Name: reliably engineering and controlling stable optogenetic gene circuits in mammalian cells
Uploaded: 2021-07-06T14:00:00
Description: Watch this Scientific Journal Video about Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells at JoVE.com

We would like to thank Bal&#225;zsi lab for comments and suggestions, Dr. Karl P. Gerhardt and Dr. Jeffrey J. Tabor for helping us construct the first LPA, and Dr. Wilfried Weber for sharing the LOV2-degron plasmids. This work was supported by the National Institutes of Health [R35 GM122561 and T32 GM008444]; The Laufer Center for Physical and Quantitative Biology; and a National Defense Science and Engineering Graduate (NDSEG) Fellowship. Funding for open access charge: NIH [R35 GM122561].
Author contributions: M.T.G. and G.B. conceived the project. M.T.G., D.C., and L.G., performed the experiments. M.T.G., D.C., L.G., and G.B. analyzed the data and prepared the manuscript. G.B. and M.T.G. supervised the project.

1. Gene circuit design
<ol>
	<li>Select genetic components to combine into a single gene circuit/plasmid (e.g., mammalian DNA integration sequence motifs32, light-responsive elements33, or functional genes34).</li>
	<li>Using any genetic engineering and/or molecular cloning software, store the DNA sequences for later use and reference, annotate each sequence, and examine all the necessary features (e.g., START codons, regulatory, or translated sequ...

Readers of this article can gain insight into the steps vital for characterizing optogenetic gene circuits (as well as other gene expression systems), including 1) gene circuit design, construction, and validation; 2) cell engineering for introducing gene circuits into stable cell lines (e.g., Flp-FRT recombination); 3) induction of the engineered cells with a light-based platform such as the LPA; 4) initial characterization of light induction assays via fluorescence microscopy; and 5) final gene expression characterizat...

Similar to other engineering disciplines, synthetic biology aims to standardize protocols, allowing tools with highly reproducible functions to be utilized for exploring questions relevant to biological systems1,2. One domain in synthetic biology where many control systems have been built is the area of gene expression regulation3,4. Gene expression control can target both protein levels and variability (noise or coefficient of variation, CV = &#963;/&#181;, measured as the standard deviation over the mean), which are crucial cellular characteristics due to their roles in physiological and pathological cellular states5,6,7,8. Many synthetic systems that can control protein levels and noise4,9,10,11,12 have been engineered, creating opportunities to standardize protocols across tools.
One novel set of tools that can control gene networks that has recently emerged is optogenetics, enabling the use of light to control gene expression13,14,15,16,17. Similar to their chemical predecessors, optogenetic gene circuits can be introduced into any cell type, ranging from bacteria to mammals, allowing expression of any downstream gene of interest18,19. However, due to the rapid generation of novel optogenetic tools, many systems have emerged that vary in genetic circuit architecture, mechanism of expression (e.g., plasmid-based vs. viral integration), and light supplying control equipment11,16,20,21,22,23,24,25. Therefore, this leaves room for standardization of optogenetic features such as gene circuit construction and optimization, method of system utilization (e.g., integration vs. transient expression), experimental tools used for induction, and analysis of results.
To make progress on standardizing optogenetic protocols in mammalian cells, this protocol describes an experimental pipeline to engineer optogenetic systems in mammalian cells using a negative feedback (NF) gene circuit integrated into HEK293 cells (human embryonic kidney cell line) as an example. NF is an ideal system to demonstrate standardization since it is highly abundant26,27,28 in nature, allowing protein levels to be tuned and noise minimization to occur. In brief, NF allows for precise gene expression control by a repressor reducing its own expression sufficiently fast, thereby limiting any change away from a steady state. The steady state can be altered by an inducer that inactivates or eliminates the repressor to allow for more protein production until a new steady state is reached for each inducer concentration. Recently, an engineered NF optogenetic system was created that can produce a wide-dynamic response of gene expression, maintain low noise, and respond to light stimuli allowing the potential for spatial gene expression control11. These tools, known as light-inducible tuners (LITers), were inspired by earlier systems that allowed gene expression control in living cells4,10,29,30 and were stably integrated into human cell lines to ensure long-term gene expression control.
Here, using the LITer as an example, a protocol is outlined for creating light-responsive gene circuits, inducing gene expression with a Light Plate Apparatus (LPA, an optogenetic induction hardware)31, and analyze responses of the engineered, optogenetically-controllable cell lines to custom light stimuli. This protocol allows users to utilize the LITer tools for any functional gene they wish to explore. It can also be adapted for other optogenetic systems with diverse circuit architectures (e.g., positive feedback, negative regulation, etc.) via integrating the methods and optogenetic equipment outlined below. Similar to other synthetic biology protocols, the video recordings and optogenetic protocols outlined here can be applied in single-cell studies in diverse areas, including but not limited to cancer biology, embryonic development, and tissue differentiation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL PCR tubes</td>
			<td>Eppendorf</td>
			<td>951010006</td>
			<td>reagent for carrying out PCR</td>
		</tr>
		<tr>
			<td>0.25% Trypsin EDTA 1X</td>
			<td>Thermo Fisher Scientific</td>
			<td>MT25053CI</td>
			<td>reagent for splitting &#38; harvesting mammalian cells</td>
		</tr>
		<tr>
			<td>0.5-10 &#956;L Adjustable Volume Pipette</td>
			<td>Eppendorf</td>
			<td>3123000020</td>
			<td>tool used for pipetting reactions</td>
		</tr>
		<tr>
			<td>100-1000 &#956;L Adjustable Volume Pipette</td>
			<td>Eppendorf</td>
			<td>3123000039</td>
			<td>tool used for pipetting reactions</td>
		</tr>
		<tr>
			<td>20-200 &#956;L Adjustable Volume Pipette</td>
			<td>Eppendorf</td>
			<td>3123000055</td>
			<td>tool used for pipetting reactions</td>
		</tr>
		<tr>
			<td>2-20 &#956;L Adjustable Volume Pipette</td>
			<td>Eppendorf</td>
			<td>3123000039</td>
			<td>tool used for pipetting reactions</td>
		</tr>
		<tr>
			<td>5 mL Polystyene Round-Bottom Tube w/ Cell Strainer Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>reagent for flow cytometry</td>
		</tr>
		<tr>
			<td>5702R Centrifuge, with 4 x 100 Rotor, 15 and 50 mL Adapters, 120 V</td>
			<td>Eppendorf</td>
			<td>22628113</td>
			<td>equipment for mammalian culture work</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Denville Scientific</td>
			<td>GR140-500</td>
			<td>reagent for gel electrophoresis</td>
		</tr>
		<tr>
			<td>Aluminum Foils for 96-well Plates</td>
			<td>VWR&#174;</td>
			<td>60941-126</td>
			<td>tool used for covering plates in light-induction experiments</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma Aldrich</td>
			<td>A9518-5G</td>
			<td>reagent for selecting bacteria with correct plasmid</td>
		</tr>
		<tr>
			<td>Analog vortex mixer</td>
			<td>Thermo Fisher Scientific</td>
			<td>02215365PR</td>
			<td>tool for carrying PCR, transformation, or gel extraction reactions</td>
		</tr>
		<tr>
			<td>Bacto Dehydrated Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0140010</td>
			<td>reagent for growing bacteria</td>
		</tr>
		<tr>
			<td>BD LSRAria</td>
			<td>BD</td>
			<td>656700</td>
			<td>tool for sorting engineered cell lines into monoclonal populations</td>
		</tr>
		<tr>
			<td>BD LSRFortessa</td>
			<td>BD</td>
			<td>649225</td>
			<td>tool for characterizing engineered cell lines</td>
		</tr>
		<tr>
			<td>BSA, Bovine Serum Albumine</td>
			<td>Government Scientific Source</td>
			<td>SIGA4919-1G</td>
			<td>reagent for IF incubation buffer</td>
		</tr>
		<tr>
			<td>Cell Culture Plate 12-well, Clear, flat-bottom w/lid, polystyrene, non-pyrogenic, standard-TC</td>
			<td>Corning</td>
			<td>353043</td>
			<td>plate used for growing monoclonal cells</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>VWR</td>
			<td>22628113</td>
			<td>instrument for mammalian cell culture</td>
		</tr>
		<tr>
			<td>Chemical fume hood</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>instrument for carrying out IF reactions</td>
		</tr>
		<tr>
			<td>Clear Cell Culture Plate 24 well flat-bottom w/ lid</td>
			<td>BD</td>
			<td>353047</td>
			<td>plate used for growing monoclonal cells</td>
		</tr>
		<tr>
			<td>CytoOne T25 filter cap TC flask</td>
			<td>USA Scientific</td>
			<td>CC7682-4825</td>
			<td>container for growing mammalian cells</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fischer Scientific</td>
			<td>BP231-100</td>
			<td>reagent used for freezing down engineered mammalian cells</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Thermo Fisher Scientific</td>
			<td>15-585-011</td>
			<td>reagent for gel electrophoresis</td>
		</tr>
		<tr>
			<td>Falcon 96 Well Clear Flat Bottom TC-Treated Culture Microplate, with Lid</td>
			<td>Corning</td>
			<td>353072</td>
			<td>container for growing sorted monoclonal cells</td>
		</tr>
		<tr>
			<td>FCS Express</td>
			<td>De Novo Software:</td>
			<td>N/A</td>
			<td>software for characterizing flow cytometry data</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Regular, USDA 500 mL</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td>reagent for growing mammalian cells</td>
		</tr>
		<tr>
			<td>Fisherbrand Petri Dishes with Clear Lid - Raised ridge; 100 x 15 mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td>equipment for growing bacteria</td>
		</tr>
		<tr>
			<td>Gibco&#160;DMEM, High Glucose</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-965-092</td>
			<td>reagent for growing mammalian cells</td>
		</tr>
		<tr>
			<td>Hs00932330_m1 KRAS isoform a Taqman Gene Expression Assay</td>
			<td>Life Technologies</td>
			<td>4331182</td>
			<td>qPCR Probe</td>
		</tr>
		<tr>
			<td>Hygromycin B (50 mg/mL), 20 mL</td>
			<td>Life Technologies</td>
			<td>10687-010</td>
			<td>reagent for selecting cells with proper gene circuit integration</td>
		</tr>
		<tr>
			<td>iScript Reverse Transcription Supermix</td>
			<td>Bio-Rad Laboratories</td>
			<td>1708890</td>
			<td>reagent for converting RNA to cDNA</td>
		</tr>
		<tr>
			<td>Laboratory Freezer -20 &#176;C</td>
			<td>VWR</td>
			<td>76210-392</td>
			<td>equipment for storing experimental reagents</td>
		</tr>
		<tr>
			<td>Laboratory Freezer -80 &#176;C</td>
			<td>Panasonic</td>
			<td>MDF-U74VC</td>
			<td>equipment for storing experimental reagents</td>
		</tr>
		<tr>
			<td>Laboratory Refrigerator +4 &#176;C</td>
			<td>VWR</td>
			<td>76359-220</td>
			<td>equipment for storing experimental reagents</td>
		</tr>
		<tr>
			<td>LB Broth (Lennox) , 1 kg</td>
			<td>Sigma-Aldrich</td>
			<td>L3022-250G</td>
			<td>reagent for growing bacteria</td>
		</tr>
		<tr>
			<td>LIPOFECTAMINE 3000</td>
			<td>Life Technologies</td>
			<td>L3000008</td>
			<td>reagemt for transfecting gene circuits into mammalian cells</td>
		</tr>
		<tr>
			<td>MATLAB 2019</td>
			<td>MathWorks</td>
			<td>N/A</td>
			<td>software for analyzing experimental data</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Acros Organics</td>
			<td>413775000</td>
			<td>reagent for immunofluorescence reaction</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes, Polypropylene 1.7 mL</td>
			<td>VWR</td>
			<td>20170-333</td>
			<td>plasticware container</td>
		</tr>
		<tr>
			<td>Mr04097229_mr EGFP/YFP Taqman Gene Expression Assay</td>
			<td>Life Technologies</td>
			<td>4331182</td>
			<td>qPCR Probe</td>
		</tr>
		<tr>
			<td>MultiTherm Shaker</td>
			<td>Benchmark Scientific</td>
			<td>H5000-HC</td>
			<td>equipment for bacterial transformation</td>
		</tr>
		<tr>
			<td>NanoDrop Lite Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-NDL-US-CAN</td>
			<td>equipment for DNA/RNA concentration measurement</td>
		</tr>
		<tr>
			<td>NEB Q5 High-Fidelity DNA polymerase 2x Master Mix</td>
			<td>NEB</td>
			<td>M0492S</td>
			<td>reagent for PCR of gene circuit fragments</td>
		</tr>
		<tr>
			<td>NEB10-beta Competent E. coli (High Efficiency)</td>
			<td>New England Biolabs (NEB)</td>
			<td>C3019H</td>
			<td>bacterial cells for amplifying gene circuit of interest</td>
		</tr>
		<tr>
			<td>NEBuilder HiFi DNA Assembly Master Mix</td>
			<td>New England Biolabs (NEB)</td>
			<td>E2621L</td>
			<td>reagent for combining gene circuit fragements</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti-E inverted microscope with a DS-Qi2 camera</td>
			<td>Nikon Instruments Inc.</td>
			<td>N/A</td>
			<td>instrument for quantifying gene expression</td>
		</tr>
		<tr>
			<td>NIS-Elements</td>
			<td>Nikon Instruments Inc.</td>
			<td>N/A</td>
			<td>software for characterizing fluorescence microscopy data</td>
		</tr>
		<tr>
			<td>oligonucleotides</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>reagent used for PCR of gene circuit components</td>
		</tr>
		<tr>
			<td>Panasonic MCO-170 AICUVHL-PA cellIQ Series CO2 Incubator with UV and H2O2 Control</td>
			<td>Panasonic</td>
			<td>MCO-170AICUVHL-PA</td>
			<td>instrument for growing mammalian cells</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16% Electron Microscopy Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710-S</td>
			<td>reagent</td>
		</tr>
		<tr>
			<td>PBS, Dulbecco&#39;s Phosphate-Buffered Saline (D-PBS) (1x)</td>
			<td>Invitrogen</td>
			<td>14190144</td>
			<td>reagent for mammalian cell culture,reagent for IF incubation buffer</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL), 100x</td>
			<td>Fisher Scientific</td>
			<td>15140-122</td>
			<td>reagent for growing mammalian cells</td>
		</tr>
		<tr>
			<td>primary ERK antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4370S</td>
			<td>primary ERK antibody for immunifluorescence</td>
		</tr>
		<tr>
			<td>primary KRAS antibody</td>
			<td>Sigma-Aldrich</td>
			<td>WH0003845M1</td>
			<td>primary KRAS antibody for immunifluorescence</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>reagent kit for purifying gene circuit plasmids</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit (50)</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>reagent kit for purifying gene circuit fragments</td>
		</tr>
		<tr>
			<td>QuantStudio 3 Real-Time PCR System</td>
			<td>Eppendorf</td>
			<td>A28137</td>
			<td>equipment for qRT-PCR</td>
		</tr>
		<tr>
			<td>Relative Quantification App</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>software for quantifying RNA/cDNA amplificaiton</td>
		</tr>
		<tr>
			<td>RNeasy Plus Mini Kit</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td>kit for extracting RNA of engineered mammalian cells</td>
		</tr>
		<tr>
			<td>Secondary ERK antibody</td>
			<td>Cell Signaling Technology</td>
			<td>8889S</td>
			<td>secondary ERK antibody for immunifluorescence</td>
		</tr>
		<tr>
			<td>secondary KRAS antibody</td>
			<td>Invitrogen</td>
			<td>A11005</td>
			<td>secondary KRAS antibody for immunifluorescence</td>
		</tr>
		<tr>
			<td>Serological Pipets 5.0 mL</td>
			<td>Olympus Plastics</td>
			<td>12-102</td>
			<td>reagents used for setting up a variety of chemical reactions</td>
		</tr>
		<tr>
			<td>SmartView Pro Imager System</td>
			<td>Major Science</td>
			<td>UVCI-1200</td>
			<td>tool for imaging correct PCR bands</td>
		</tr>
		<tr>
			<td>SnapGene Viewer (free) or SnapGene</td>
			<td>SnapGene</td>
			<td>N/A</td>
			<td>software DNA sequence design and analysis</td>
		</tr>
		<tr>
			<td>Stage top incubator</td>
			<td>Tokai Hit</td>
			<td>INU-TIZ</td>
			<td>tool for carrying PCR, transformation, or gel extraction reactions</td>
		</tr>
		<tr>
			<td>TaqMan Fast Advanced Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444557</td>
			<td>reagent for PCR of gene circuit fragments</td>
		</tr>
		<tr>
			<td>TaqMan Human GAPD (GAPDH) Endogenous Control (VIC/MGB probe), primer limited, 2500 rxn</td>
			<td>Life Technologies</td>
			<td>4326317E</td>
			<td>qPCR Probe</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Bio-Rad</td>
			<td>1851148</td>
			<td>tool for carrying PCR, transformation, or gel extraction reactions</td>
		</tr>
		<tr>
			<td>VisiPlate-24 Black, Black 24-well Microplate with Clear Bottom, Sterile and Tissue Culture Treated</td>
			<td>PerkinElmer</td>
			<td>1450-605</td>
			<td>plate used for light-induction experiments</td>
		</tr>
		<tr>
			<td>VWR Disposable Pasteur Pipets, Glass, Borosilicate Glass Pipet, Short Tip, Capacity=2 mL, Overall Length=14.6 cm</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td>reagent for mammalian cell culture</td>
		</tr>
		<tr>
			<td>VWR Mini Horizontal Electrophoresis Systems, Mini10 Gel System</td>
			<td>VWR</td>
			<td>89032-290</td>
			<td>equipment for DNA gel electrophoresis</td>
		</tr>
		<tr>
			<td>Flp-In 293</td>
			<td>Thermo Fisher Scientific</td>
			<td>R75007</td>
			<td>Engineered cell line with FRT site</td>
		</tr>
	</tbody>
</table>

reliably engineering and controlling stable optogenetic gene circuits in mammalian cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Gene circuit assembly and stable cell line generation within this article were based on commercial, modified HEK-293 cells containing a transcriptionally active, single stable FRT site (Figure 1). The gene circuits were constructed into vectors that had FRT sites within the plasmid, allowing for the Flp-FRT integration into the HEK-293 cell genome. This approach is not limited to Flp-In cells, as FRT sites can be added to any cell line of interest anywhere in the genome using DNA editing tec...

Watch this Scientific Journal Video about Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells at JoVE.com

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

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.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

The standardized optogenetic protocol is designed for labs interested in using light as the stimulus to control cellular properties in engineered mammalian cell systems. This protocol is easy to implement in labs otherwise unfamiliar with optogenetics and provides methods for precise spatial temporal control of engineered biological systems. This method has a broad range of application in specific research areas where spatiotemporal information may be important including cancer biology, systems biology, and tissue differentiation.To begin with, select the genetic components to combine bind into a single gene circuit or plasmid. For example, mammalian DNA integration sites, light responsive elements, or functional genes. After assembling and examining all the necessary features such as start codons, regulatory or translated sequences, use any genetic engineering and molecular cloning software, annotate and store the DNA sequences for later use and as a reference.Validate the primers computationally for plasmid construction through the built-in molecular cloning software features, for example, sequence alignment. To generate a stable cell line, transfect the design gene circuits and the desired cells using a liposome mixture of the gene circuit DNA with appropriate recombinase. After cells are 80 to 100%confluent, freeze the cells in a mix of 45%old media, 45%fresh media, and 10%DMSO.Transfer the remaining cells to a sterile tube and perform single-cell sorting to isolate monoclonal cells into a 96-well plate. About two to three weeks after monoclonal sorting, the wells within the 96-well plate should have foci. When 50 to 60%confluency is achieved, split the cells into a 12-well plate.Add the firmware to the assembled LPA circuit using a microcontroller programmer. Next, combine 3D printed parts in the assembled circuit board using the mounting plate, the circuit board, the LED spacer, the plate adapter, a 24-well plate, a plate lid, mounting bolts, wing nuts and stacking components. To begin an optogenetic experiment, use the Iris software available on the Tabor Lab website to program an SD card for the LPA and explore appropriate light conditions.Next, enter intensity values with the technical replicates for the desired experimental outline. Add about 75, 000 cells per well in a 24-well black plate in a total volume of 500 microliters, then place the cells in a humidified incubator with 5%carbon dioxide to allow them to settle for two to six hours. After transferring the cells in the tissue culture hood at the end of the incubation, remove the plastic lid and add an adhesive foil strip to the top of the plate for minimizing the light transfer between the wells so that the light can only enter through the LED located at the bottom of the well.Place the plate in the fitted 3D parts of the LPA, then cover the plate with the 3D printed LPA lid which fits over the device screws on each corner. Plug the device into the power source and push the reset button on the LPA device to ensure that the updated LPA experiment settings are applied and incubate the cells. After incubation, inside the tissue culture hood, remove the foil strip from the plate and let the plate sit for one to two minutes to prevent condensation from forming on the plastic lid.Next, put the original plastic lid back on the plate and image the cells with the appropriate phase contrast or fluorescence microscope. After 24 to 72 hours post-induction, followed by trypsinization, transfer the entire contents of each well of about 500 microliters to the labeled tubes equipped with the cell strainer. Then using appropriate flow cytometry instrument software, create a forward and side scatter gate to capture the single cells of the appropriate size and granularity to exclude debris and cellular clumps.Once the gate is set, capture approximately 10, 000 cells with the appropriate gate and adjust cell number depending on the amount of cellular data required and repeat capturing experimental cells in each tube. After induction and trypsinization, transfer the entire contents of each well of about 500 microliters to the labeled tubes. Centrifuge the cells for five minutes at 400 times G.After discarding the supernatant, move the samples to a chemical fume hood.Resuspend the cells in 750 to 1, 000 microliters of 4%PFA diluted in PBS and pipette up and down several times to break the cell clumps. After 15 minutes of incubation, add 750 to 1, 000 microliters of PBS and again pipette up and down several times, then pellet down the cells for five minutes at 400 times G at room temperature. After discarding the supernatant, move the samples to a chemical fume hood and resuspend the cells in 750 to 1, 000 microliters of ice cold methanol as demonstrated and allow the cells to sit for 30 minutes in a minus 20 degree Celsius freezer.After the incubation, add 750 to 1, 000 microliters of PBS while by pipetting up and down several times. Then after centrifuging the cells for five minutes at 400 times G, discard the supernatant and move the samples to a chemical fume hood. Resuspend the cells in 100 microliters or other suitable dilution of labeled primary antibody and pipette up and down six to eight times, then incubate for one hour at room temperature.Next, add 750 to 1, 000 microliters of the incubation buffer and pipette solution up and down several times, then centrifuge the cells for five minutes at 400 times G.Next, resuspend the cells in 500 microliters of PBS and break the clumps by pipetting up and down several times. Transfer the entire contents of each tube to labeled tubes with cells strainers. Bring the cells to appropriate flow cytometry instruments with lasers of the correct wavelengths.The optical calibration was performed for the devices. Using the acquired image, compensation values for LED were calculated, background signal was subtracted, and pixel intensity was deduced. The coefficient of variation was minimal between 96 wells.The calibration software demonstrated the LED variation by location and created a gray scale adjustment so that each LED is normalized to the same intensity. Gene expression was induced in engineered lighter cells via fluorescence microscopy at different light pulse durations on the LPA and the cells were imaged for Brightfield and GFP expression. Using flow cytometry, the cells were induced at different pulse length values and showed a dose response of gene expression quantified at the population level of over fourfold change from uninduced states.The negative feedback achieved over fivefold gene expression noise reduction as shown by comparing various light intensity values for the lighter system versus vivid or light on. The qPCR analysis showed that the engineered lighter cells expressed RNA levels in a dose-responsive manner with over tenfold induction from uninduced states, matching the flow cytometry quantification. The engineered lighter gene circuit showed increasing amounts of blue light that resulted in increased KRAS protein levels and phosphorylated ERK levels.Several functional assays may be performed including growth, cell motility, wound healing, proliferation, or invasion assays. These methods could provide specific insights into the mechanisms of cancer biology, tissue differentiation, or drug resistance.

reliably-engineering-controlling-stable-optogenetic-gene-circuits

Research

JoVE Journal

Bioengineering

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Syringe-injectable Mesh Electronics for Stable Chronic Rodent Electrophysiology

Implantable brain electrophysiology probes are valuable tools in neuroscience due to their ability to record neural activity with high spatiotemporal resolution from shallow and deep brain regions. Their use has been hindered, however, by mechanical and structural mismatches between the probes and brain tissue that commonly lead to micromotion and gliosis with resulting signal instability in chronic recording experiments. In contrast, following the implantation of ultraflexible mesh electronics via syringe injection, the mesh probes form a seamless, gliosis-free interface with the surrounding brain tissue that enables stable tracking of individual neurons on at least a year timescale. This protocol details the key steps in a typical mouse neural recording experiment using syringe-injectable mesh electronics, including the fabrication of mesh electronics in a standard photolithography-based process possible at many universities, loading mesh electronics into standard capillary needles, stereotaxic injection in vivo, connection of the mesh input/output to standard instrumentation interfaces, restrained or freely moving recording sessions, and histological sectioning of brain tissue containing mesh electronics. Representative neural recordings and histology data are presented. Investigators familiar with this protocol will have the knowledge necessary to incorporate mesh electronics into their own experiments and take advantage of the unique opportunities afforded by long-term stable neural interfacing, such as studies of aging processes, brain development, and the pathogenesis of brain disease.

Mesh electronics probes seamlessly integrate and provide stable, long-term, single-neuron level recording within the brain. This protocol uses mesh electronics for in vivo experiments, involving the fabrication of mesh electronics, loading into needles, stereotaxic injection, input/output interfacing, recording experiments, and histology of tissue containing mesh probes.

Implantable brain electrophysiology probes are valuable tools in neuroscience due to their ability to record neural activity with high spatiotemporal ...