Lentiviral Systems for Controlled Mammalian Expression of Unnatural Fluorescent Protein Probes

 描述(申请人提供)：本R03小额资助计划的目标是开发慢病毒系统，以优化非天然荧光蛋白生物传感器的表达，为氧化还原信号和氧化还原生物学的研究提供便利的新研究工具。最近，我们实验室发明了一种新的方法来产生基于反应的基因编码的荧光探针，将非天然氨基酸引入循环排列的荧光蛋白中。该方法在制备优良的荧光探针方面是非常有效的。 灵敏度和选择性。特别是，我们已经创造了第一个遗传编码的荧光传感器，用于硫化氢(H_2S)和过氧亚硝酸盐(ONOO-)，这两个重要的细胞氧化还原信号分子。这些探测器引起了人们的极大关注。然而，该方法需要在同一哺乳动物细胞中同时导入和表达多个基因，以实现非天然氨基酸的基因掺入。现在，在难以转化的细胞中使用这些非天然的荧光探针在技术上具有挑战性。为此，我们建议设计慢病毒载体，以提供高效的基因转导和非自然荧光探针在各种哺乳动物细胞中的优化表达。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。小分子可诱导启动子将被用来实现个体遗传成分的受控表达。这项工作的完成有望为许多生物学研究开辟一条新的途径，这些研究可能会受益于这些强大的荧光工具。利用慢病毒载体转导的哺乳动物细胞和动物并表达这些荧光传感器，将阐明新的生物学。此外，慢病毒系统可能有助于开发荧光分析来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R03项目将产生重要的研究方法和试剂，以促进生物和生物医学科学的发展。

 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.