Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

1(electronic tones)Bioluminescent optogenetics is a platform technology in which genetically encoded light sources can visualize and control biological functions and networks within and between cells. Bioluminescence reaches every light-sensing domain expressed in the target cell population without any tissue and cell damage by fiber optics and extended physical light exposure. To begin, start preparing the Luciferin stock by dissolving five milligrams of lyophilized CTZ in 250 microliters of respective solvent.

Ensure dissolution of CTZ along the walls by pipetting. While protecting the vial from direct light, prepare 50 microliter aliquots in 0.5 milliliter black microcentrifuge tubes and store the aliquots at minus 80 degrees Celsius for future use. Perform all further manipulations in a light tight room in a laminar flow hood illuminated by red light.

To perform single bioluminescence light stimulation, prepare a working solution of Luciferin in a cell culture medium, shortly before adding to the cells at a final concentration of 100 micromolar. Add Luciferin-containing medium to the cells and incubate for the desired duration of light stimulation. Turn off the red light.

After waiting for a few seconds until eyes have adjusted to complete darkness, monitor light emission by eye. Document the light emission by taking a photograph. Terminate the light stimulation by replacing the Luciferin-containing medium with the culture medium, then wash the cells with the culture medium once or twice to eliminate all Luciferin depending on experiment sensitivity.

To avoid cell loss, plate the cells on PDL-coated dishes and incubate the cells. To perform repeated bioluminescence light stimulation, set up the live cell imaging chamber by creating a light tight compartment around the live cell imaging microscope using a box and black sheets. Ensure covering all the light sources present inside the room.

Set up the perfusion system with the desired solution for intake and the chamber output leading to a waste container. Depending upon the number of repeat stimulations, aliquot the prepared working Luciferin imaging solution into the microcentrifuge tubes. Place a coverslip with transfected cells in the chamber.

While keeping the pump running, remove the inlet tube of the pump from the intake beaker. Quickly immerse the inlet tube in Luciferin solution, keeping the transition time short to avoid any air void in the tubing. As soon as the Luciferin solution has been taken up, place the inlet tube back into the intake beaker.

Repeat removing and immersing the inlet tube in Luciferin as many times as needed at the intervals of several minutes to hours depending on the physiological pattern to which the cells are supposed to be exposed. Then keep the cells in a light protected environment for 16 to 24 hours for transcription or the duration until the effect of light stimulation is assessed. To perform the bioluminescence activation in vivo, prepare Luciferin by taking a CTZ vial out from minus 80 degrees Celsius, then let it warm to room temperature in the light protected area.

For each vial containing 500 micrograms of CTZ, add 250 microliters of sterile water using a pipette and put the rubber stopper back on the vial. Incubate the reconstituted glass vial in a water bath tempered at 55 degrees Celsius for a few minutes to completely dissolve the powder. Transfer the solution into a black microcentrifuge tube and rinse the walls to retrieve all CTZ.

After removing the amount of solution needed, store the remaining solution at four degrees Celsius. After preparing, reconstituting, and aliquoting the vehicle in a similar way, preform the bioluminescence light stimulation by removing the volume of Luciferin or vehicle needed for the size of the animal and application route chosen. Inject the animal with Luciferin or vehicle as designed.

For desired recombinase activation during a specific behavioral paradigm, inject the animal just before the behavioral testing. For phasic transcription, inject the animal repeatedly over days and collect the data from the bioluminescence stimulated animal as designed. The fast light and activity regulated expression system allowed transcription of the reporter gene with increased intracellular calcium ions and light.

The blue LED led to robust reporter fluorescence and the FLuc expression was determined by luminescence measurements after Luciferin addition. In an in vivo imaging system, both LED and CTZ robustly increased the FLuc expression, while the presence of the transcription factor component alone resulted in considerable background signal, possibly due to spontaneous proteolysis. NanoLuc was employed for optogenic regulation through the CRY/CIB dimerization and photosensitive transcription factor EL222.

The bioluminescence induced by the addition of hCTZ to HEK-293 cells and its removal after 15 minutes was more efficient than 20 minutes of LED exposure for CRY/CIB and EL222. No significant differences in the transcription efficacy were observed between the two systems when co-transfected with hCTZ, although the fusion proteins of CRY/CIB were more efficient than EL222. CRY/CIB showed consistently higher background levels compared to EL222 independent of hCTZ concentration.

The bioluminescence of a photosensitive split Cre recombinase based on the VVD LOV protein iCreV was tested. The results from the CTZ application revealed that the CTZ presence robustly increased the expression over the background and is similar to the LED application. Using bioluminescence to activate not only iron moving photoreceptors, but any photosensory domains adds another dimension to light stimulation expanding the manipulation of cell functions across temporal and spatial scales.

Bioluminescent optogenetics has been applied in cells and model organisms. These protocols can be customized to test a broad range of directed hypotheses in vitro and in vivo.