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

• 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
• 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

 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R 03小额赠款提案的目的是开发慢病毒系统，用于优化非天然荧光蛋白生物传感器的表达，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。最近，我们的实验室发明了一种新的方法，通过将非天然氨基酸引入循环排列的荧光蛋白中来产生基于反应的遗传编码荧光探针。这种方法在衍生具有优异的荧光特性的荧光探针方面是非常有效的。 灵敏度和选择性。特别是，我们已经创建了第一个基因编码的硫化氢（H2S）和过氧亚硝酸盐（ONOO-），两个重要的细胞氧化还原信号分子的荧光传感器。这些探测器备受关注。然而，该方法需要在相同的哺乳动物细胞中同时引入和表达多个基因，以实现非天然氨基酸的遗传掺入。现在，在难以检测的细胞中使用这些非天然荧光探针在技术上具有挑战性。为此，我们建议工程慢病毒载体，以提供有效的基因转导和优化表达的非天然荧光探针在各种哺乳动物细胞。我们将优化慢病毒系统，以提高病毒包装效率和增强基因表达。小分子诱导型启动子将用于实现个体遗传组分的受控表达。这项工作的完成有望为许多可能受益于这些强大的荧光工具的生物学研究开辟新的途径。新的生物学将阐明使用哺乳动物细胞和动物转导的慢病毒载体和表达这些荧光传感器。此外，慢病毒系统可能有助于荧光检测的发展，用于筛选相关酶的抑制剂或激活剂，可能导致新的治疗方法。总之，这个小型研究R 03项目将产生重要的研究方法和试剂，以加速生物和生物医学科学。