已经是GFP+。 然后将细胞转移至Th 2培养物。 在Th 2条件下再培养4天结束时，1/2的细胞为T-bet阴性。 纯化T-bet阴性和阳性细胞，并利用避免发夹结构导致的困难的方法对掺入的shRENA进行PCR扩增。 使用ABI仪器对所得到的大量扩增的shRNA进行深度测序。 在T-bet阴性细胞中发现了超过20个shRNAs的独特表达，在T-bet阳性细胞中发现了类似数量的shRNAs。 技术方面被证明是可重复的使用PSR扩增与一个外部和一个内部引物。 目前，在两种测定中得分的那些正在单独测试，以确定它们是否会阻断T-bet表达或灭绝。 这个屏幕有望揭示许多令人兴奋的新信息磷酸酶在Th 1和Th 2分化的作用，并概述新的途径。