GFP-Based Assays to Probe Transcriptional Controls(RMI)

基于 GFP 的转录控制检测 (RMI)

• 批准号: 7021190
• 负责人: RICHARD ALAN KATZ
• 金额: $ 21.13万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7021190
• 关键词: DNA replication; amidohydrolases; biotechnology; cell cycle; cell line; chemical stability; clinical research; drug screening /evaluation; enzyme inhibitors; gene induction /repression; genetic regulation; genetic transcription; green fluorescent proteins; high throughput technology; human tissue; messenger RNA; protein biosynthesis; small interfering RNA; small molecule; technology /technique development

DESCRIPTION (provided by applicant): Two (2) green fluorescence protein (GFP)-based assays will be developed to probe mechanisms of transcriptional and post-transcriptional control. The first assay will utilize cells that contain a GFP gene that is silenced by epigenetic mechanisms. Treatment of these cells with small molecules that are known histone deacetylase (HDAC) inhibitors (HDIs), results in robust reactivation of GFP. This finding indicates that the silencing mechanisms likely include (but are not limited to) histone deacetylation. HDIs have been recognized as anti-cancer drugs and high throughput screens (HTSs) of chemical libraries using this GFP cell-based assay should identify new HDIs, as well as compounds that target other proteins that are required for maintenance of silencing. Preliminary results include several proof of-principle experiments that address specificity, sensitivity and background of the assay. The proposed experiments will test the adaptability of the assay to a multi-well format. Pilot screens with known compounds (HDIs) and chemical libraries will be carried out. GFP-silent cells will also be challenged with siRNAs that will knockdown the expression of HDACs and this treatment is also predicted to reactivate the silent GFP gene. Additional experiments will also be carried out to assess the feasibility of a genome-wide siRNA HTS to identify other genes involved in the maintenance of epigenetic silencing. A second GFP based assay will be used to probe the pathway of posttranscriptional control that coordinates DNA replication and histone biosynthesis, through histone mRNA stability. In this assay, a fusion transcript will be generated encoding GFP and the 3'-untranslated region of histone mRNA. It is predicted that inhibition of DNA synthesis will result in specific degradation of the GFP fusion transcript. Chemicals or siRNAs that interrupt this regulatory pathway are predicted to stabilize the fusion transcript and allow continued GFP expression. Feasibility studies will be carried out to validate this assay. As both assays are GFP-based, optimized conditions for GFP detection in a HTS format will be applicable to both systems. Both of these assays address fundamental aspects of cell regulation (epigenetic silencing, cell cycle control) that have been implicated in cancer development. The assays have the potential to reveal new lead compounds, as well as new gene targets for therapeutics.

描述（由申请人提供）：将开发两（2）种基于绿色荧光蛋白（GFP）的检测试剂盒，以探测转录和转录后控制机制。第一个试验将利用含有通过表观遗传机制沉默的GFP基因的细胞。用已知的组蛋白脱乙酰酶（HDAC）抑制剂（HDI）的小分子处理这些细胞，导致GFP的稳健再活化。这一发现表明沉默机制可能包括（但不限于）组蛋白脱乙酰化。HDI已被公认为抗癌药物，使用这种基于GFP细胞的测定法对化学文库进行高通量筛选（HTS）应鉴定新的HDI，以及靶向维持沉默所需的其他蛋白质的化合物。初步结果包括几个证明原理的实验，解决特异性，灵敏度和背景的测定。拟定的实验将测试测定对多孔格式的适应性。将对已知化合物（HDI）和化学库进行中试筛选。GFP沉默细胞也将受到siRNA的攻击，siRNA将敲低HDAC的表达，并且这种处理也被预测会重新激活沉默的GFP基因。还将进行其他实验以评估全基因组siRNA HTS鉴定参与维持表观遗传沉默的其他基因的可行性。第二个基于GFP的试验将用于探测转录后控制途径，该途径通过组蛋白mRNA稳定性来协调DNA复制和组蛋白生物合成。在该测定中，将产生编码GFP和组蛋白mRNA的3 '-非翻译区的融合转录物。据预测，DNA合成的抑制将导致GFP融合转录物的特异性降解。预测中断该调节途径的化学品或siRNA稳定融合转录物并允许GFP持续表达。将进行可行性研究以验证该测定。由于两种检测试剂盒均基于GFP，因此HTS形式的GFP检测的优化条件将适用于两种系统。这两种测定都解决了与癌症发展有关的细胞调节的基本方面（表观遗传沉默，细胞周期控制）。这些分析有可能揭示新的先导化合物，以及新的治疗基因靶点。