Analyzing Cytokine- and TCR-Mediated Lymphocyte Responses by RNAi

为了更深入地了解淋巴细胞中细胞因子决定的和基于免疫球蛋白/T细胞受体的信号转导的遗传调控，人们努力使用RNA干扰(RNAi)技术作为筛选工具。已获得选定的shRNAs和siRNAs文库。利用Amaxa转基因技术将siRNAs导入静止的CD4T细胞中，在测试系统中，JAK3等激酶的表达和功能得到了显著的抑制。目前，我们已经组装了蛋白酪氨酸激酶文库和蛋白酪氨酸磷酸酶文库，将测试其抑制或增强初始CD4T细胞诱导Th2表型的能力。为此，我们开发了“重组”小鼠，忠实地表达DS-Red作为IL-13的替代品，以及其他表达Am-Cyan作为IL-4的替代品和不稳定的DS-Red作为IL-13的替代品。因此，可以测试细胞对替代表达的诱导或抑制，从而可以避免重新刺激。我们预计既要克隆已经被选择的细胞(即，根据实验方案，要么是被诱导的，要么是被抑制的)，并确定它们已经整合了什么shRNA，或者使用批量诱导的或被抑制的细胞，对大量选择的群体表达的shRNA进行微阵列分析。

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.