Rapid Optimization of a Light-Inducible System to Control Mammalian Gene Expression

This project aims to develop a high throughput method of optimizing and characterizing a multi-component optogenetic tool called LACE for mammalian gene expression. To begin, place a cell culture flask containing HEK 293 T cells inside a biosafety cabinet. Use a serological pipette to aspirate the spent media.

Pipette 10 milliliters of DPBS to one corner of the flask and gently swirl to wash the cells. Then aspirate the wash solution and dispose both the DPBS and the aspirator into the waste container. Now add 1.5 milliliters of 0.05%Trypsin-EDTA to the flask to cover the surface, and incubate.

Gently tap the flask to loosen the cells from the surface. Next, pipette 8.5 milliliters of fresh DMEM into the flask. Aspirate the solution up and down until no aggregates are visible to resuspend the cells.

Then transfer nine milliliters of the cell suspension into a 15-milliliter conical tube. Add nine milliliters of fresh DMEM to the flask. Incubate the suspension until confluency under 5%carbon dioxide at 37 degrees Celsius.

Now, mix 10 microliters of the suspended cells with 10 microliters of trypsin dye at a one-to-one ratio to calculate cell concentration using a hemocytometer. Seed approximately 35, 000 cells in 100 microliters into each well of a high-performance number-1.5, black, 96-well, glass-bottom plate. Place the plate into an incubator set to 37 degrees Celsius and 5%carbon dioxide for 24 hours.

For transfection, first aliquot 11 microliters of warm serum-free DMEM into a 1.5-milliliter, micro-centrifuge tube for one well and 10%excess. Prepare various plasmid mass ratios of CRY2-enhanced green fluorescent protein to CIBN-guide RNA/Aliquot 110 nanograms per well of the prepared solutions into each tube containing the serum-free medium aliquots. Then pipette an additional 11 microliters of serum-free DMEM for each well into separate tubes for transfection reagent dilution.

Add the diluted transfection reagent to the DNA medium mixture and incubate to form the transfection complexes. Then pipette 20 microliters of the prepared transfection complex to each well of the 96-well plate containing the HEK 293 T cells. Wrap the 96-well plate in aluminum foil and place it into an incubator set at 37 degrees Celsius under 5%carbon dioxide for 24 hours.

To begin the activation process, modify the microcontroller input code to illuminate the desired wells using the specified script lines. Set the light-emitting diode intensity to 9.27 milliwatts per square centimeter using the code lines indicated. Adjust the pulse length to one second, then set the pulse frequency to 0.067 hertz, equivalent to every 15 seconds.

Spray the 3D-printed lid of the optoPlate with 70%ethanol. Then allow it to dry in a biosafety cabinet. Now, turn on a red light lamp in the darkroom.

Place the 96-well plate into the biosafety cabinet. Replace the plate lid with the dried 3D-printed lid. Place the 96-well plate onto the LED array to assemble the activation apparatus.

Place the fully assembled LED array apparatus into an incubator set at 37 degrees Celsius and 5%carbon dioxide for 24 hours. Connect the microcontroller, light-emitting diode and fan ports to a power source. To perform flow cytometry, run the system startup program on the site expert software.

Load two milliliters of deionized water into the sample loader. Run quality control using the provided quality control beads. Set up and specify the sample wells for acquisition.

Create the plots and tables for side scatter versus forward scatter, side scatter height versus side scatter area, side scatter area versus FITC-A, and the FITC-A statistics table. Now, snap the V-bottom 960-well plate into the plate loader of the cytometer. Select an untransfected well and click on Initialize, followed by Run.

Adjust the side scatter and FITC voltages to center the population of interest on the SSC-A versus FSC-A plot. Click on Run, then select a CMV-enhanced green fluorescent protein transfected well. Adjust the FITC voltage to include both autofluorescent and fluorescing cells in the SSC-A versus FITC-A plot.

Create a polygon to gate the healthy cell population. A second polygon to gate for doublet discrimination on the SSC-H versus SSC-A plot. Create a polygon gate to distinguish fluorescing cells from non-fluorescing ones using the gate from the untransfected cells as a reference.

Auto-record samples at 60 microliters per minute until either 200 seconds have elapsed or 10, 000 events have been reached. Then export the mean FITC values as a CSV file and analyze the data. Clean the flow cytometer by selecting the daily clean option.

Fluorescence imaging showed that at a mass ratio of one-to-nine blue light activation induced lower maximal eGFP expression compared to the five-to-five ratio, which also exhibited increased leakiness in the dark condition. Flow cytometry showed that over 95%of gated events in P1 were singlets across all conditions. And untransfected cells had less than 0.1%eGFP-positive population.

The CMV-eGFP control had approximately 99%eGFP-positive cells, while 2pLACE transfected cells had approximately 57%The CMV-eGFP transfected cells showed strong constitutive eGFP expression under both light and dark conditions, while untransfected cells showed no visible fluorescence. It also confirmed successful transfection of 2pLACE and activation of eGFP expression under blue light with lower expression than CMVeGFP. A threefold increase in eGFP expression was observed in light-activated 2pLACE samples compared to dark conditions.

The percentage of eGFP-positive cells in 2pLACE transfected samples was approximately 60%Three-to-seven mass ratio produced the highest mean fluorescence intensity and dynamic range. This protocol enables the high throughput evaluation of samples and technical replicate using the optoPlate to program varying light conditions and pulse frequencies.