Analyzing Cytokine- and TCR-Mediated Lymphocyte Responses by RNAi

为了更深入地了解淋巴细胞中由细胞因子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，已经开始努力使用RNA干扰（RNAi）技术作为筛选工具。已获取选定的shrna和sirna文库。使用Amaxa转染技术将sirna引入静息CD4 T细胞已经实现，并且在测试系统中，已经获得了令人印象深刻的Jak3等激酶的表达和功能抑制。目前，我们已经组装了一个蛋白酪氨酸激酶文库和一个蛋白酪氨酸磷酸酶文库，将测试其抑制或增强初始CD4 T细胞诱导Th2表型的能力。为此，我们开发了一种“重组”小鼠，它忠实地表达DS-Red作为IL-13的替代品。因此，可以对细胞进行诱导或抑制DS-Red表达的测试，从而避免再刺激。我们期望克隆已被选择的细胞（即诱导或抑制，取决于实验方案），并确定它们纳入了哪些shrna，或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。为了测试该方法的总体可行性，我们使用了一个小鼠GeneNet慢病毒siRNA文库进行了全基因组siRNA筛选，该文库包含从System Biosciences Inc.购买的39,000个基因，旨在通过IL-4诱导M12.4.1 B淋巴瘤细胞中的CD23。文库通过感染成功导入M12.4.1细胞，根据感染细胞对嘌呤霉素的耐药情况剔除未感染细胞。然后用IL-4刺激感染的M12细胞，通过荧光细胞分选将表达CD23的细胞与CD23阴性细胞分离。对阴性细胞进行再刺激；再次分离表达细胞和非表达细胞。第三轮后，收集CD23+和CD23-细胞，利用载体侧翼序列的引物进行PCR扩增慢病毒siRNA插入，并用Affymetrix基因芯片对扩增的序列进行微阵列分析。一系列候选序列在CD23阴性细胞中显著富集，可能反映了那些更可能在非CD23表达者中发现的sirna，因此，通过推断，它们在阻止细胞表达CD23方面发挥了一定作用。这些基因包括：Jak1、Map4k2（一种Map激酶、knase、kinase kinase）、syk和一种非受体型蛋白酪氨酸磷酸酶。

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.