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Review
. 2013 Sep 23:4:131.
doi: 10.3389/fendo.2013.00131.

Evolution of BRET Biosensors from Live Cell to Tissue-Scale In vivo Imaging

Abhijit De  1 , Akshi Jasani, Rohit Arora, Sanjiv S Gambhir
Affiliations
Review

Evolution of BRET Biosensors from Live Cell to Tissue-Scale In vivo Imaging

Abhijit De et al. Front Endocrinol (Lausanne). .

Abstract

Development of bioluminescence resonance energy transfer (BRET) based genetic sensors for sensing biological functions such as protein-protein interactions (PPIs) in vivo has a special value in measuring such dynamic events at their native environment. Since its inception in the late nineties, BRET related research has gained significant momentum in terms of adding versatility to the assay format and wider applicability where it has been suitably used. Beyond the scope of quantitative measurement of PPIs and protein dimerization, molecular imaging applications based on BRET assays have broadened its scope for screening pharmacologically important compounds by in vivo imaging as well. In this mini-review we focus on an in-depth analysis of engineered BRET systems developed and their successful application to cell-based assays as well as in vivo non-invasive imaging in live subjects.

Keywords: bioluminescence resonance energy transfer; cell-based assay; fluorescent proteins; luciferase; optical imaging; protein–protein interactions.

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Figures

Figure 1
Figure 1
Various new generation of BRET pairs reported using mutant Renilla luciferase proteins. (A) Represents TagRFP-RLuc8 pair using normal coelenterazine (Clz) substrate. The bottom chart represents normalized amplitude vs. wavelength measured from the purified fusion protein added with Clz substrate. (B) Represents the same pair using the coelenterazine-v (Clz-v) substrate analog. (C) Represents TurboFP-RLuc8 pair using the Clz-v substrate. The chart represents normalized amplitude vs. wavelength measured from purified protein with Clz-v substrate. Note that in majority cases [e.g., (A) or (C)], typical normalized amplitude emission of the donor is higher than the acceptor emission output, whereas in some cases [e.g., (B)], due to efficient transfer of energy, normalized amplitude of acceptor emission is higher than the donor emission. (D) Represents western blot analysis of various BRET fusion proteins made by combining mutant Renilla luciferases (such as RLuc8 and RLuc8.6) with mOrange, TagRFP, and TurboFP fluorescence acceptors. As control, RLuc8 (RL8) protein of 35 kDa size was also shown on the right most lane. (E) BRET ratios (denoted as A/D ratio on Y-axis) are calculated by measuring the donor and acceptor Emmax from each of these fusions in the presence of indicated Clz (or its analog) substrate from mammalian cells expressing the proteins. Spectral resolutions (difference between the donor and acceptor emission peaks) are also plotted on the Z-axis (as dark circles). One can observe an inverse relationship between the BRET ratio measured and the spectral resolution of the BRET partners which is more or less linear in fashion. In some cases, the BRET ratio was seen to be >1 which is theoretically not possible. This is because the values mentioned here are the raw measured values without accounting for the Cf value. Once the Cf value is added into the equation, the BRET ratios drop below 1. Note that either Clz-h or Clz-v substrate analogs can be used against the same fusion protein to fine tune the spectral resolution. (F) Spectral profile measured from live mammalian cells over-expressing RLuc8, mOrange-RLuc8, TagRFP-RLuc8, and TurboFP-RLuc8 fusion proteins. Cells over-expressing these proteins were exposed to live cell substrate EnduRenTM (Endu) and emission profiles were imaged using IVIS spectrum imaging system loaded with 20 nm band-pass filters between 460 and 720 nm. This arrangement provides a multiplexing opportunity, where the same donor protein can be combined with multiple acceptors, whose Emmax can be resolved by band-pass filter sets as indicated on the chart.
Figure 2
Figure 2
Bioluminescence resonance energy transfer performance in deep tissue imaging experiments. (A) Upper panel represents mouse images comparing improvements in the signal output from lungs. Mammalian cells engineered for equivalent over-expression of donor alone (RLuc8 or RLuc8.6) or BRET proteins (mOrange-RLuc8 or TurboFP-RLuc8.6) as marked, were compared. Note the photon output values in the reference color scale bars. Highest signal output from same number of cells placed within lungs was noted with TurboFP-RLuc8.6 BRET protein imaged with Clz substrate. (B) Schematic illustration of the most successful BRET format tested for monitoring the rapamycin induced FRB-FKBP12 association. (C) Representative bioluminescence images of nude mice with accumulated mammalian cells in the lungs which stably over-express FRB and FKBP12 interacting partners fused to RLuc8.6 and TurboFP respectively. Cells (3 × 106 in 150 μL PBS) were injected through the tail vein, resulting in significant trapping in the lungs. One group of mice (n = 8) was injected 2 h before cell injection with 40 μg rapamycin dissolved in 20 μL DMSO and further diluted in 130 μL PBS administered through the tail vein. A second group of mice (n = 8) was injected with DMSO (20 in 130 μL PBS). Two hours after cell injection, the mice were injected i.v. with Clz substrate and sequentially imaged using open/donor/acceptor filters. Substrate-only control mice (n = 4) were used for background subtraction. The figure is partially represented with permission from PNAS (7).
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