Analyzing Cytokine- and TCR-Mediated Lymphocyte Responses by RNAi

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。已经在具有嘌呤霉素抗性元件的慢病毒载体中制备了由所有小鼠磷酸酶组成的shRNA文库。 单位科学家选择分析磷酸酶在外周CD 4 T细胞分化为Th 1细胞中的重要性。 利用的模型利用了在该单位中制备的指示小鼠，其中GFP表达标志着主Th 1转录调节因子T-bet的存在。 当T细胞在体外分化为Th 1细胞时，它们表达非常大量的GFP，并且很容易检测到，并且可以通过细胞分选纯化。 进行的初始筛选旨在确定在Th分化过程中哪些磷酸酶对于T-bet的表达或消退是重要的。 将幼稚CD 4 T细胞暴露于与磷酸酶互补的所有shRNA（超过1000个成员）的文库。shRNA在慢病毒中，使得它们可以被引入幼稚CD 4 T细胞中。 然后在Th 1条件下使细胞分化4天，在IL-2中静止并暴露于嘌呤霉素，以消除未掺入和表达文库成员的细胞。 在这个阶段，绝大多数细胞已经是GFP+。 然后将细胞转移至Th 2培养物。 在Th 2条件下再培养4天结束时，1/2的细胞为T-bet阴性。 纯化T-bet阴性和阳性细胞，并利用避免发夹结构导致的困难的方法对掺入的shRENA进行PCR扩增。 使用ABI仪器对所得的大量扩增的shRNA进行深度测序。 在T-bet阴性细胞中发现了超过20个shRNAs的独特表达，在T-bet阳性细胞中发现了类似数量的shRNAs。 技术方面被证明是可重复的使用PSR扩增与一个外部和一个内部引物。 我们现在已经鉴定了三种催化PI-3激酶途径中的反应的磷酸酶和一种与TCR信号传导相关的磷酸酶，所述TCR信号传导增强Th 1引发或抑制xuch引发。 我们将利用过表达或低表达研究来验证这些作用，然后分析这些磷酸酶在体内的作用。

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. An shRNA library consisting of all the mouse phosphatases has been prepared in lentivirus vectors with a puromycin resistance element. Unit scientists have chosen to analyze the importance of phosphatases in peripheral CD4 T cell differentiation to Th1 cells. The model utilized takes advantage of an indicator mouse prepared in the Unit in which GFP expression marks the presence of the master Th1 transcriptional regulator T-bet. When T cells differentiate in vitro ino Th1 cells, they express veery large amounts of GFP and are easily detectable and can be purified by cell sorting. The initial screen carried out was aimed at determining what phosphatases were important for the expression or the extinction of T-bet during the Th differentiation process. Nave CD4 T cells were exposed to a library of all shRNAs complementary to phosphatases (over 1000 members), The shRNAs were in lentiviruses so that they could be introduced into nave CD4 T cells. The cels were then differentiated under Th1 conditions for 4 days, rested in IL-2 and exposed to puromycin, to eliminate cells that had not incorporated and expressed a member of the library. At this stage the great majority of the cells were already GFP+. The cells were then shifted to a Th2 culture. At the end of four additional days of culture under Th2 conditions, 1/2 of the cells were T-bet negative. The T-bet negative and positive cells were purified and the incorporated shRENAs were PCR amplified, utilizing a method to avoid the difficulties resulting from the hairpin. The resulting large set of amplified shRNAs were subjected to deep sequencing using an ABI instrument. More than 20 shRNAs were found to be uniquely expressed in the T-bet negative cells and a similar number in the T-bet positives. The technical aspects were were shown to be reproducible using PSR amplification with one extewrnal and one internal primer. We have now identified three phosphastases that catalyze reactions in the PI-3kinase pathway and one related to TCR signaling that either enhance Th1 priming or suppress xuch priming. We will undertake validation of these effects utilizing over or underexpression studies and then will analyze the effects of these phosphatases in vivo.