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

基本信息

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
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/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

项目摘要

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基于细胞因子确定和免疫球蛋白/ T细胞受体信号传导的遗传调节，已经进行了使用RNA干扰（RNAI）技术作为筛查工具的努力。已经获得了精选的shRNA和siRNA库。已经实现了使用AMAXA转染技术将siRNA引入静止的CD4 T细胞中，并且在测试系统中，已经获得了对激酶（例如JAK3）的表达和功能的令人印象深刻的抑制作用。当前，我们已经组装了蛋白质酪氨酸激酶文库和一个蛋白酪氨酸磷酸酶文库，该蛋白磷酸酶库将因其抑制或增强通过幼稚的CD4 T细胞抑制或增强Th2表型的能力进行测试。为此，我们开发了一种“重组”的老鼠，该老鼠忠实地将DS-RED表示为IL-13的代理。因此，可以测试细胞以诱导或抑制DS-RED表达，从而避免重新刺激。我们预计已选择的两个克隆细胞（即诱导或抑制，取决于实验方案），并确定它们已掺入的shRNA或使用散装诱导或抑制的细胞，对块状群体表达的SHRNA进行微阵列分析。为了测试这种方法的总体可行性，已经使用来自System Biosciences Inc.的39,000个基因的小鼠Genenet慢病毒siRNA库进行了全基因组siRNA筛选的初步努力。针对IL-4的M12.4.1 BLymphoma细胞中CD23的诱导诱导CD23。该文库通过感染成功地在M12.4.1细胞中引入，并根据感染细胞对嘌呤霉素的耐药性消除。然后用IL-4刺激感染的M12细胞，通过基于荧光的细胞分选从CD23阴性细胞中分离出表达CD23的细胞。负细胞被重新刺激；再次分离表达和非表达细胞。第三轮后，收集CD23+和CD23-细胞，使用PCR使用来自载体中侧翼序列的引物和使用Affymetrix基因芯片的微阵列分析评估的放大序列，该溶液siRNA插入通过PCR放大。发现一系列候选序列在CD23阴性细胞中富集了一系列候选序列，大概反映了这些siRNA在非CD23-表达器中更有可能发现的siRNA，因此，通过推断，它们在防止细胞表达CD22的情况下起着一定作用。其中包括：JAK1，MAP4K2（MAP激酶，KNASE，激酶激酶），SYK和非受体型蛋白酪氨酸磷酸酶。