An Improved Bioluminescence Resonance Energy Transfer Strategy for Imaging Intracellular Events in Single Cells and Living Subjects

Abstract

Bioluminescence resonance energy transfer (BRET) is currently used for monitoring various intracellular events, including protein-protein interactions, in normal and aberrant signal transduction pathways. However, the BRET vectors currently used lack adequate sensitivity for imaging events of interest from both single living cells and small living subjects. Taking advantage of the critical relationship of BRET efficiency and donor quantum efficiency, we report generation of a novel BRET vector by fusing a GFP 2 acceptor protein with a novel mutant Renilla luciferase donor selected for higher quantum yield. This new BRET vector shows an overall 5.5-fold improvement in the BRET ratio, thereby greatly enhancing the dynamic range of the BRET signal. This new BRET strategy provides a unique platform to assay protein functions from both single live cells and cells located deep within small living subjects. The imaging utility of the new BRET vector is shown by constructing a sensor using two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. This new BRET vector should facilitate high-throughput sensitive BRET assays, including studies in single live cells and small living subjects. Applications will include anticancer therapy screening in cell culture and in small living animals. [Cancer Res 2007;67(15):7175-83]

Identification of transmembrane domains that regulate spatial arrangements and activity of prokineticin receptor 2 dimers

“…PKR2 was tagged at the intracellular C-terminus with the BRET donor or acceptor molecules, Rluc8 or Venus, which are more stable variants of the classic Rluc and YFP, respectively (De et al, 2007). To ascertain if the C-terminal tagging of the receptor with either Rluc8 or Venus did not alter its function, we compared the activity of the tagged variants to the untagged WT PKR2 in regard to the G αs and G αq pathways.…”

Functional Selective Oxytocin-derived Agonists Discriminate between Individual G Protein Family Subtypes

“…1c). We then investigated OTR coupling to G␣ q , G␣ i1 , G␣ i2 , G␣ i3 , G␣ oA , and G␣ oB with the G␣ q -97-Rluc, G␣ i1 -91-Rluc, G␣ i2 -91-Rluc, G␣ i3 -91-Rluc, G␣ oA -91-Rluc8, and G␣ oB -91-Rluc8 constructs; Rluc8 (27) constructs were used to characterize G␣ oA and G␣ oB functional selectivity because the agonist-promoted BRET variation was more pronounced and less variable with them than with the Rluc. 4 To avoid possible variations in the BRET signal resulting from fluctuation in the relative expression level of donors and acceptors, we set up transfection conditions in which comparable protein expression levels were maintained constant (see supplemental Fig.…”

Integrating the biological characteristics of oncolytic viruses and immune cells can optimize therapeutic benefits of cell-based delivery

“…20,22,23 Among the molecular imaging methods described to date, in vivo bioluminescence imaging 19,22 has emerged as one of the most versatile and sensitive methods for in vivo studies of biology and therapeutic responses in animal models. As such bioluminescence imaging has been used to advance our understanding of a number of chemotherapeutic agents 24,25 and biological therapies 26,27 for cancer and other diseases. 28 The combination of cytokine induced killer (CIK) cells as the carrier vehicle and oncolytic vaccinia virus as its biological payload fall into the category of cell-based combination therapies that may offer additive or synergistic therapeutic benefits.…”