Name: Rapid Optimization of a Light-Inducible System to Control Mammalian Gene Expression (Video) | JoVE Journal
Uploaded: 2025-11-04T14:15:00.000+00:00
Duration: 9 min 8 s
Description: Learn to optimize light-inducible gene expression in HEK293T cells using the LACE optogenetic tool and a 3D-printed LED array. This high-throughput method enables precise control of mammalian gene expression.

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5 minutes

This work is supported by the Translational Research Institute through NASA Cooperative Agreement NNX16AO69A and by the Good Foods Institute. This project was supported by the UC Davis Flow Cytometry Shared Resource Laboratory with technical assistance from Bridget McLaughlin, Jonathan Van Dyke, and Ashley Karajeh, with funding from the NCI P30 CA093373 (Comprehensive Cancer Center) and S10OD018223 (Beckman Coulter &#8220;Cytoflex&#8221; cytometer).

1. Plating HEK293T cells in a 96-well format
<ol>
	<li>In a biosafety cabinet, collect a bleach waste container, serological pipettes, pipette-aid, Dulbecco&#39;s Phosphate Buffered Saline (DPBS), Dulbecco-modified Eagle&#39;s Medium (DMEM) with 10% fetal bovine serum (FBS), and a T-75 cell culture flask with confluent HEK293T cells. Prewarm the media to 37 &#176;C.</li>
	<li>In a T-75 flask, check that the confluency is about 80%-100% confluent through an inverted microscope.</li>
	<li>In the biosafety cabinet, use a serological pipette to aspirate the spent media from the cell culture flask. Avoid touching the surface or plastic of the flask. Dispose of the spent media and aspirator in the waste container.</li>
	<li>Gently wash the cells with about 10 mL of DPBS by aspirating it into a corner of the flask and gently swirling it around the surface. Aspirate the DPBS from the flask and dispose of the wash and the aspirator in the waste container.</li>
	<li>Add 1.5 mL of 0.05% Trypsin-EDTA to cover the surface of the flask and incubate at room temperature for 1 min. Gently tap the flask to loosen the cells from the surface.</li>
	<li>Add 8.5 mL of fresh DMEM into the flask. Resuspend and mix cells with a serological pipette until all aggregates have been broken up by aspirating up and down.</li>
	<li>Collect 9 mL of cell suspension into a 15 mL conical tube.
	<ol>
		<li>Add 9 mL of fresh DMEM into the flask and place it into an incubator at 37 &#176;C and 5% CO2</li>
	</ol>
	</li>
	<li>Using a hemocytometer, mix 10 &#181;L of suspended cells with 10 &#181;L of Trypan blue dye at a 1:1 ratio to calculate the concentration of cells. Use this value to prepare enough cells to seed on the plate.</li>
	<li>Seed ~35,000 cells in 100 &#181;L for each well of a high-performance #1.5 black 96-well glass bottom plate and place in an incubator at 37 &#176;C and 5% CO2 for 24 h.</li>
</ol>
2. 2pLACE transfection optimization
<ol>
	<li>For one well and 10% excess, aliquot 11 &#181;L of warm serum-free DMEM (SFM) into a 1.5 mL microcentrifuge tube.</li>
	<li>Using a CRY2-eGFP: CIBN-gRNA plasmid mass ratios equal to 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7: 3, 8:2, and 9:1, aliquot 110 ng for each well of total DNA to the aliquots of SFM (Table 1).</li>
	<li>As a positive control, aliquot the same mass of CMV-eGFP plasmid to different aliquots of SFM.</li>
	<li>For each well, aliquot 11 &#181;L of SFM for transfection reagent dilution.</li>
	<li>Prepare the transfection reagent at a 1:3 mass (&#181;g) to volume (&#181;L) ratio of DNA and transfection reagent.</li>
	<li>Add diluted transfection reagent to the diluted DNA and incubate for 12 min at room temperature to create the transfection complexes.</li>
	<li>Add ~20 &#181;L of the transfection complex to each well. Ensure that it does not touch the walls of the well.</li>
	<li>Wrap the plate in aluminum foil and place it in an incubator at 37 &#176;C and 5% CO2 for 24 h.</li>
</ol>
3. 2pLACE activation
<ol>
	<li>Modify the microcontroller input code to illuminate the desired wells using these settings (Supplementary Coding File 1, Lines 147 - 158).
	<ol>
		<li>LED Intensity: 9.27 mW/cm2 (Supplementary Coding File 1, Lines 200 - 215).</li>
		<li>Pulse Length: 1 s (Supplementary Coding File 1, Lines 280 - 290).</li>
		<li>Pulse Frequency: 0.067 Hz (every 15 s) (Supplementary Coding File 1, Lines 264 - 278). 
		&#8203;NOTE: Wavelength settings are determined by the types of LEDs installed.</li>
	</ol>
	</li>
	<li>Spray the 3D printed lid of the OptoPlate with 70% ethanol and let it dry in a biosafety cabinet.</li>
	<li>Turn on a darkroom red light lamp and place the 96-well plate into a biosafety cabinet, and replace the lid with the dried 3D printed lid. 
	NOTE: One can optionally view the CMV-eGFP cells using fluorescence microscopy to estimate transfection efficiency.</li>
	<li>Take the 96-well plate and place it onto the LED array to construct the LED array apparatus. Ensure that the plate snaps into place.</li>
	<li>Connect the microcontroller, LED, and fan ports to a power source.</li>
	<li>Carefully spray wires down with 70% ethanol and wipe dry.</li>
	<li>Place the LED array apparatus into an incubator at 37 &#176;C and 5% CO2 for 24 h. Ensure that the LEDs turn on as intended and that the wires are not taut when the incubator is sealed.</li>
</ol>
4. Flow cytometry preparation
<ol>
	<li>While in a red-light environment, take out the OptoPlate and place the plate into a biosafety cabinet 
	NOTE: One can optionally view the cells by fluorescence microscopy to visually confirm light induction.</li>
	<li>Carefully aspirate all media from each well.</li>
	<li>Detach the cells using 30 &#181;L of 0.05% Trypsin-EDTA and incubate for 1 min at room temperature.</li>
	<li>Mix each well with 100 &#181;L cold FACS buffer (1% FBS in PBS) until single-celled.</li>
	<li>Transfer each sample into a V-bottom 96-well plate.</li>
	<li>Centrifuge the V-bottom 96-well plate at 164 x g for 10 min at room temperature.</li>
	<li>Aspirate 80 &#181;L of supernatant without disturbing the pellet.</li>
	<li>Add 200 &#181;L of cold FACS buffer to resuspend the pellet.</li>
	<li>Wrap the V-bottom 96-well plate in aluminum foil for light insulation.</li>
	<li>Place the plate into a styrofoam box with ice for transportation.</li>
</ol>
5. Flow cytometry gating and data collection
<ol>
	<li>Turn on the flow cytometer using the switch in the back, and open the respective flow cytometry software on the computer.</li>
	<li>Run the system startup program and load 2 mL of DI water in the sample loader (Supplementary Figure 1A).</li>
	<li>Run quality control (QC) using the provided QC beads (Supplementary Figure 1B).</li>
	<li>Ensure the flow cytometer is in plate sampling mode (Supplementary Figure 1C).</li>
	<li>Set up and specify sample wells (Supplementary Figure 1D).</li>
	<li>Create the following scatter plots and tables (Supplementary Figure 2A).
	<ol>
		<li>Side Scatter (SSC-A) vs Forward Scatter (FSC-A).</li>
		<li>SSC-H vs SSC-A.</li>
		<li>SSC-A vs FITC-A.</li>
		<li>FITC-A statistics table (Supplementary Figure 2B).</li>
	</ol>
	</li>
	<li>Snap the V-bottom plate into the plate loader of the cytometer.</li>
	<li>Select an untransfected well and click on Initialize, and then click on Run.Ensure volume sample rate is 10 &#181;L/min (Supplementary Figure 2C).</li>
	<li>Adjust SSC and FITC voltages to center the population of interest on the SSC-A vs FSC-A plot (Supplementary Figure 2C).
	<ol>
		<li>Create a polygon to gate the healthy cell population of interest (Supplementary Figure 2D).</li>
		<li>Create a polygon to gate for doublet discrimination on the SSC-H vs SSC-A plot.</li>
		<li>Adjust FITC voltage to place untransfected cells towards the left of the SSC-A vs FITC-A plot (Supplementary Figure 2D).</li>
	</ol>
	</li>
	<li>Repeat for the remaining untransfected wells and record Mean FITC-A data.</li>
	<li>Select a CMV-eGFP transfected well and click on Run.</li>
	<li>Adjust FITC voltage to capture both autofluorescing and fluorescing cells in the SSC-A vs FITC-A plot.
	<ol>
		<li>Create a polygon to gate the fluorescing cells against the non-fluorescing cells while referencing the initial gate in untransfected cells (Supplementary Figure 2E).</li>
	</ol>
	</li>
	<li>Auto-record samples at 60 &#181;L/min until 200 s have been reached and/or events have reached 10,000 (Supplementary Figure 2F).</li>
	<li>Export Mean FITC as a CSV file and analyze the data.</li>
	<li>Clean the flow cytometer through the Daily Clean option (Supplementary Figure 2G).</li>
</ol>
6. LED intensity optimization
<ol>
	<li>Edit the microcontroller script to test desired LED intensities (Supplementary Coding File 1, Lines 200-215). 
	NOTE: Equivalent LED intensities with respect to the values in the microcontroller script are reported elsewhere38. Self-calibration may be necessary when using different LEDs.</li>
	<li>Ensure the script contains the following values in (Supplementary Coding File 1, Lines 200-215) as designed in Table 2, and upload the code to the microcontroller.</li>
	<li>Repeat steps 1-5.</li>
</ol>

The authors declare no competing interests.

This protocol demonstrates the optogenetic control of a multicomponent transient gene expression in mammalian cells, allowing other researchers to rapidly and simultaneously characterize modular optogenetic systems with a fluorescent reporter.
In this case, a two-plasmid light-inducible system was optimized for the expression of eGFP in HEK293T cells. For successful co-transfection, it is essential to optimize seeding densities and mass ratios for the highest transfection efficiency. This is e...

Synthetic biology tools such as inducible gene expression systems have provided significant contributions to biological research. Their ability to regulate gene expression in a tunable, reversible, and precise manner can improve control of protein production1,2,3,4,5,6, cell morphology7,8,9,10,11,12, metabolic pathways13,14,15,16,17,18, and other targets for biomanufacturing and therapeutic applications. Chemical inducible systems can have residual19,20 or off-target effects. Chemical additives can also be very expensive and difficult to scale up industrially due to additional downstream purification processes21,22,23. Light-inducible gene expression systems can offer a scalable and precise method for biomanufacturing practices24,25,26,27.
Many light-inducible gene expression systems have been developed as useful synthetic biology tools in a variety of applications28,29,30,31,32,33,34,35,36 and wavelengths of blue35,37,38, red/far-red12,39,40, or green41 light. In the case of CRISPR-based optogenetic gene regulation, modifying the amount of individual guide RNAs (gRNAs) delivered can affect the mRNA expression of endogenous genes42, and will need to be optimized for different cell lines. Transactivation proteins such as VP6437,42,43,44, VP1639,40,44,45,46, p6544, or fusions of them47 can also be used, increasing the modularity of optogenetic systems. In order to investigate complex and intertwined biological pathways12,40,48,49,50,51,52,53, different combinations of these known components can be tested. Additionally, different cell lines can show differences in induction with the same system52. Different induction levels can lead to insufficient gene expression or sensitivity in the precise control of gene expression, requiring rigorous testing to find an optimal optogenetic system in novel or more biologically relevant cell lines. With a growing pace and widening applicability of optogenetic gene regulation, it is imperative to rapidly characterize these different systems.
Current methods in characterizing optogenetic tools either use stable or transient expression of genes12,54. Generating stably expressing cell lines and optimizing system dynamics for maximum expression can be time-consuming and therefore costly. Transient expression enables quick and valuable insights, especially when testing multicomponent optogenetic systems. Here, we used the blue light activated CRISPR-dCas9 effector (LACE)37 system, composed of cryptochrome 2 (CRY2) and cryptochrome-interacting basic-helix-loop-helix N-terminal (CIBN). It has been used to control gene expression in mammalian cells such as HEK293T (human embryonic kidney cells)37,38, CHO-DG44 (Chinese hamster ovary cells)55, and C2C12 (mouse myoblast cells)38. Its modular nature is ideal for quickly characterizing system performance through transient expression and high-throughput methods. For example, multicomponent systems like LACE may need optimization of mass ratios to improve induction, a step not typically employed in optogenetic system characterization.
This protocol details the rapid optimization and characterization of two plasmid LACE (2pLACE)38 system. In this setup, 2pLACE controls expression of a fluorescent reporter, eGFP (enhanced green fluorescent protein), in HEK293T cells. Activation is performed in black glass-bottom 96-well plates using an OptoPlate-96, a 3D printed LED array designed and constructed by Lukasz Bugaj and colleagues56,57,58,59. This protocol specifically optimizes maximal expression with different mass ratios of the two-plasmid system. It also includes user-friendly code to control different light intensities to investigate the tunability of the system and its full dynamic range. Flow cytometry is used for data collection and analysis. Protocols for generating plasmid constructs and 3D printing materials for the LED array can be found in previous publications54,56. These methods highlight a quick, high-throughput pipeline for characterizing light-inducible gene expression systems.

<table><tbody><tr><td>1.5 mL microcentrifuge tubes</td><td>VWR</td><td>10025-724</td><td></td></tr><tr><td>10 mL Reagent Reservoirs</td><td>VWR</td><td>77395-252</td><td></td></tr><tr><td>10 mL serological pipettes</td><td>VWR</td><td>75816-100</td><td></td></tr><tr><td>1000 &amp;mu;L filter tips</td><td>VWR</td><td>76322-154</td><td></td></tr><tr><td>15 mL&amp;nbsp; High-Performance Centrifuge Tubes, flat cap</td><td>VWR</td><td>89039-664</td><td></td></tr><tr><td>1931-C Optical Power Meter</td><td>Newport</td><td>1931-C</td><td></td></tr><tr><td>2 mL serological pipettes</td><td>VWR</td><td>75816-104</td><td></td></tr><tr><td>20 &amp;mu;L filter tips</td><td>VWR</td><td>76322-134</td><td></td></tr><tr><td>200 &amp;mu;L filter tips</td><td>VWR</td><td>76322-150</td><td></td></tr><tr><td>50 mL High-Performance Centrifuge Tubes, flat cap</td><td>VWR</td><td>89039-656</td><td></td></tr><tr><td>96-well v-bottom plates</td><td>BRANDplates</td><td>781661</td><td></td></tr><tr><td>A21, LED Red light bulb</td><td>Bluex Bulbs</td><td>N/A</td><td>red light source</td></tr><tr><td>Arduino IDE Software</td><td>Arduino</td><td>N/A</td><td></td></tr><tr><td>Biosafety Cabinet, Class II</td><td>NuAire</td><td>N/A</td><td></td></tr><tr><td>Bright-Line Hemocytometer</td><td>Hausser Scientific</td><td>3110</td><td></td></tr><tr><td>Cell counting slides</td><td>BioRad</td><td>1450016</td><td></td></tr><tr><td>Cell Culture Plate 96-well, #1.5H glass bottom plate</td><td>Cellvis</td><td>P96-1.5H-N</td><td></td></tr><tr><td>CytExpert Software</td><td>Beckman Coulter</td><td>N/A</td><td></td></tr><tr><td>CytoFLEX Ready to Use Daily QC Fluorospheres</td><td>Beckman Coulter</td><td>C65719</td><td>4 &amp;deg;C</td></tr><tr><td>CytoFLEX-S Flow Cytometer (4 violet, 2 blue, 4 Yellow Green, 3 Red channels)</td><td>Beckman Coulter</td><td>C09766</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline powder, no calicum, no magnesium</td><td>Fisher Scientific</td><td>21600069</td><td>Room Temperature</td></tr><tr><td>Eppendorf Centrifuge 5804</td><td>Eppendorf</td><td>&amp;nbsp;022622501</td><td>v-bottom plate centrifuge step</td></tr><tr><td>Eppendorf Centrifuge 5810R</td><td>Eppendorf</td><td>22625501</td><td></td></tr><tr><td>Eppendorf Research plus, 8-channel, variable volume, 30 - 300 &amp;mu;L</td><td>Eppendorf</td><td>3125000052</td><td></td></tr><tr><td>Eppendorf Research plus, single-channel, variable volume, 100 - 1000 &amp;mu;L</td><td>Eppendorf</td><td>3123000063</td><td></td></tr><tr><td>Eppendorf Research plus, single-channel, variable volume, 2 - 20 &amp;mu;L</td><td>Eppendorf</td><td>3123000039</td><td></td></tr><tr><td>Eppendorf Research plus, single-channel, variable volume, 20 - 200 &amp;mu;L</td><td>Eppendorf</td><td>3123000055</td><td></td></tr><tr><td>General Purpose Laboratory Labeling Tape</td><td>VWR</td><td>89097-920</td><td></td></tr><tr><td>Gibco DMEM, powder, high glucose</td><td>Fisher Scientific</td><td>12100061</td><td>4 &amp;deg;C</td></tr><tr><td>Gibco Trypan Blue Solution, 0.4%</td><td>Fisher Scientific</td><td>15-250-061</td><td>Room Temperature</td></tr><tr><td>Gibco Value Heat Inactivated FBS</td><td>Fisher Scientific</td><td>A5256901</td><td>-20 &amp;deg;C</td></tr><tr><td>Gibco, Trypsin-EDTA (0.05%), with EDTA, Animal Origin, 1X, Phenol Red, 500 mL</td><td>Gibco</td><td>25300062</td><td>-20 &amp;deg;C</td></tr><tr><td>HEK293T Cells</td><td>ATCC</td><td>CRL-11268</td><td>-80 &amp;deg;C, Liquid Nitrogen</td></tr><tr><td>Lab markers, Micronova</td><td>VWR</td><td>89205-944</td><td></td></tr><tr><td>Metal Desk Lamp</td><td>Simple Designs</td><td>N/A</td><td>Lamp for red light</td></tr><tr><td>OptoPlate-96</td><td>LABmaker</td><td>N/A</td><td></td></tr><tr><td>Pipet-aid XP</td><td>Drummond</td><td>4-000-101</td><td></td></tr><tr><td>Plasmid: CIBN-gRNA</td><td>N/A</td><td>N/A</td><td>-20 &amp;deg;C</td></tr><tr><td>Plasmid: CMV-eGFP</td><td>N/A</td><td>N/A</td><td>-20 &amp;deg;C</td></tr><tr><td>Plasmid: CRY2-eGFP</td><td>N/A</td><td>N/A</td><td>-20 &amp;deg;C</td></tr><tr><td>PolyJet DNA In Vitro Transfection Reagent</td><td>Fisher Scientific</td><td>NC1536117</td><td>4 &amp;deg;C</td></tr><tr><td>T-75 Cell Culture Flask</td><td>VWR</td><td>10062-860</td><td></td></tr><tr><td>Water Jacketed CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>VWR</td><td>10810-884</td><td></td></tr></tbody></table>

rapid optimization of a light-inducible system to control mammalian gene expression

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

Inducible gene expression tools can open novel applications in human health and biotechnology, but current options are often expensive, difficult to reverse, and have undesirable off-target effects. Optogenetic systems use light-responsive proteins to control the activity of regulators such that expression is controlled with the "flip of a switch". This study optimizes a simplified light activated CRISPR effector (2pLACE) system, which provides tunable, reversible, and precise control of mammalian gene expression. The OptoPlate-96 enables high-throughput screening via flow cytometry for single-cell analysis and rapid optimization of 2pLACE. This study demonstrates how to use the 2pLACE system with the OptoPlate-96 in HEK293T cells to identify the optimal component ratios for maximizing dynamic range and to find the blue light intensity response curve. Similar workflows can be developed for other mammalian cells and for other optogenetic systems and wavelengths of light. These advancements enhance the precision, scalability, and adaptability of optogenetic tools for biomanufacturing applications.

Adopting workflow elements from previous literature37,38,55, we added high-throughput simultaneous illumination from the OptoPlate-96 and demonstrated a pipeline to rapidly optimize a light-inducible gene expression system in mammalian cells (Figure 1).
For inducible gene expression systems, high dynamic ranges are a critical performance marker, in addition to absolute on...

This study describes a method for high-throughput experiments using a 3D-printed LED array to optimize light-inducible gene expression in HEK293T cells.

Inducible gene expression tools can open novel applications in human health and biotechnology, but current options are often expensive, difficult to ...

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.

rapid-optimization-light-inducible-system-to-control-mammalian-gene

Research

JoVE Journal

Bioengineering

This video demonstrates the tetracycline, or Tet-On, gene expression system in a mouse model. In the presence of doxycycline, a tetracycline derivative, the Tet-On system consisting of reverse tetracycline-controlled transactivator protein becomes activated and expresses the gene of interest.

Inducible Tet-On Regulatable System-Based Gene Expression In Vivo: A Tetracycline-Induced Gene Expression System for Target Gene Transcription Activation in Mouse Model

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

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Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

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

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Light-Controlled Fermentations for Microbial Chemical and Protein Production

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Learn to optimize light-inducible gene expression in HEK293T cells using the LACE optogenetic tool and a 3D-printed LED array. This high-throughput method enables precise control of mammalian gene expression.

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

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