Lentiviral Systems for Controlled Mammalian Expression of Unnatural Fluorescent Protein Probes

用于非天然荧光蛋白探针的受控哺乳动物表达的慢病毒系统

基本信息

批准号：
8873037
负责人：
Huiwang Ai
金额：
$ 7.6万
依托单位：
UNIVERSITY OF CALIFORNIA RIVERSIDE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-01 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8873037
关键词：
Address Amino Acids Amino Acyl-tRNA Synthetases Animals Applications Grants Attention Biological Biological Assay Biology Biosensor Cardiovascular system Cells Communities Coupled Detection Development Diabetes Mellitus Disease Doxycycline Engineering Enzymes Evolution Family Fluorescence Fluorescent Probes Gene Expression Gene Order Gene Proteins Gene Transfer Genes Genetic Genetic Code Grant Hydrogen Sulfide Image Immune Individual Inflammatory Investigation Laboratories Lentivirus Vector Life Link Malignant Neoplasms Mammalian Cell Methods Mission Neurologic Neurons Organism Oxidation-Reduction Pathologic Processes Peroxonitrite Process Production Proteins Reaction Reagent Reporter Research Research Methodology Science Signal Transduction Signaling Molecule Stem cells Stroke System Technology Toxic effect Transduction Gene Transfection Transfer RNA United States National Institutes of Health Viral Viral Packaging Viral Vector Whole Organism Work base high throughput screening human disease improved inhibitor/antagonist interest neuronal cell body novel strategies novel therapeutics promoter protein expression public health relevance screening sensor small molecule tool trafficking

项目摘要

DESCRIPTION (provided by applicant): The objective of this R03 Small Grant proposal is to develop lentiviral systems for optimized expression of unnatural fluorescent protein biosensors, affording convenient new research tools for investigation on redox signaling and redox biology. Recently, our laboratory has invented a new approach to generate reaction-based genetically encoded fluorescent probes by introducing unnatural amino acids into circularly permuted fluorescent proteins. This approach is very effective in deriving fluorescent probes with excellent sensitivity and selectivity. In particular, we have created the first genetically encoded fluorescet sensors for hydrogen sulfide (H2S) and peroxynitrite (ONOO-), two important cell redox signaling molecules. These probes have attracted much attention. However, the method requires simultaneous introduction and expression of multiple genes in the same mammalian cells to achieve the genetic incorporation of unnatural amino acids. It is now technically challenging to use these unnatural fluorescent probes in cells that are difficult to transfect. To this end, we propose to engineer lentiviral vectors to afford efficient gene transduction and optimized expression of unnatural fluorescence probes in various mammalian cells. We will optimize the lentiviral systems for elevated viral packing efficiency and enhanced gene expression. Small molecule-inducible promoters will be utilized to achieve controlled expression of individual genetic components. The completion of this work is expected to open a new avenue for many biological studies that may benefit from these powerful fluorescent tools. New biology will be elucidated using mammalian cells and animals transduced with the lentiviral vectors and expressing these fluorescent sensors. Furthermore, the lentiviral systems may help the development of fluorescent assays for screening inhibitors or activators of relevant enzymes, potentially leading to new therapeutics. In summary, this small research R03 project will generate important research methodology and reagents to accelerate biological and biomedical sciences.

描述（由申请人提供）：该R03小额资助提案的目标是开发用于非天然荧光蛋白生物传感器的优化表达的慢病毒系统，为氧化还原信号传导和氧化还原生物学的研究提供方便的新研究工具。通过将非天然氨基酸引入循环排列的荧光蛋白中来生成基于反应的基因编码荧光探针的新方法，该方法对于衍生具有优异性能的荧光探针非常有效。特别是，我们创建了第一个针对硫化氢（H2S）和过氧亚硝酸盐（ONOO-）这两种重要的细胞氧化还原信号分子的基因编码荧光传感器，但该方法需要同时引入。以及在同一哺乳动物细胞中表达多个基因以实现非天然氨基酸的基因掺入，目前在难以转染的细胞中使用这些非天然荧光探针在技术上具有挑战性。我们建议设计慢病毒载体，以在各种哺乳动物细胞中提供有效的基因转导和非天然荧光探针的优化表达。我们将优化慢病毒系统，以提高病毒包装效率并增强小分子诱导型启动子以实现受控。这项工作的完成预计将为许多生物学研究开辟一条新途径，这些生物学研究可能会受益于这些强大的荧光工具，并且将使用转染的哺乳动物细胞和动物来阐明新的生物学。此外，慢病毒系统可能有助于开发用于筛选相关酶抑制剂或激活剂的荧光测定法，从而有可能产生新的治疗方法。总之，这个小型研究 R03 项目将产生重要的研究方法和试剂。加速生物和生物医学科学的发展。