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。在带有嘌呤霉素抗性元件的慢病毒载体中，构建了由所有磷酸酶组成的shRNA文库。单位科学家选择分析磷酸酶在外周CD4T细胞分化为Th1细胞中的重要性。所使用的模型利用在单元中准备的指示器鼠标，其中GFP表达标记主Th1调节T-bet的存在。当T细胞在体外分化为Th1细胞时，它们表达大量的GFP，并且很容易被检测到，并可以通过细胞分选得到纯化。初步筛选的目的是确定在Th分化过程中，哪些磷酸酶对T-bet的表达或消失是重要的。将NAVE CD4T细胞暴露于与磷酸酶互补的所有shRNA文库(超过1000个成员)，这些shRNAs存在于慢病毒中，因此它们可以被导入到NAVE CD4T细胞中。然后细胞在Th1条件下分化4天，在IL-2中休息，并暴露在嘌呤霉素中，以剔除没有整合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+。然后这些细胞被转移到Th2培养上。在Th2条件下额外培养4天后，1/2的细胞T-bet阴性。提纯T-bet阴性和阳性细胞，并对掺入的shRENAs进行PCR扩增，避免了发夹带来的困难。使用ABI仪器对得到的大量扩增shRNA进行深度测序。有20多个shRNA在T-bet阴性细胞中唯一表达，在T-bet阳性细胞中有类似数量的shRNA表达。用1个外引和1个内引物进行PSR扩增，技术方面是重复性的。目前，那些在两种检测中都得分的人正在单独进行测试，以确定它们是会阻止T-bet的表达还是会消失。这个屏幕有望揭示关于磷酸酶在Th1和Th2分化中的作用的许多令人兴奋的新信息，并勾勒出新的途径。

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 phosphatases has been prepared in a lentivirus vector 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 regulatory 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. Currently those that scored in both assays, are being tested individually to determine whether they will block T-bet expression or extinction. This screen promises to reveal much exciting new information regarding the role of phosphatases in Th1 and Th2 differentiation and to outline new pathways.