We would like to thank former Weiss Lab members that led or contributed to developing the poly-transfection method and its application to cell classifiers: Jeremy Gam, Bre DiAndreth, and Jin Huh; other Weiss lab members who have contributed to further method development/optimization: Wenlong Xu, Lei Wang, and Christian Cuba-Samaniego; Prof. Josh Leonard and group members, including Patrick Donahue and Hailey Edelstein, for testing poly-transfection and providing feedback; and Prof. Nika Shakiba for inviting this manuscript and providing feedback. We would also like to thank the National Institutes of Health [R01CA173712, R01CA207029, P50GM098792]; National Science Foundation [1745645]; Cancer Center Support (core) Grant [P30CCA14051, in part] from the NCI, and National Institutes of Health [P50GM098792] for funding this work.

NOTE: Table 1 and Table 2 serve as significant references for this protocol. Table 1 shows reagent scaling for reactions, and Table 2 shows DNA ratio arithmetic for an example poly-transfection described in the protocol (upper half) and for a possible follow-up experiment (lower half).
1. Preparing cells for transfection
<ol>
	<li>Ensure that the culture of human embryonic kidney (HEK293) cells is 60%-80% confluent before initiating the protocol. To do this, seed 1 x 106 cells in a 100 mm x 15 mm tissue culture Petri dish 2 days prior, and incubate at 37 &#176;C with 5% CO2. 
	NOTE:&#160;Although our protocol focuses on HEK293 cells, other cell types may be substituted.</li>
	<li>Prepare the media and cells for transfections as described below.
	<ol>
		<li>Pre-warm at least 20 mL of a solution of Dulbecco&#39;s modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% non-essential amino acids (NEAA; see Table of Materials) to 37 &#176;C. Pre-warm at least 2.4 mL of Trypsin and 2.4 mL of phosphate-buffered saline (PBS) to 37 &#176;C as well. Pre-warm reduced serum medium to ~16 &#176;C. 
		NOTE: All tissue culture work should be performed with care in a biosafety cabinet.</li>
	</ol>
	</li>
	<li>Resuspend the cells in the DMEM solution as described below.
	<ol>
		<li>Aspirate and dispose of the current media. Dispense 2 mL of PBS onto the HEK293 cell culture to wash the cells. Aspirate and dispose of the PBS.</li>
		<li>Dispense 2 mL of Trypsin onto the HEK293 cell culture. Place the Petri dish in an incubator at 37 &#176;C for 3 min or until the cells no longer adhere to the dish. Return the dish to the biosafety cabinet and dilute the cell solution by dispensing 8 mL of DMEM solution into the plate.</li>
		<li>Mix the solution by gently pipetting up and down several times. Aspirate all the media and place it in a 15 mL conical tube.</li>
	</ol>
	</li>
	<li>Centrifuge the cells at 300 x g for 3 min to pellet them. Aspirate the media (taking care not to aspirate the cells) and discard it. Resuspend the cells in 5 mL of DMEM solution, mixing by gently pipetting up and down.</li>
	<li>Estimate&#160;the current cell concentration using an automated cell counter (listed in the Table of Materials). Seed six wells (in this example) in a 24-well plate with 1 x 105 cells (for a seeding density of 50,000 cells/cm2).</li>
	<li>Add the DMEM solution up to 500 &#181;L (add DMEM solution to the wells first), and then label one well per treatment as the following: no color control, mKO2 control, TagBFP control, NeonGreen control, all color control, and poly-transfection 1.&#160;TagBFP, mKO2, and NeonGreen control wells are single color controls for all fluorescent proteins included in the poly-transfection.</li>
</ol>
2. Performing transfection
<ol>
	<li>Prepare tubes for each DNA aggregate. Set aside 1.5 mL microcentrifuge tubes and label the tubes as: no color control, mKO2 control, TagBFP control, NeonGreen control, all color control, poly-transfection mix 1, and poly-transfection mix 2.
	<ol>
		<li>Add 36 &#181;L of reduced serum medium to the no color control, mKO2 control, TagBFP control, NeonGreen control, and all color control tubes. Add 18 &#181;L of reduced serum medium to each of the poly-transfection mix 1 and poly-transfection mix 2 tubes. 
		NOTE: The plasmid concentrations are assumed to be 150 ng/&#181;L.</li>
		<li>Add 600 ng of filler plasmid to the no color control tube. Add 300 ng of mKO2 and 300 ng of filler plasmid to the mKO2 color control tube. Add 300 ng of TagBFP and 300 ng of filler plasmid to the TagBFP color control tube.</li>
		<li>Add 300 ng of constitutive NeonGreen plasmid and 300 ng of filler plasmid to the NeonGreen color control tube. Add 100 ng each of mKO2, TagBFP, and constitutive NeonGreen, as well as 300 ng of filler plasmid, to the all color control tube.</li>
		<li>Add 150 ng of mKO2 to the poly-transfection mix 1 tube. Add 75 ng of reporter NeonGreen plasmid and 75 ng of filler plasmid to the poly-transfection mix 1 tube.</li>
		<li>Add 150 ng of TagBFP to the poly-transfection mix 2 tube. Add 75 ng of L7ae plasmid and 75 ng of filler plasmid to the poly-transfection mix 2 tube.</li>
	</ol>
	</li>
	<li>Create the transfection master mix in a 1.5 mL microcentrifuge tube by combining 216 &#181;L of reduced serum medium with 9.48 &#181;L of transfection reagent (see Table 1 for reagent ratios and reaction scaling). Mix well by pipetting up and down, and set aside.</li>
	<li>Add 1.58 &#181;L of enhancer reagent to each of the no color control, single color control, and all color control tubes. Add 79 &#181;L of enhancer reagent to each of the poly-transfection mix tubes. Mix each tube individually by pipetting vigorously.</li>
	<li>Add the transfection master mix to each tube containing DNA.
	<ol>
		<li>Add 37.58 &#181;L of transfection master mix to each of the no color control, single color control, and all color control tubes. Mix each tube individually by pipetting vigorously.</li>
		<li>Add 18.79 &#181;L of transfection master mix to each of the poly-transfection mix tubes. Mix each tube individually by pipetting vigorously.</li>
	</ol>
	</li>
	<li>Dispense the transfection mixes into the wells.
	<ol>
		<li>Pipette 65.97 &#181;L of each transfection mix for the no color, single color, and all color controls into the corresponding wells.</li>
		<li>Pipette 32.98 &#181;L of the poly-transfection mix 1 into the poly-transfection well and swirl the plate quickly but gently in a tight figure-eight pattern along a flat surface to distribute the complexes effectively. Then, pipette 32.98 &#181;L of the poly-transfection mix 2 into the same poly-transfection well and swirl the plate in the same fashion.</li>
	</ol>
	</li>
	<li>Place the plate in an incubator at 37 &#176;C, with 5% CO2 and without shaking, for a period of 48 h. 
	NOTE: To increase cell viability, the cell media can be replaced every 6 h following transfection (though this is not always necessary, and with HEK293 cells and its derivatives, one must be careful not to detach the cells from the plate when changing the media).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td colspan="3">Amount</td>
			<td colspan="3">Scaling</td>
		</tr>
		<tr>
			<td>Reduced serum medium for DNA mixture</td>
			<td colspan="3">36 &#956;L per control tube, 18 &#956;L per poly-transfection tube&#160;</td>
			<td colspan="3">0.05 &#956;L Reduced serum medium/ng DNA per tube, with 10-20% extra volume to account for pipetting</td>
		</tr>
		<tr>
			<td>DNA</td>
			<td colspan="3">300-600 ng per tube</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>P3000</td>
			<td colspan="3">1.58 &#956;L per control tube, 0.79 &#956;L per poly-transfection tube</td>
			<td colspan="3">0.0022 &#956;L P3000/ng DNA per tube, with 10-20% extra</td>
		</tr>
		<tr>
			<td>Reduced serum medium for Lipo master mix</td>
			<td colspan="3">36 &#956;L per control tube, 18 &#956;L per poly-transfection tube</td>
			<td colspan="3">0.05 &#956;L reduced serum medium/ng total DNA, with 10-20% extra volume to account for pipetting</td>
		</tr>
		<tr>
			<td>Transfection and enhancer reagent</td>
			<td colspan="3">1.58 &#956;L per control tube, 0.79 &#956;L per poly-transfection tube</td>
			<td colspan="3">0.0022 &#956;L Lipofectamine 3000/ng DNA, with 10-20% extra</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Reagent scaling for transfections. The table indicates the correct ratio of reagent to include for the DNA quantity included in a single well. This can be used to scale reactions effectively and form master mixes. Quantities of reagent have been scaled to include a 20% excess.
3. Preparing cells for flow cytometry
<ol>
	<li>Pre-warm at least 4.2 mL of the DMEM solution to 37 &#176;C. Pre-warm at least 4.2 mL of Trypsin and 4.2 mL of PBS to 37 &#176;C. Keep fluorescence-activated cell sorting (FACS) buffer solution at 4 &#176;C until ready to use.</li>
	<li>Resuspend the cells in the FACS buffer solution (PBS supplemented with 1% BSA, 5 mM ethylenediaminetetraacetic acid [EDTA], and 0.1% sodium azide [NaN3], to reduce clumping; see Table of Materials) as described below.
	<ol>
		<li>Aspirate and dispose of the current media in each well. Dispense 5 mL of PBS into each well to wash the cells. Aspirate and dispose of the PBS.</li>
		<li>Dispense 5 mL of Trypsin into each well. Place the plate in an incubator at 37 &#176;C for 3 min, or until the cells no longer adhere to the dish. Return the plate to the biosafety cabinet and dilute the cell solutions by dispensing 5 mL of DMEM solution into each well.</li>
		<li>For each well, mix the solution by gently pipetting up and down several times. For each well, aspirate all the media and place it in a 15 mL conical tube.</li>
	</ol>
	</li>
	<li>Centrifuge the cells at 300 x g for 3 min to pellet them. Aspirate the media, taking care not to aspirate the cells and discard it. In each tube, resuspend the cells in 5 mL of FACS buffer solution, mixing by gently pipetting up and down.</li>
	<li>Pass each cell suspension solution through a strainer (to remove clumps) into separate flow cytometry conical tubes. Keep these tubes on ice for no more than 1 h, and perform flow cytometry as soon as possible.</li>
</ol>
4. Performing flow cytometry
NOTE: Operating a flow cytometer requires proper training and knowledge of the necessary tasks. As software and equipment may vary and users should be trained generally, this section refers to specific operations that are useful to perform.
<ol>
	<li>First, examine the cells transfected with the filler plasmid control (no color control) to select for cell characteristics and avoid anomalies (including aggregates, debris, etc.). While there are many combinations of parameters to distinguish cells, use the following three general options as a good way to visualize distinguishing features.
	<ol>
		<li>Look at the side scatter area (log or linear scale per preference/cell type) versus the forward scatter area (linear scale).</li>
		<li>Look at the side scatter height (log scale) versus the side scatter width (linear scale).</li>
		<li>Look at the forward scatter width (linear scale) versus the forward scatter height (linear scale).</li>
	</ol>
	</li>
	<li>Next, look at the single color controls. Use the all color control to tune the instrument voltages, such that the signals from each fluorescent protein are normalized to equivalent arbitrary units (a.u.) of fluorescence. Next, run the single color controls for each fluorescent protein, which are used to set the compensation matrix, allowing for bleed-through correction. 
	NOTE: Ideally, the full dynamic range of the fluorescence values should be visible. Further normalization of fluorescent protein signals can be done via conversion to standardized units (e.g., molecules of equivalent fluorescein [MEFLs; see Beal et al.15]). To enable MEFL conversion during analysis, run rainbow calibration beads. Such calibration is also useful for reducing instrument-to-instrument and day-to-day signal variation16.</li>
	<li>Run the poly-transfection sample tube. 
	NOTE: Where possible, it is recommended to run 1,000 x 10^ (^ = mixes) cells, as higher-dimensional poly-transfections need to be subdivided into more bins during analysis, and each bin needs enough cells (ideally &#62;10) to make statistically significant comparisons.</li>
</ol>
5. Performing post-experiment analysis
<ol>
	<li>Initially, use data from the controls (and, if applicable, the beads) to ensure accurate results. Use one of the available software tools to perform live cell gating (using gates outlined above), compensation, and autofluorescence correction. 
	NOTE: We typically use either custom MATLAB code (e.g., https://github.com/jonesr18/MATLAB_Flow_Analysis or Cytoflow17), which has both a graphical user interface and a python library suitable for the pre-processing phase and for poly-transfection analysis.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Method</td>
			<td>Complex</td>
			<td>ng Fluorescent Marker</td>
			<td>ng L7ae (fraction)</td>
			<td>ng Reporter (fraction)</td>
			<td>ng Filler DNA (fraction)</td>
			<td>Total (ng)</td>
		</tr>
		<tr>
			<td>Poly-transfection 1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>600</td>
		</tr>
		<tr>
			<td>&#8594;</td>
			<td>1</td>
			<td>150</td>
			<td>75 (&#189;)</td>
			<td></td>
			<td>75 (&#189;)</td>
			<td>300</td>
		</tr>
		<tr>
			<td>&#8594;</td>
			<td>2</td>
			<td>150</td>
			<td></td>
			<td>75 (&#189;)</td>
			<td>75 (&#189;)</td>
			<td>300</td>
		</tr>
		<tr>
			<td>Poly-transfection 2</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>600</td>
		</tr>
		<tr>
			<td>&#8594;</td>
			<td>1</td>
			<td>150</td>
			<td>25 (1/6)</td>
			<td></td>
			<td>125 (5/6)</td>
			<td>300</td>
		</tr>
		<tr>
			<td>&#8594;</td>
			<td>2</td>
			<td>150</td>
			<td></td>
			<td>125 (5/6)</td>
			<td>25(1/6)</td>
			<td>300</td>
		</tr>
	</tbody>
</table>
Table 2:&#160;DNA amounts for poly-transfection demonstrated in the protocol, and an example follow-up experiment with tuned plasmid ratios. The upper half of the table shows the composition of plasmids used in a simple poly-transfection experiment. The lower half shows the composition of an updated experiment that adjusts the plasmid ratios to better subsample a hypothetical concentration space, where the gene expression modulator is at a more optimal 1:5 ratio relative to its reporter, yielding more transfected cells to sample around this ratio.

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R.W. is a co-founder of Strand Therapeutics and Replay Bio; R.W. and R.J. filed a provisional patent related to a cell type classifier.

Rapid prototyping methods such as computer-aided design (CAD), breadboarding, and 3D printing have revolutionized mechanical, electrical, and civil engineering disciplines. The ability to quickly search through many possible solutions to a given challenge greatly accelerates progress in a field. We believe that poly-transfection is an analogous technology for biological engineering, enabling rapid prototyping of genetic circuits. Additionally, other rapid prototyping technologies require hands-on sequential iteration of ...

The field of mammalian synthetic biology has rapidly progressed, from developing simple sense-and-respond parts in cultured cell lines to the optimization of complex networks of genes to address real-world challenges in diagnostics and therapeutics1. These sophisticated circuits are capable of sensing biological inputs from microRNA profiles to cytokines to small molecule drugs, and implementing logic processing circuits including transistors, band-pass filters, toggle switches, and oscillators. They have also shown promising results in animal models of diseases like cancer, arthritis, diabetes, and many more1,2,3,4,5. However, as the complexity of a circuit grows, optimizing the levels of each of its components becomes increasingly challenging.
One particularly useful type of genetic circuit is a cell classifier, which can be programmed to sense and respond to cellular states. Selective production of protein or RNA outputs in specific cellular states is a powerful tool to guide and program differentiation of cells and organoids, identify and destroy diseased cells and/or undesirable cell types, and regulate the function of therapeutic cells1,2,3,4,5. However, creating circuits in mammalian cells that can accurately classify cell states from multiple cellular RNA and/or protein species has been highly challenging.
One of the most time-consuming steps of developing a cell classification circuit is to optimize the relative expression levels of individual component genes, such as sensors and processing factors, within the circuit. To speed up circuit optimization and allow for the construction of more sophisticated circuits, recent work has used mathematical modeling of cell classifier circuits and their components to predict optimal compositions and topologies6,7. While this has shown powerful results so far, mathematical analysis is limited by the need to systematically characterize the input-output behavior of component genes in the circuit, which is time-consuming. Further, a myriad of context-dependent problems can emerge in complex genetic circuits, causing the behavior of a full circuit to defy predictions based on individual part characterizations8,9.
To more rapidly develop and test complex mammalian circuits such as cell state classifiers, our lab developed a technique called poly-transfection10, an evolution of plasmid co-transfection protocols. In co-transfection, multiple plasmid DNA species are complexed together with a positively charged lipid or polymer reagent, then delivered to cells in a correlated manner (Figure 1A). In poly-transfection, plasmids are separately complexed with the reagent, such that the DNA from each transfection complex is delivered to cells in a de-correlated manner (Figure 1B). Using this method, cells within the transfected population are exposed to numerous combinations of ratios of two or more DNA payloads carrying different circuit components.
To measure the ratios of circuit components delivered to each cell, each transfection complex within a poly-transfection contains a constitutively expressed fluorescent reporter that serves as a proxy for cellular uptake of the complex. Filler DNA that does not contain any elements active within a mammalian cell is used to tune the relative amount of the fluorescent reporter and circuit components delivered to a cell in a single transfection complex and is discussed in more detail in the discussion. An example of filler DNA used in the Weiss lab is a plasmid containing a terminator sequence, but no promotor, coding sequence, etc. Cells with different ratios of circuit components can then be compared to find optimal ratios for gene circuit function. This in turn yields useful predictions for choosing promoters and other circuit elements to achieve optimal gene expression levels when combining circuit components into a single vector for genetic integration (e.g., a lentivirus, transposon, or landing pad). Thus, instead of choosing ratios between circuit components based on intuition or via a time-consuming trial and error process, poly-transfection evaluates a wide range of stoichiometries between genetic parts in a single-pot reaction.
In our lab, poly-transfection has enabled the optimization of many genetic circuits, including cell classifiers, feedback and feedforward controllers, and bistable motifs. This simple but powerful method significantly speeds up design cycles for complex genetic circuits in mammalian cells. Poly-transfection has since been used to characterize several genetic circuits to reveal their multi-dimensional input-output transfer functions at high resolution10, optimize an alternate circuit topology for cell state classification11, and accelerate various published12,13 and ongoing projects.
Here we describe and depict the workflow for using poly-transfection to rapidly optimize a genetic circuit (Figure 2). The protocol shows how to generate high-quality poly-transfection data and avoid several common errors in the poly-transfection protocol and data analysis (Figure 3). It then demonstrates how to use poly-transfection to characterize simple circuit components and, in the process, benchmark poly-transfection results against co-transfection (Figure 4). Finally, the results of poly-transfection show optimization of the cancer classifier circuit (Figure 5).

<table><tbody><tr><td>15mL Corning Falcon conical tubes</td><td>ThermoFisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>24-well petri dish</td><td>Any company of choice</td><td></td><td>(Non-pyrogenic, Sterile, RNase, DNase, DNA and Pyrogen Free)</td></tr><tr><td>Bovine serum albumin</td><td>NEB</td><td>B9000S</td><td></td></tr><tr><td>Centrifuge</td><td>Any company of choice</td><td></td><td>Capable of exposing 15mL Falcon tubes to 300 rcf</td></tr><tr><td>Countess 3 Automated Cell Counter</td><td>ThermoFisher Scientific</td><td>AMQAX2000</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides</td><td>ThermoFisher Scientific</td><td>C10228</td><td></td></tr><tr><td>Cytoflow</td><td>Non-commercial software package</td><td></td><td>https://cytoflow.readthedocs.io/en/stable/#&amp;nbsp;</td></tr><tr><td>DMEM</td><td>VWR</td><td>10-013-CV</td><td>Use the correct media for your cell type</td></tr><tr><td>EDTA&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>03690-100ML</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma Aldrich</td><td>F4135</td><td></td></tr><tr><td>HEK cells</td><td>ATCC</td><td>CRL-1573</td><td>Use the relevant cell type for your experiments. HEK cells tend to transfect very efficiently.</td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>CRL-12401</td><td>Use the relevant cell type for your experiments.</td></tr><tr><td>Lipofectamine 3000 and P3000 enhancer</td><td>ThermoFisher Scientific</td><td>L3000001</td><td>Use the correct reagent for your cell type; transfection and enhancer reagent</td></tr><tr><td>LSRFortessa flow cytometer</td><td>BD Biosciences</td><td>N/A</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Microcentrifuge Tubes, 1.5 mL</td><td>Any company of choice</td><td></td><td></td></tr><tr><td>Opti-MEM</td><td>ThermoFisher Scientific</td><td>31985070</td><td>reduced serum medium</td></tr><tr><td>Phosphate buffered saline</td><td>ThermoFisher Scientific</td><td>70011044</td><td></td></tr><tr><td>Rainbow calibration beads</td><td>Spherotech</td><td>URCP-100-2H</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>S2002</td><td></td></tr><tr><td>Trypsin</td><td>VWR</td><td>25-053-CI</td><td></td></tr></tbody></table>

rapid development of cell state identification circuits with poly-transfection

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit components remains challenging and labor-intensive. To accelerate this process, our lab developed poly-transfection, a high-throughput extension of traditional mammalian transfection. In poly-transfection, each cell in the transfected population essentially performs a different experiment, testing the behavior of the circuit at different DNA copy numbers and allowing users to analyze a large number of stoichiometries in a single-pot reaction. So far, poly-transfections that optimize ratios of three-component circuits in a single well of cells have been demonstrated; in principle, the same method can be used for the development of even larger circuits. Poly-transfection results can be easily applied to find optimal ratios of DNA to co-transfect for transient circuits or to choose expression levels for circuit components for the generation of stable cell lines.
Here, we demonstrate the use of poly-transfection to optimize a three-component circuit. The protocol begins with experimental design principles and explains how poly-transfection builds upon&#160;traditional co-transfection methods. Next, poly-transfection of cells is carried out and followed by flow cytometry a few days later. Finally, the data is analyzed by examining slices of the single-cell flow cytometry data that correspond to subsets of cells with certain component ratios. In the lab, poly-transfection has been used to optimize cell classifiers, feedback and feedforward controllers, bistable motifs, and many more. This simple but powerful method speeds up design cycles for complex genetic circuits in mammalian cells.

In Figure 1, we compare co-transfection to poly-transfection. In a co-transfection, all plasmids are delivered in the same transfection mix, resulting in high correlation between the amount of each plasmid any single cell receives (Figure 1A). While the number of total plasmids delivered to each cell varies significantly, the fluorescence of the two reporter proteins in the individual cells across the population is well-correlated, indicating that the two plasmi...

Watch this Scientific Journal Video about Rapid Development of Cell State Identification Circuits with Poly-Transfection at JoVE.com

Rapid Development of Cell State Identification Circuits with Poly-Transfection

Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis.

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit ...

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Research

JoVE Journal

Bioengineering

1.5K Views.  Massachusetts Institute of Technology. Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis. 

Video: Rapid Development of Cell State Identification Circuits with Poly-Transfection

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Genetics

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

Medicine

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Immunology and Infection

Engineering Intracellular Protein Sensors in Mammalian Cells

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells to sense and respond to these biomarkers may help the understanding of molecular mechanisms underlying pathologies, as well as to develop new cell-based therapies. While several systems that detect extracellular proteins have been developed, a modular framework that can be easily re-engineered to sense different intracellular proteins was missing.
Here, we describe a protocol to implement a modular genetic platform that senses intracellular proteins and activates a specific cellular response. The device operates on intracellular antibodies or small peptides to sense with high specificity the protein of interest, triggering the transcriptional activation of output genes, through a TEV protease (TEVp)-based actuation module. TEVp is a viral protease that selectively cleaves short cognate peptides and is widely used in biotechnology and synthetic biology for its high orthogonality to the cleavage site. Specifically, we engineered devices that recognize and respond to protein-biomarkers of viral infections and genetic diseases, including mutated huntingtin, NS3 serine-protease, Tat and Nef proteins to detect Huntington&#8217;s disease, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections, respectively. Importantly, the system can be hand tailored for the desired input-output functional outcome, such as fluorescent readouts for biosensors, stimulation of antigen presentation for immune response, or initiation of apoptosis to eliminate unhealthy cells.

Here, we present a protocol for engineering genetically-encoded intracellular protein sensor-actuator(s). The device specifically detects target proteins through intracellular antibodies (intrabodies) and responds by switching on gene transcriptional output. A general framework is built to rapidly replace intrabodies, enabling rapid detection of any desired protein, without altering the general architecture.

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Biology

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

Comparative Analysis of Forward and Reverse Transfection Methods on mPSCs

An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.

Author Spotlight: Advancements in Synthetic Genetic Devices for Stem Cell Fate Manipulation and Cellular Therapy Development

The present protocol describes a method for reverse poly-transfection of mouse embryonic stem cells during culture with 2i and LIF media. This method yields higher viability and efficiency than traditional forward transfection protocols, while also enabling one-pot optimization of plasmid ratios.

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical ...

Developmental Biology

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.

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, ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.