Chromatin Insulator Function and Nuclear Organization

染色质绝缘体功能和核组织

• 批准号: 7733963
• 负责人: Elissa Lei
• 金额: $ 27.99万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY 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/7733963
• 关键词: Antibodies; Automobile Driving; Binding; Biological Assay; Candidate Disease Gene; Cell Line; Cells; Chromatin; Chromatin Loop; Complex; DNA Sequence; Disruption; Double-Stranded RNA; Drosophila genus; Enhancers; Fluorescence-Activated Cell Sorting; Gene Expression; Genes; Genetic Screening; Genome; Green Fluorescent Proteins; Gypsies; Image; Immunofluorescence Immunologic; Indirect Immunofluorescence; Libraries; Liquid substance; Maintenance; Methods; Monitor; Mutation; Nuclear; Nuclear Matrix; Organism; Population; Proteins; RNA Interference; Range; Reporter; Screening procedure; Signal Transduction; Standards of Weights and Measures; Structure; System; Testing; Transcript; Transcriptional Regulation; Work; base; enhanced green fluorescent protein; interest; medical schools; mutant; novel; promoter; stable cell line

Cell based assays for insulator body localization are currently being utilized and/or developed to answer basic mechanistic questions. Localization of insulator bodies can be determined by standard indirect immunofluorescence using antibodies specific for gypsy insulator proteins. In addition, eight independent stable cell lines that express a fluorescent insulator protein have been produced and clonally selected. Prior to immunofluorescence or monitoring of GFP signal, cells are first subjected to dsRNA knockdown for transcripts of interest to determine whether the proteins they encode are required for maintenance of insulator bodies. Candidates of interest are factors involved in RNA silencing, which themselves are subject to degradation using this method, as well as novel factors found to copurify with insulator proteins. As a complimentary assay to insulator body localization, we are utilizing transcriptional reporter cell lines under the control of two distinct insulator sequences. Cell lines harboring either gypsy or Fab-8 insulator sequence in between the OpIE2 enhancer and a minimal promoter driving the enhanced green fluorescent protein gene (EGFP) are examined by fluorescence activated cell sorting (FACS) following dsRNA knockdown of transcripts of interest (Ciavatta et al., 2007). Unlike immunofluorescence, FACS analysis is quantitative and indicates the full range of insulator activities in the cell population. Stably transfected cells expressing a GFP-tagged insulator protein can be used to identify novel factors required for insulator body integrity using high throughput genome wide dsRNA screening. With the assistance of the Drosophila RNAi Screening Center at Harvard Medical School, a library of 21,000 dsRNAs corresponding to all annotated Drosophila genes will be applied to the cells using automated liquid handling. Automated imaging will be utilized to identify RNAs that no longer support GFP reporter localization to 20-25 nuclear foci indicating disruption of insulator body formation. This screen should identify factors required specifically for proper insulator localization as well as general nuclear organization.

目前正在利用和/或开发基于细胞的绝缘体定位测定方法来回答基本的机理问题。 绝缘体的定位可以通过使用吉普赛绝缘子蛋白特异的抗体来确定标准间接免疫荧光。 此外，已经产生并选择了八种表达荧光绝缘子蛋白的独立稳定细胞系。 在进行免疫荧光或监测GFP信号之前，首先要对感兴趣的转录本进行DSRNA敲低，以确定是否需要编码的蛋白质才能维持绝缘体的体系。 感兴趣的候选者是RNA沉默中涉及的因素，RNA沉默本身可能会使用这种方法降解，并且发现与绝缘子蛋白共腐蚀的新因素。 作为绝缘体本地化的免费测定，我们在两个不同的绝缘子序列的控制下利用转录报告基细胞系。 OPIE2增强子和驱动增强绿色荧光蛋白基因（EGFP）的最小启动子之间具有吉普赛或FAB-8绝缘子序列的细胞系通过荧光激活的细胞分选（FACS）在DSRNA敲低转录后（Ciavatta等人，2007年）。 与免疫荧光不同，FACS分析是定量的，并指示细胞群中的绝缘活性范围。 表达GFP标记的绝缘子蛋白的稳定转染的细胞可用于鉴定使用高吞吐量基因组宽DSRNA筛选所需的新型因素。 在哈佛医学院的果蝇RNAi筛查中心的协助下，使用自动化的液体处理将与所有与所有注释的果蝇基因相对应的21,000个DSRNA库将其应用于细胞。 自动化成像将用于确定不再支持GFP报告基因定位到20-25核灶的RNA，表明破坏绝缘体体形的形成。 该屏幕应具体确定适当的绝缘体定位以及一般核组织所需的因素。