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.