Targeting High-risk Neuroblastoma using high through put siRNA screening

使用高通量 siRNA 筛选靶向高风险神经母细胞瘤

• 批准号: 7733242
• 负责人: Javed KHAN
• 金额: $ 18.13万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/7733242
• 关键词: Animal Model; Apoptosis; Biological Assay; Cell Count; Cell Cycle; Cell Line; Cells; Childhood; Data; Devices; Down-Regulation; Electronics; Gene Silencing; Genes; Genome; Gold; Growth; Human; Isotopically-Coded Affinity Tagging; Label; Life; MYCN gene; Malignant Childhood Neoplasm; Malignant Neoplasms; Measures; Methods; Microelectrodes; Microscope; Monitor; Neuroblastoma; Normal tissue morphology; Pathway interactions; Patients; Personal Satisfaction; Pharmaceutical Preparations; Phase; Preclinical Testing; Proteins; Publications; Publishing; RNA Interference; Recommendation; Resources; Risk; Screening procedure; System; Technology; Testing; Time; Translating; Xenograft Model; Xenograft procedure; antigene; base; disorder risk; gene repression; indexing; programs; research study; size

We will utilize two of the cell lines, one MYCN not-amplified and one MYCN amplified cell line that can be grown in a xenograft model. Among other assays we will use the Real-Time Cell Electronic Sensing System (RT-CES system; Acea Biosciences, CA, USA) as well as the BD Pathway 96-well confocal microscope. The RT-CES is an electronic device that allows a label-free, cell based monitoring of cell number and size in real time without the requirement for manipulating the cells. The data is collected from living cells in real-time in a 96-well format, over the course of the entire experiment. Each well incorporates a gold circle-on-line microelectrode array, where the cells attach and the impendence of each well is measured (Cell Index) which is proportional to the cell number. The BD Pathway microscope allows for high content screening of siRNA gene silencing and monitors cell number, apoptosis, and cell cycle parameters. We have tested several drugs and siRNAs with these methods and find it a robust measure of cell number. The most promising targets and the appropriate siRNA will be further evaluated in the two xenograft animal models as outlined above. All positive hits will be further screened in a wider panel of NB Xenografts which will be part of a panel of the Pediatric Preclinical Testing Program (PPTP) which is currently being utilized as a pipeline to screen new drugs and make recommendation for human pediatric phase 1/2 trials (http://ctep.cancer.gov/resources/child.html. We have successfully optimized the high through put siRNA screening using a set of approximately 400 apoptosis related genes and the findings are being prepared for publication.

我们将利用两条细胞系，一种未扩增的MYCN，以及可以在异种移植模型中生长的MYCN扩增细胞系。在其他测定中，我们将使用实时电池电子感应系统（RT-CES系统； ACEA Biosciences，CA，USA）以及BD途径96孔室共聚焦显微镜。 RT-CES是一种电子设备，可以实时对单元格数和大小进行基于细胞的基于标签的监视，而无需操纵细胞。在整个实验过程中，数据以96孔格式实时从活细胞中收集。每个井都结合了一个金圆在线微电极阵列，其中细胞附着和每个孔的强迫率（细胞指数）与单元格成正比。 BD途径显微镜可以对siRNA基因沉默的高度筛选，并监测细胞数，凋亡和细胞周期参数。我们已经用这些方法测试了几种药物和siRNA，并发现它是细胞数量的强大度量。如上所述，将在两个异种移植动物模型中进一步评估最有希望的目标和适当的siRNA。 All positive hits will be further screened in a wider panel of NB Xenografts which will be part of a panel of the Pediatric Preclinical Testing Program (PPTP) which is currently being utilized as a pipeline to screen new drugs and make recommendation for human pediatric phase 1/2 trials (http://ctep.cancer.gov/resources/child.html. We have successfully optimized the high通过将siRNA筛选使用一组大约400个相关基因的筛查，并准备了这些发现以供出版。