RNAi Screening in Hematopoietic Cells

造血细胞中的 RNAi 筛选

• 批准号: 8556015
• 负责人: Iain Fraser
• 金额: $ 41.83万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8556015
• 关键词: Address; Bioinformatics; Biological; Biological Assay; Biology; Bioluminescence; Biosensor; Cell Line; Cell Lineage; Cell model; Cells; Collaborations; Computer software; Coupled; Data; Data Set; Development; Dose; Double-Stranded RNA; Endotoxins; Evaluation; Feedback; Frequencies; Gene Targeting; Genes; Genetic Screening; Genetic Transcription; Genomics; Goals; Hematopoietic; Human; Immune; Immune Cell Activation; Immune response; Individual; Infection; Inflammatory; Informatics; Interferon Type I; Interferons; Kinetics; Ligands; Lipids; Literature; Luciferases; MAP Kinase Gene; MAP3K7IP1 gene; Macrophage Activation; Mammalian Cell; Methodology; MicroRNAs; Microscopy; Modeling; Molecular; Murid herpesvirus 1; Mus; Nucleic Acids; Output; Pathway Analysis; Pathway interactions; Pattern; Performance; Phase; Phenotype; Post-Translational Protein Processing; Process; Protein Kinase; Proteins; Proteomics; Protocols documentation; RNA Interference; Reporter; Research; Research Personnel; Resistance; Robotics; Salmonella; Screening Result; Screening procedure; Seeds; Septic Shock; Signal Pathway; Signal Transduction; Small Interfering RNA; Small RNA; Stimulus; System; TNF gene; TRAF6 gene; Technology; Transcript; Transfection; Universities; Validation; Vendor; Viral; Work; adapter protein; base; cell type; clinically relevant; cytokine; gene function; genome wide association study; genome-wide; information processing; insight; macrophage; monocyte; novel; p65; pathogen; population based; programs; promoter; protein expression; receptor; response; small molecule; tool; transcription factor

The discovery of RNA interference (RNAi) and the major advances in the understanding of small RNA biology in the past decade have provided researchers with an invaluable tool for wide-scale and rapid genetic screening. A central goal of our research program has been to develop methodology for efficient application of RNA interference (RNAi) screening technology in hematopoietic cell lineages, and to implement genome-wide RNAi screens in both human and mouse hematopoietic cells to interrogate the mechanistic basis of immune cell responses to pathogenic stimuli. Our current efforts are focused on macrophages as they form the first line of defense against numerous bacterial and viral pathogens and characterization of these initial encounters are central to the LSB-wide efforts to generate quantitative models of host-pathogen interactions. We have developed assays in macrophage/monocyte cell lines with both microscopy-based 'high content' single cell readouts, and also bioluminescence-based population assays using luciferase reporters driven by inflammatory gene promoters. While the majority of our screening data has come from using reporters for the NF-κB component p65/relA and for TNFα transcription, we are also developing reporters for MAPK and IRF activation and for additional inflammatory cytokines. Together, this panel of reporters will provide a comprehensive range of biosensors for evaluation of the macrophage activation profile in response to various pathogenic inputs. Effective delivery of siRNA into hematopoietic cells remains a significant obstacle to the implementation of effective siRNA screens. We have developed highly efficient lipid-based transfection protocols in 384-well format for both mouse (RAW264.7) and human (THP1) cell lines where we can routinely achieve 85-95% knockdown of target protein. We have also confirmed our assays conform to the plate uniformity criteria established by the NCGC small molecule screening group, finding no significant edge effects with either assay which has permitted the use of full 384-well plates in our primary screens. We have identified reproducible positive siRNA controls for a range of TLR ligands, including the TLR receptors, receptor-associated adapter proteins and protein kinases involved in the activation of the MAPK and NF-κB signaling pathways. Considering that the response to dsRNA during pathogen infection is very robust in macrophages, and that a strong subsequent interferon response would be problematic for the interpretation of effects resulting from gene-specific siRNA knockdown, it was important to evaluate our described protocols for any non-specific macrophage response to transfected siRNA. We found no significant elevation in type-I interferon from either the RAW or THP-1 reporter cell lines during our assay window of 48 to 72 hr post-transfection. Last year, we described completion of the experimental phase of primary two genome wide screens to identify genes involved in the human and mouse LPS response. As the best characterized TLR stimulus, data from these screens will provide a valuable comparison of the endotoxin response in mouse and human cells, with important potential clinical relevance in the context of septic shock and endotoxin tolerance. In 2012, we completed the statistical analysis of the primary screen data. We used the freely available CellHTS2 software package to process our primary data and calculate z-scores for the deviation of each gene from the normalized median for each screen readout. We selected approximately 600 primary hits for both human and mouse screens based on a combination of phenotypic strength and connections to the core TLR and NF-κB signaling pathways identified using a variety of pathway analysis software applications. Recent studies have shown that off target effects (driven by miRNA-like targeting of 3UTRs in unintended target genes by the seed sequence of an siRNA) are quite prevalent in RNAi screens. To reduce the propagation of these unwanted effects in our secondary screens, we chose to use six non-pooled siRNAs from alternative vendors (three each from Ambion and Qiagen). Secondary screens are ongoing using this approach. During the analysis of our primary screening data, we curated a set of 128 genes comprising a canonical set of TLR signaling components including TLR receptors, proximal signaling components, NF-κB and MAPK pathway proteins, transcription factors, cytokines and negative regulators. We found that approximately 26 of these targets were strong hits in the human LPS primary screen, while a further 15 were weak hits. This led us to ask whether this hit frequency was reasonable, and whether unexpected negatives are determined more by insufficient KD or by cell-type variability in components required for signaling (since canonical pathways are often derived from studies in a range of model cell types). We screened the LPS response for six additional individual siRNAs for every gene in this set, which we would expect to give effective depletion of the target gene by at least one siRNA. Analysis of the data set identified a small number of core pathway components as strong hits, presumably due to insufficient KD in the primary screen, including TRAM, TRAF6 and TAB1. However, the total number of strong canonical pathway hits in the secondary screen was actually reduced to around 25 genes, suggesting that many components that influence TLR signaling do so in a cell type specific manner. This is consistent with recent informatic analyses of screens done with the same pathogenic stimulus but in different cell systems, which suggest that while common pathways are identified in these parallel screens, the specific genes most sensitive to siRNA KD are often non-overlapping. However, in our assay of LPS induced TNF transcription, we do see a noteworthy pattern where the strongest phenotypes cluster in the initial receptor and adapter proteins and the TNF transcriptional enhanceosome components, while the phenotypes are weaker in the intermediate steps when signaling branches through the NF-κB and MAPK pathways. The signaling pathways and transcription factor responses induced in macrophages upon TLR stimulation are regulated by feedback loops that modulate the kinetics and magnitude of gene transcription. Among these, NF-κB has been a paradigm for a signal- responsive transcription factor that operates in a feedback regulatory network. Despite the considerable literature on NF-κB activation and function, there remains a lack of data on NF-κB single cell dynamics in macrophage cells responding to pathogenic stimuli. The development of a mouse macrophage cell line expressing GFP tagged p65/relA for the above siRNA screen provides an opportunity for us to address this. Moreover, the coupled TNFα promoter-driven transcriptional reporter provides a unique secondary readout that allows evaluation of the consequences of NF-κB activation at a single cell level. We have initiated several collaborations using this novel assay platform. We are working with Mia Sung and Gordon Hager at the NCI to study how macrophages interpret different LPS doses in the context of NF-κB activation and TNFα transcriptional output. We are also using the cells as models for single cell infection studies with murine cytomegalovirus (mCMV) in collaboration with Peter Ghazal at the University of Edinburgh, and for Salmonella studies with Clare Bryant at the University of Cambridge.

在过去的十年中，RNA干扰（RNAI）的发现以及对小RNA生物学的理解的主要进步为研究人员提供了一种无价的广泛和快速基因筛查工具。我们的研究计划的一个核心目的是开发在造血细胞谱系中有效应用RNA干扰（RNAI）筛查技术的方法，并在人和小鼠造血细胞中实施全基因组RNAi筛查，以询问免疫细胞反应对病原性刺激的机械基础。 我们目前的努力集中在巨噬细胞上，因为它们构成了针对众多细菌和病毒病原体的第一道防线，并且这些初始相遇的表征是LSB范围范围内为产生宿主 - 病原相互作用的定量模型的核心。 我们已经在巨噬细胞/单核细胞系中开发了基于显微镜的“高含量”单细胞读数的测定法，也开发了使用荧光素酶报告子，以及受炎症基因启动子驱动的荧光素酶报道器。虽然我们的大多数筛选数据都来自用于NF-κB成分p65/rela和TNFα转录的记者，但我们还在开发用于MAPK和IRF激活的记者，以及其他炎症细胞因子。 总之，这组记者将提供一系列的生物传感器，以评估巨噬细胞激活曲线，以响应各种致病性输入。有效地将siRNA传递到造血细胞中仍然是实施有效siRNA筛查的重要障碍。我们已经为小鼠（RAW264.7）和人（THP1）细胞系开发了高效的基于脂质的转染方案，以384孔的格式开发了我们可以常规地实现目标蛋白的85-95％敲低的细胞系。我们还证实了我们的测定法符合NCGC小分子筛选组建立的板均匀性标准，发现任何一种测定都没有明显的边缘效应，这可以在我们的主要屏幕中使用完整的384孔板。我们已经确定了一系列TLR配体的可再现阳性siRNA对照，包括TLR受体，受体相关的衔接蛋白和参与MAPK和NF-κB信号通路激活的蛋白激酶。考虑到病原体感染期间对DSRNA的反应在巨噬细胞中非常健壮，并且强烈的随后干扰素反应对于解释基因特异性siRNA敲低产生的影响是有问题的，因此评估我们所描述的任何对任何非特异性巨噬细胞响应对转染SiRNA的巨噬细胞响应的方案很重要。 在转染后48至72小时，我们发现I型干扰素从RAW或THP-1报告基细胞系中没有显着升高。 去年，我们描述了主要两个基因组宽筛选的实验阶段的完成，以鉴定与人和小鼠LPS反应有关的基因。 作为TLR刺激的最佳特征，这些筛选的数据将对小鼠和人类细胞中内毒素反应的有价值比较，在败血性休克和内毒素耐受性的背景下具有重要的潜在临床相关性。 2012年，我们完成了主要屏幕数据的统计分析。 我们使用免费可用的CellHTS2软件包来处理我们的主要数据，并计算每个屏幕读数中每个基因偏离标准化中位数的Z分数。 我们根据使用各种途径分析软件应用程序确定的表型强度以及与核心TLR和NF-κB信号通路的连接的组合，选择了大约600个主要的命中。最近的研究表明，在RNAi筛选中，OFF目标效应（由siRNA的种子序列在意外靶基因中的miRNA样靶向3 UTR驱动）非常普遍。为了减少次要屏幕中这些不良效应的传播，我们选择使用替代供应商的六个非填充sirnas（来自Ambion和Qiagen的三个）。使用这种方法正在进行次要屏幕。 在分析主要筛选数据的过程中，我们策划了一组128个基因，其中包括一组TLR信号成分，包括TLR受体，近端信号成分，NF-κB和MAPK途径蛋白，转录因子，细胞因子和负调节剂。我们发现，其中大约26个目标在人类LPS主屏幕上是强烈的命中，而另外15个是弱命中率。这导致我们询问该命中频率是否合理，以及是否通过KD不足或通过信号传导所需组件的细胞类型变异确定意外的负面因素（因为经典途径通常是从一系列模型细胞类型中的研究中得出的）。 我们为本集中的每个基因筛选了六个额外单独的siRNA的LPS响应，我们希望至少有一个siRNA对目标基因的有效耗竭。对数据集的分析将少量的核心途径组件确定为强命中，这可能是由于主要屏幕中的KD不足，包括TRAM，TRAF6和TAB1。 但是，次级屏幕中强典型途径的总数实际上减少到25个基因，这表明许多影响TLR信号传导的组件以特定于细胞类型的方式进行。这与对使用相同病原刺激进行的筛选的最新信息分析是一致的，但是在不同的细胞系统中，这表明尽管在这些平行筛选中鉴定出公共途径，但对siRNA KD最敏感的特定基因通常是不重叠的。然而，在我们对LPS诱导的TNF转录的测定中，我们确实看到了一种值得注意的模式，其中最初的表型群集在初始受体和衔接子蛋白中群中簇以及TNF转录增强体成分，而表型在通过NF-κB和映射途径的信号传动分支时，表型在中间步骤中较弱。 TLR刺激后在巨噬细胞中诱导的信号通路和转录因子反应受调节基因转录动力学和大小的反馈回路调节。其中，NF-κB一直是在反馈调节网络中运行的信号响应转录因子的范例。尽管有有关NF-κB激活和功能的大量文献，但仍缺乏响应致病性刺激的巨噬细胞中NF-κB单细胞动力学的数据。在上述siRNA屏幕上表达GFP标记为P65/RELA的小鼠巨噬细胞系的开发为我们提供了解决此问题的机会。 此外，耦合的TNFα启动子驱动的转录报告基因提供了独特的二级读数，可以评估单个细胞水平上NF-κB激活的后果。我们使用这个新颖的测定平台开始了几次合作。 我们正在与NCI的MIA Sung和Gordon Hager合作，研究巨噬细胞如何在NF-κB激活和TNFα转录输出的背景下解释不同的LPS剂量。 我们还将这些细胞用作与爱丁堡大学彼得·加扎尔（Peter Ghazal）合作的鼠巨细胞病毒（MCMV）进行单细胞感染研究的模型，并与剑桥大学的克莱尔·布莱恩特（Clare Bryant）合作。