RNAi Screening in Hematopoietic Cells

自2008年8月成立分子和细胞生物学小组以来，已经聘请了实验室人员，第一代版本的机器人筛查平台投入使用，在准备在单核/巨噬细胞系中运行我们的第一个RNAi筛查方面取得了重大进展。 化验进展： 我们计划使用基于显微镜的高含量单细胞读数的分析和基于生物发光的群体分析来运行筛查，这些分析使用由炎症基因启动子驱动的荧光素酶报告。我们设计并构建了双启动子慢病毒载体，允许来自单个病毒的两个基因表达。这导致了RAW264.7小鼠巨噬细胞系的创建，该细胞系表达两种用于高含量筛选的荧光生物传感器。第一次读出表达了GFP与由其内源启动子驱动的RelA NFkB转录因子的融合。这种蛋白质在未受刺激的细胞中分裂到细胞质，并在促进巨噬细胞激活的广泛配体的反应下移位到细胞核。使用内源性RELA启动子有助于更准确地复制以前使用内源RELA观察到的振荡胞浆/核易位。第二个生物传感器使用小鼠肿瘤坏死因子α启动子来驱动红色荧光蛋白mCherry的表达，该蛋白融合到一个不稳定的PEST序列中，从而减少了蛋白质在细胞中的半衰期。后一种修饰在动力学实验中提供了更动态的肿瘤坏死因子α启动子活性读数。我们已经建立了一个克隆细胞系，在第一个小时内表现出强大的relA易位，随后在12小时后，肿瘤坏死因子α启动子驱动的mCherry表达显著增加，对广泛的Toll样受体(TLR)配体做出反应。我们已经使用类似的策略创建了THP1人类单核细胞系，表达由人肿瘤坏死因子α启动子驱动的萤火虫荧光素酶和由泛素启动子驱动的肾虫荧光素酶。构成的Renilla表达式为基于总体的读数中的细胞数目变异性提供了一个有价值的归一化因子。因此，TLR刺激后该细胞系中的萤火虫/肾比提供了一种衡量肿瘤坏死因子α启动子活性的方法，我们看到不同的TLR配体的反应显著且可重复性地增加。有趣的是，这位记者在这个THP1细胞系中对内毒素的反应(2-4小时)比在原始细胞系中的mCherry(12-16小时)更快。尽管荧光素酶分析的较高灵敏度可能与此有关，但这一行为可能反映了人和小鼠肿瘤坏死因子α启动子的反应动力学上的一些差异，这一发现可能具有临床意义。 SiRNA传递的优化： 有效地将siRNA送入造血细胞仍然是实施有效的siRNA筛选的重大障碍。为了避免在全基因组筛查中出现高频率的假阴性，重要的是建立一种可重复性的方法，实现80%的靶基因敲除(Kd)。我们利用了这样一个事实，即上述RAW和THP1报告细胞系的创建在它们表达的报告中提供了方便的控制siRNA靶标。在RAW和THP1株系中分别检测GFP和肾形荧光素酶KD的能力提供了两个优点。首先，它允许我们分析蛋白质而不是mRNA KD(siRNA传递最常见的验证方法)，提供了有效筛查所需终点的更直接测量。其次，在384孔格式下运行两种分析的能力允许更广泛的实验条件矩阵，这提高了确定最佳给药方案的机会。使用这种方法，我们正处于验证所描述的RAW和THP1细胞系的384-Well格式的基于脂质的高效转染法的最后阶段。 机器人工作流： 之前购买的机器人设备用于高内容筛查：可容纳&gt；150微板的细胞培养箱、Tecan evo150液体处理机和BD Path生物成像仪，并通过Thermo Catalyst Express机械臂将三台机器进行机器人集成。我们已经使用上述原始细胞系为384孔板中的筛子建立了机器人工作流程。这将允许在12小时以上的时间内记录来自相同活细胞的rela-GFP和TNFα启动子-mCherry读数。我们预测，能够从相同的细胞中对多种表型进行评分，并在同一RNAi屏幕上比较单个细胞和群体数据，这将是所描述的分析平台的一个有价值的功能。 一旦我们完成了对检测重复性的控制，我们希望在接下来的几个月内进行初步筛选，针对以前由其他实验室使用小鼠体内突变方法确定的TLR信号通路基因进行siRNA。我们已经购买了全基因组siRNA文库，并设想在明年初进行大规模筛选。此外，我们正在与Scott Martin、Natasha Caplen和Chris Austin合作，努力在国家化学基因组中心建立一个跨NIH RNAi筛查小组。我们将为NCGC RNAi小组提供上述一种或两种细胞系，以便使用他们的机器人基础设施运行并行筛选。这将为两组中的协议提供重要的验证，并增加对每个数据集中重叠的屏幕点击的信心。

Since establishing the Molecular and Cell Biology group in August 2008, lab personnel have been hired, a first generation version of the robotic screening platform brought into service, and significant progress made in preparation for running our first RNAI screens in monocyte/macrophage cell lines. Assay development: We plan to run screens using assays with both a microscopy-based 'high content' single cell readout, and also bioluminescence-based population assays using luciferase reporters driven by inflammatory gene promoters. We have designed and constructed dual promoter lentiviral vectors that permit the expression of two genes from a single virus. This has led to the creation of a RAW264.7 mouse macrophage cell line expressing two fluorescent biosensors for high content screening. The first readout expresses a GFP fusion with the RelA NFkB transcription factor driven by its endogenous promoter. This protein partitions to the cytoplasm in unstimulated cells, and translocates to the nucleus in response to a wide range of ligands that promote macrophage activation. Use of the endogenous RelA promoter facilitates a more accurate reproduction of the oscillatory cytosol/nuclear translocation previously observed with endogenous RelA. The second biosensor uses the murine TNF alpha promoter to drive expression of the red fluorescent protein mCherry, fused to a destabilizing PEST sequence that reduces the protein's half-life in the cell. This latter modification provides a more dynamic readout of TNF alpha promoter activity in kinetic experiments. We have established a clonal cell line exhibiting robust RelA translocation in the first hour, followed by a significant increase in TNF alpha promoter-driven mCherry expression after 12 hours in response to a wide range of Toll-like receptor (TLR) ligands. We have used a similar strategy to create a THP1 human monocyte cell line expressing firefly luciferase driven by the human TNF alpha promoter and also renilla luciferase driven by the ubiquitin promoter. The constitutive renilla expression provides a valuable normalization factor for cell number variability in a population-based readout. Thus, the firefly/renilla ratio in this cell line after TLR stimulation provides a measure of TNF alpha promoter activity, and we see a significant and reproducible increase in response to various TLR ligands. Interestingly, this reporter responds more quickly to LPS in this THP1 cell line (2-4 hr) than does mCherry in the RAW cell line (12-16 hr). Although the higher sensitivity of the luciferase assay may contribute to this, this behavior likely reflects some difference in the response kinetics of the human and mouse TNF alpha promoters, a finding that may have clinical relevance. Optimization of siRNA delivery: Effective delivery of siRNA into hematopoietic cells remains a significant obstacle to the implementation of effective siRNA screens. It is important to establish a reproducible method that can achieve >80% knockdown (KD) of target genes in order to avoid a high frequency of false negatives in genome-wide screens. We have taken advantage of the fact that the creation of the RAW and THP1 reporter cell lines described above provide convenient control siRNA targets in the reporters they express. The ability to assay GFP and renilla luciferase KD in the RAW and THP1 lines respectively provides two advantages. First, it allows us to assay for protein rather than mRNA KD (the most common validation method for siRNA delivery), providing a more direct measure of the required endpoint needed for an effective screen. Second, the ability to run both assays in 384-well format allows for a more extensive matrix of experimental conditions, which improves the chance of identifying an optimal delivery protocol. Using such an approach, we are in the final stages of validating highly efficient lipid-based transfection protocols in 384-well format for both of the described RAW and THP1 cell lines. Robotic workflows: Robotic equipment was purchased previously for high content screens: a cytomat incubator with capacity for >150 microplates, a Tecan evo150 liquid handler and a BD pathway bioimager, with robotic integration of the three machines through a Thermo catalyst express robotic arm. We have established robotic workflows for screens in 384-well plates using the RAW cell line described above. This will permit the recording of both the RelA-GFP and TNF alpha promoter-mCherry readouts from the same live cells over a 12+ hr time course. We predict that ability to score multiple phenotypes from the same cells, and to compare both single cell and population data in the same RNAi screen will be a valuable feature of the described assay platform. Once we have completed controls for assay reproducibility we expect to run preliminary screens within the next few months with siRNAs targeting TLR signaling pathway genes previously identified by other laboratories using murine in vivo mutagenesis methods. We have purchased whole genome siRNA libraries, and envision running large-scale screens by early next year. In addition, we are collaborating with Scott Martin, Natasha Caplen and Chris Austin in their efforts to establish a trans-NIH RNAi screening group at the National Chemical Genomics Center. We will provide one or both of the cell lines described above for the NCGC RNAi group to run parallel screens using their robotic infrastructure. This will provide an important validation of protocols in both groups, as well as increasing confidence in screen hits that overlap in each dataset.