This work was supported by the Israel Science Foundation [Grants 1721/12, 1368/12, and 1444/16] (to A.P). A.P. is affiliated with Brettler Center and David R. Bloom Center, School of Pharmacy, The Hebrew University of Jerusalem.

1. Ligating the Gene of Interest into the Repressible Site of Vector
<ol>
	<li>Obtain an inducible vector such as pcDNA5/FRT/TO or pcDNA4/TO.</li>
	<li>Analyze the gene of interest for any potentially susceptible restriction sites in the middle of the gene that are also featured in the multiple cloning site of the chosen vector by in silico DNA analysis software4.</li>
	<li>Using standard overlap PCR techniques4, flank the gene of interest with two selected restr...

<ol>
	<li>Ooi, A., Wong, A., Esau, L., Lemtiri-Chlieh, F., Gehring, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Guide+to+Transient+Expression+of+Membrane+Proteins+in+HEK-293+Cells+for+Functional+Characterization.">A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization.</a> Frontiers in Physiology. 7, 300 (2016).</li><li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+A+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: A vehicle for the expression of recombinant proteins.</a> Journal of Pharmacological and Toxicological Methods. 51 (3), 187-200 (2005).</li><li>Xu, X., Nagarajan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+sequence+of+the+Chinese+hamster+ovary+(CHO)-K1+cell+line.">The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line.</a> Nature Biotechnology. 29 (8), 735-741 (2011).</li><li>Hazan, A., Kumar, R., Matzner, H., Priel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pain+receptor+TRPV1+displays+agonist-dependent+activation+stoichiometry.">The pain receptor TRPV1 displays agonist-dependent activation stoichiometry.</a> Scientific reports. 5, 12278 (2015).</li><li>Kumar, R., Hazan, A., Basu, A., Zalcman, N., Matzner, H., Priel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+Residue+in+the+TRPV1+Vanilloid+Binding+Pocket+Regulates+Deactivation+Kinetics.">Tyrosine Residue in the TRPV1 Vanilloid Binding Pocket Regulates Deactivation Kinetics.</a> Journal of Biological Chemistry. 291 (26), 13855-13863 (2016).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and bioanalytical chemistry. 397 (8), 3173-3178 (2010).</li><li>Preuss, A. K., Connor, J. A., Vogel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transfection+induces+different+intracellular+calcium+signaling+in+CHO+K1+versus+HEK+293+cells.">Transient transfection induces different intracellular calcium signaling in CHO K1 versus HEK 293 cells.</a> Cytotechnology. 33 (1-3), 139-145 (2000).</li><li>Dalton, A. C., Barton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over-expression+of+secreted+proteins+from+mammalian+cell+lines.">Over-expression of secreted proteins from mammalian cell lines.</a> Protein Science. 23 (5), 517-525 (2014).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. -. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Bohlen, C. J., Priel, A., Zhou, S., King, D., Siemens, J., Julius, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bivalent+tarantula+toxin+activates+the+capsaicin+receptor,+TRPV1,+by+targeting+the+outer+pore+domain.">A bivalent tarantula toxin activates the capsaicin receptor, TRPV1, by targeting the outer pore domain.</a> Cell. 141 (5), 834-845 (2010).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pflu&#776;gers Archiv European journal of physiology. 391 (2), 85-100 (1981).</li><li>Jones, J., Nivitchanyong, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+tetracycline-responsive+recombinant+protein+production+and+effect+on+cell+growth+and+ER+stress+in+mammalian+cells.">Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells.</a> Biotechnology and Bioengineering. 91 (6), 722-732 (2005).</li><li>Raphemot, R., Weaver, C. D., Denton, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+for+small-molecule+modulators+of+inward+rectifier+potassium+channels.">High-throughput screening for small-molecule modulators of inward rectifier potassium channels.</a> J Vis Exp. (71), e4209 (2013).</li></ol>

Transfection is a widely used protocol for protein expression and research, with many different variations to improve expression consistency and stability. Transient transfection reagents offer a simple, easy to use protocol where the cell and protein of interest can be analyzed within hours to overnight from the time of transfection. Unfortunately this approach can be unpredictable when the mode of analysis requires a consistent level of protein expression, such as single channel recordings in electrophysiology<sup clas...

Heterologously expressing systems is one of the most widely used techniques to study a multitude of cellular functions1. Their low endogenous protein profile, minimal maintenance requirements, reliable growth, and ability to take up and express foreign DNA have made cell lines such as Human Embryonic Kidney (HEK293) and Chinese Hamster Ovary (CHO) almost essential to biological research2,3. Areas of research using heterologous systems include membrane proteins, intracellular signaling, and enzymatic activity. Following transfection of foreign DNA into the cell, many different forms of analysis can be performed, including electrophysiology, ratiometric calcium imaging, western blot, etc.4,5.
Due to the wide array of potential applications for heterologous expression systems, many different reagents and products have been developed to utilize these cells and their qualities6. DNA delivery systems that transiently or permanently integrate foreign DNA into cells to study exogenous protein has become one of the most popular and useful tools for biological research. More specifically, transiently transfecting DNA into a cell is widely used as a simple, straight forward process that requires relatively little time and materials. Furthermore, the success rate of cells that undergo transfection is high7. This technique is very reliable when combined with a marker gene such as green fluorescent protein (GFP), and can be used for many different techniques such as calcium imaging and electrophysiology5. Unfortunately, though, transiently expressing DNA into host cells comes with some major pitfalls, not in the least that the expression level per cell is unreliable. The number of copies of plasmid DNA taken up per cell is uncontrollable, thus the expression between individual experiments can vary greatly2. This issue becomes significant when either trying to replicate physiological conditions, or performing precise data collection techniques.
As a solution to the complications mentioned above, stable transfection protocols have been designed in which a gene of interest can be inserted into the genome of a cell under the tight control of an inducible promoter, such as a tetracycline repressor expression system, ensuring a single copy of the plasmid integrates into the genome of each cell and is only expressed after induction of the transcription mechanism, for example, in the presence of doxycycline. While this solves the obstacles of inconsistent protein expression levels, this method loses the convenience of quick and relatively simple protocol of transient transfections. Establishing a stable cell line takes at least a few weeks in which one must calibrate a killing curve set by specific antibiotics to maintain the protein expression and ensure integration of the vector and skillfully select and grow cell colonies. Overall this takes significantly more time and effort with a lower success rate&#8203;8.
Here, we introduce an intermediate protocol that draws on the strengths of both of the popular transfection options to provide a simple and effective way to control expression levels in any inducible cell line. While maintaining cells with an inducible tet system, we transiently transfect our gene of interest,&#160;Transient Receptor Potential cation channel subfamily V member 1 (TRPV1), ligated into a vector that can homologously combine with the repressor system. In this way, the gene can be introduced into the cells without beginning to express. Only with the addition of doxycycline does the gene begin to express, allowing us to calibrate the levels of protein expression according to the technique or levels observed in physiological conditions. Our protocol also avoids lengthy complications associated with generating a stably expressing cell line. We begin by showing the changing levels of TRPV1 activation in calcium imaging from un-induced through four hours of induction and how the rise in intracellular calcium levels correlates. We then duplicate the protocol in the whole cell configuration of the patch clamp technique, showing the increasing current with increasing time of induction. Finally, we present examples of single channel electrophysiology recordings, and show that this technique is especially useful for controlled expression when looking for precise data collection based on individual units of the protein. Through our protocol, we offer a convenient way to control protein expression in heterologous systems while avoiding lengthy cell culture complications, thus providing a way to control conditions between experiments and provide more replicable results.

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			<td height="21" style="height:21px;width:216px;">P1000 micropipette puller</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">P-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MF-900 Microforge</td>
			<td style="width:153px;">NARISHIGE</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ValveBank perfusion sysytem</td>
			<td style="width:153px;">AutoMate Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Digidata&#174; 1440A Low-noise Data Acquisition System</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axopatch 200B Amplifier</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pCLAMP 10.6 Software</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">micromanipulator</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">MP-225</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To quickly create an inducible expression model, we made use of HEK293 cells that express Tetracycline Repressor protein (TR) (e.g., T-REx-293) and vectors that contain tetracycline operator sequences (TetO) between the CMV promoter and the multiple cloning site (e.g., pcDNA4/TO). When transfected into TREx-293 cells, the expression of the gene of interest in pcDNA4/TO is repressed, as TR binds to TetO.&#160;Adding doxycycline to the medium prevents TR-TetO interaction, ...

Watch this Scientific Journal Video about Controllable Ion Channel Expression through Inducible Transient Transfection at JoVE.com

Controllable Ion Channel Expression through Inducible Transient Transfection

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 ...

The overall goal of this procedure is to provide a convenient way to control ion channels expression in heterologous systems while avoiding lengthy cell culture complications. Thus providing a way to control conditions between experiments and generate more replicable results. This method can help facilitate research in the ion channel field.Such as characterizing channel properties in the pharmacology. Since the current methods are either complex or show inconsistent protein expression. The main advantage of this technique is that we can achieve controlled expression levels of a wide variety of proteins tailored for individual experiments within a relatively short time.To begin this procedure, transfer TREX 293 cells to a 12 well plate prepared with 0.9 milliliters of full DMEM per well for generating 50 percent confluency. And incubate the cells overnight at 37 degrees Celsius in a CO2 incubator. Next, prepare a transfection mixture composed of DMEM, the gene of interest, shown here, TRPV1, an inert plasmid, and the lipid tranfection reagent.For electrophysiological experiments, include EGFP in mammalian expression plasmid in the transfection cocktail in order to visualize the cells that successfully undergo transfection. Incubate the transfection mixture at room temperature for 30 minutes. Then, transfect the cells by pipetting the transfection mixture dropwise onto the plated cells in the 12 well plate.Rock the plate vigorously to ensure homogenous dispersion of DNA and transfection reagent. Afterward, incubate the transfected cells overnight at 27 degrees Celsius. The next morning, if performing electrophysiology, confirm that the cells have been successfully transfected by observing the fluorescent signal of EGFP under a UV lamp.If performing electrophysiology, transfer cells from the 12 well plate to PDL coated cover slips in 500 microliters of full DMEM. If performing calcium imaging, spot approximately 20, 000 cells to PDL coated calcium imaging chambers. In this procedure, prepare a doxycycline stock solution at one milligram per milliliter in double distilled water.Protect the stock solution from light at four degrees Celsius for up to three weeks. For long term storage, keep the doxycycline stock solution at negative 20 degrees Celsius. Next, prepare fresh doxycycline solution in full DMEM.And warm it to 37 degrees Celsius. For electrophysiological recordings, pipette 500 microliters of doxycycline solution at two micrograms per milliliter to each well to make a final concentration of doxycycline at one microgram per milliliter. For calcium imaging, add 100 microliters of doxycycline solution at three micrograms per milliliter to each well to make a final concentration of doxycycline at one microgram per milliliter.Incubate the sample for the desired amount of time for induction at 37 degrees Celsius, and five percent CO2. To calibrate the protein expression through calcium imaging, prepare Ringer's solution and warm it to 37 degrees Celsius. To help disperse AM esters, a fluroescent ion indicators, such as Fura-2 in aquious soluions, prepare a solution of non-ionic F-127 in DMSO.Heat the solution to 27 degrees Celsius until the non-ionic F-127 is dissolved. Then store the non-ionic F-127 solution at room temperature and warm it to 37 degrees Celsius again before use. After that, prepare a Fura-2 AM loading solution from the Ringer's solution, supplemented with two to three micromolar Fura-2 AM, 02 milligrams per milliliter non-ionic F-127, and 10 millimolar D-glucose.Then aspirate the medium from the cells in the chambers and replace it with Fura-2 AM loading solution. Incubate the cells for 60 minutes at room temperature in the dark. Afterward, aspirate the Fura-2 AM solution.And wash the cells with Ringer's and 10 millimolar D-glucose solution to remove extra cellular dye. Leave 200 microliters of Ringer's and 10 millimolar D-glucose solution in each. And incubate it for 30 minutes at room temperature in the dark.After 30 minutes, place the chamber on the stage above the microscope nose piece, and secure it with holder. Then calculate and prepare a working concentration of the TRPV1 specific agonist capsaisin according to the final desired concentration. Next, turn on the lamp, camera and microscope, and select Fura-2 filter to detect emission at 510 nanometer wavelength.Adjust for the desired magnification and focus the cells in the selected field by manipulating these parameters using the microscope knobs. In the dark, subtract background light and set the exposure time. Set picture sampling at the desire rate.Then, begin by recording the fluorescence response of Fura-2 loaded cells by exciting them at 340 nanometer and 380 nanometer wavelengths. The ratio of 340 to 380 signals is an indicator for intracellular calcium concentration and thus also for TRPV1 activity. Subsequently, add two micromolar capsaicin in 200 microliters of Ringer's solution to create a final saturating concentration of one micromolar capsaicin in order to evaluate the expression level of TRPV1 and analyze the TRPV1 response.To determine the ideal induction time for single channel recording, first prepare the bath and the pipette solutions. Then prepare some glass pipettes, fire-polished to a resistance of 10 to 12 megaohms. Perform whole cell patch clamp by creating a seal on the membrane of the targeted cell.Subsequently, set the holding potential to negative 40 millivolts. And rupture the membrane by applying negative pressure. Afterward, using the micromanipulator, lift the pipette with the mebrane patch away from the cell.Then position the profusion system directly next to the pipette containing the membrane patch. Lower the Bessel filter and output gain to two to five killahurtz and 10 amps respectively. Following that, profuse the saturating concentrations of agonist onto the membrane patch.Then analyze the number of single channel patches successfully recorded against the induction time applied to the cells and adjust the induction time according to the desired results. Shown here are the pseudo colored images of TREX 293 cells transiently expressing RTRPV1, before and after capsaicin application. And here are the corresponding changes of the intracellular calcium levels with time in the treated, transfected TREX 293 cells.Each graph represents an average of 50 cells. Success rate of single channel patch in TRPV1 recordings, is dependent on the induction time. Here are the current traces of patches from TREX 293 cells transiently expressing the RTRPV1 after two hours and four hours of induction.And this plot describes the number of channels in a patch after the indicated time of induction. This technique, can give us the analysis of a protein with controlled expression within 12 hours of transfection if it is performed properly. When attempting this procedure it's important to remember to standardize the transfection concentrations and induction time for different proteins.After watching this video, you should have a good understanding of how to produce a controllable expression system in a time efficient manner for the ion of your choice.

controllable-ion-channel-expression-through-inducible-transient

Research

JoVE Journal

Genetics

9.2K Views.  The Hebrew University of Jerusalem (HUJI). The overall goal of this procedure is to provide a convenient way to control ion channels expression in heterologous systems while avoiding lengthy cell culture complications. Thus providing a way to control conditions between experiments and generate more replicable results. This method can help facilitate research in the ion channel field.Such as characterizing channel properties in the pharmacology. Since the current methods are either complex or show inconsistent protein expression...