Reporter Constructs for Target Gene Discovery

用于目标基因发现的报告基因构建体

• 批准号: 8553189
• 负责人: Jayne Stommel
• 金额: $ 21.53万
• 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/8553189
• 关键词: 1-Phosphatidylinositol 3-Kinase; Biological Assay; CHEK2 gene; Cells; DNA; DNA Damage; Fluorescence; Genetic Screening; Genome; Glioma; Green Fluorescent Proteins; Image; Lentivirus Vector; Luciferases; Measurement; Measures; Molecular; Pathway interactions; Phosphorylation; Reporter; Signal Pathway; Signal Transduction; base; gene discovery; luminescence; new technology; small hairpin RNA; small molecule libraries

We are developing novel technologies for imaging signaling pathway activation for high throughput genetic screens. This will consist of luciferase and green fluorescent protein-based DNA construct that will enable the measurement of protein phosphorylation through Bi-Molecular or Intra-Molecular Fluorescence or Luminescence Complementation in cell-based assays. As a proof of principle, we are currently developing constructs to measure PI 3-kinase pathway activation via BAD and FOXO3 phosphorylation and DNA damage signaling via H2AX and CHK2 phosphorylation. These will be cloned into lentiviral vectors which will be used to transduce primary glioma cells, then the cells will be used with whole genome shRNA and chemical library screens.

我们正在开发用于成像高吞吐量遗传筛查的新技术来激活信号通路。 这将由荧光素酶和绿色荧光蛋白的DNA构建体组成，该构建体将通过基于细胞的测定中的双分子或分子内或分子内荧光或发光互补来测量蛋白质磷酸化。 作为原则的证明，我们目前正在开发通过BAD和FOXO3磷酸化和通过H2AX和CHK2磷酸化来测量PI 3-激酶途径的激活。 这些将被克隆到慢病毒载体中，该载体将用于转导原代神经胶质瘤细胞，然后将细胞与整个基因组shRNA和化学文库筛选一起使用。