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: 7732622
• 负责人: William Paul
• 金额: $ 33.9万
• 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/7732622
• 关键词: Biological Process; CD4 Positive T Lymphocytes; Candidate Disease Gene; Cell physiology; Cells; Class; Clone Cells; Cytokine Gene; Depth; Development; Gene Proteins; Genes; Genetic; Genome; Goals; Immunoglobulins; Interleukin-13; Interleukin-4; Libraries; Lymphocyte; Mediating; Methods; Microarray Analysis; Mus; Participant; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Population; Preparation; Process; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Protocols documentation; Purpose; RNA Interference; Range; Regulation; Rest; Role; Screening procedure; Set protein; Signal Pathway; Signal Transduction; System; T-Cell Receptor; Technology; Testing; Transfection; base; cytokine; insight; interest; protein expression; response; 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. 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 "recombineered" mice that faithfully expresses DS-Red as a surrogate for IL-13 and others that express Am-Cyan as a surrogate of IL-4 and destabilized DS-Red as a surrogate for IL-13. Thus, cells can be tested for induction or inhibition of surrogate 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 selected populations.

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“重组”小鼠，其忠实地表达DS-Red作为IL-13的替代物，以及表达Am-Cyan作为IL-4的替代物和不稳定的DS-Red作为IL-13的替代物的其他小鼠。 因此，可以测试细胞对替代表达的诱导或抑制，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。