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: 7592323
• 负责人: William Paul
• 金额: $ 38.58万
• 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/7592323
• 关键词: B-Cell Lymphomas; Biological Process; CD4 Positive T Lymphocytes; Candidate Disease Gene; Cell Separation; Cell physiology; Cells; Class; Clone Cells; Cytokine Gene; Depth; Development; Fluorescence; Gene Chips; Gene Proteins; Genes; Genetic; Genome; Goals; Harvest; Immunoglobulins; Infection; Interleukin-13; Interleukin-4; Libraries; Lymphocyte; Maps; Mediating; Methods; Microarray Analysis; Mus; Participant; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Polymerase Chain Reaction; Population; Preparation; Process; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Protocols documentation; Puromycin; Purpose; RNA Interference; Range; Regulation; Resistance; Rest; Role; Screening procedure; Series; Set protein; Signal Pathway; Signal Transduction; Small Interfering RNA; System; T-Cell Receptor; Technology; Testing; Transfection; base; cytokine; human SYK protein; insight; interest; prevent; protein expression; receptor; response; tool; vector

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. Selected libraries of shRNAs and siRNAs have been obtained. Introduction of siRNAs into resting CD4 T cells using Amaxa transfection technology has been achieved and, in test systems, impressive inhibition of expression and function of kinases such as Jak3 has been obtained. Currently, we have assembled a protein tyrosine kinase library and a protein tyrosine phosphatase library that will be tested for its capacity to inhibit or enhance the induction of Th2 phenotype by naive CD4 T cells. To this end, we have developed a "recombineered" mouse that faithfully expresses DS-Red as a surrogate for IL-13. Thus, cells can be tested for induction or inhibition of DS-Red expression and thus restimulation can be avoided. We anticipate both cloning cells that have been selected (i.e. either induced or suppressed, depending on the experimental protocol) and determining what shRNAs they have incorporated or using bulk induced or suppressed cells, carrying out microarray analysis of the shRNAs expressed by the bulk slected populations. As a test of overall feasibility of this approach, an initial effort at genome-wide siRNA screening has been performed with a mouse GeneNet lentiviral siRNA Library comprising 39,000 genes purchased from System Biosciences Inc. targeting the induction of CD23 in M12.4.1 B lymphoma cells by IL-4. The library was successfully introduced in M12.4.1 cells by infection and non-infected cells eliminated based on the resistance of the infected cells to puromycin. The infected M12 cells were then stimulated with IL-4 and the cells that expressed CD23 were separated from the CD23-negative cells by fluorescence-based cell sorting. The negative cells were restimulated; expressing and non-expressing cells were separated again. After a third round, the CD23+ and CD23- cells were harvested, the lentiviral siRNA inserts amplified by PCR using primers from the flanking sequences in the vector and the amplified sequences evaluated by microarray analysis using Affymetrix gene chips. A series of candidate sequences were found that were strikingly enriched in the CD23-negative cells, presumably reflecting those siRNAs more likely to be found in non-CD23-expressors and thus, by inference, that have played some role in preventing cells from expressing CD23. Among these are the genes for: Jak1, Map4k2(a Map kinase, knase, kinase kinase), syk and a non-receptor-type protein tyrosine phosphatase.

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。 已经获得了选择的shRNA和siRNA文库。 已经实现了使用Amaxa转染技术将siRNA引入静息CD 4 T细胞，并且在测试系统中，已经获得了对激酶如Jak 3的表达和功能的令人印象深刻的抑制。目前，我们已经组装了一个蛋白酪氨酸激酶库和一个蛋白酪氨酸磷酸酶库，将测试其抑制或增强初始CD 4 T细胞诱导Th 2表型的能力。 为此，我们开发了一种“重组”小鼠，其忠实地表达DS-Red作为IL-13的替代物。 因此，可以测试细胞对DS-Red表达的诱导或抑制，从而可以避免再刺激。 我们预期克隆已经选择的细胞（即诱导或抑制，取决于实验方案）并确定它们已经掺入了什么样的shRNA，或者使用大量诱导或抑制的细胞，对大量选择的群体表达的shRNA进行微阵列分析。 作为该方法的总体可行性的测试，已经用购自System Biosciences Inc.的包含39，000个基因的小鼠GeneNet慢病毒siRNA文库进行了全基因组siRNA筛选的初步努力。IL-4靶向诱导M12.4.1 B淋巴瘤细胞中的CD 23。 通过感染将该文库成功地引入M12.4.1细胞中，并基于感染细胞对嘌呤霉素的抗性消除未感染的细胞。 然后用IL-4刺激感染的M12细胞，并通过基于荧光的细胞分选将表达CD 23的细胞与CD 23阴性细胞分离。 再次刺激阴性细胞;再次分离表达和非表达细胞。 第三轮后，收获CD 23+和CD 23-细胞，使用来自载体中侧翼序列的引物通过PCR扩增慢病毒siRNA插入物，并使用Affyphase基因芯片通过微阵列分析评估扩增的序列。 发现一系列候选序列在CD 23阴性细胞中显著富集，推测反映了那些siRNA更可能在非CD 23表达者中发现，因此，通过推断，它们在阻止细胞表达CD 23中发挥了一些作用。 其中包括Jak 1、Map 4k 2（一种Map激酶、knase、激酶激酶）、syk和一种非受体型蛋白酪氨酸磷酸酶的基因。