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 restriction enzyme recognition sites (that are not found in the gene of interest), the first inserted before the start codon, and the second after the stop codon.</li>
	<li>Digest both the vector and insert with restriction enzymes (chosen in step 1.2) at enzymes&#39; working temperature for one hour, followed by addition of one unit calf intestine phosphatase (CIP) to the vector reaction only for 30 minutes in order to avoid self-ligation.</li>
	<li>Load the digested DNA onto a 1% agarose gel and separate the cleaved segments by electrophoresis9.</li>
	<li>Using UV light, cut with a blade the pieces of gel containing the desired segments of vector and insert to be ligated.</li>
	<li>Extract the DNA segments from the agarose gel pieces (using commercial available DNA extraction kits) and measure their final concentration by spectrophotometer at 260 nm.</li>
	<li>Ligate the gene of interest into the inducible selected vector using T4 ligase enzyme at room temperature for 20 minutes. In order to increase the probability of the insert ligating with the vector, use a 3:1 molar ratio of the insert: vector. For control, prepare a ligation reaction that includes only the vector.</li>
	<li>For bacterial transformation, add 10 ng of vector alone and vector + gene into 50 &#956;L competent E. coli. Incubate the tube on ice for 30 minutes followed by 45 seconds incubation at 42 &#176;C. Immediately place back on ice for two minutes and transfer the transformed bacteria to 500 &#956;L&#160;LB medium. Incubate for one hour at 37 &#176;C under agitation at 220 rpm.</li>
	<li>For selection of successfully transformed bacteria, plate 100 &#956;L&#160;of each reaction (as described in 1.9) on a prepared LB agar plate containing the suitable antibiotic (e.g., 100 &#956;g/mL ampicillin when using pCDNA4/TR).</li>
	<li>Allow to grow overnight at 37 &#176;C. Plates may be stored at 4 &#176;C for up to two weeks by tightly wrapping them in plastic paraffin film.</li>
	<li>Lift individual colonies using a 10 &#956;L tip, and allow to grow overnight in 3 mL LB + antibiotic medium at 37 &#176;C under agitation at 220 rpm. Bacteria with DNA of interest may be frozen (-20 &#176;C) for short term storage by centrifuging the LB and bacteria at 11,000 x g and aspirating out the supernatant. Long term storage requires freezing the bacteria as glycerol stocks at -80 &#176;C.</li>
	<li>Extract DNA through mini-prep and measure the final concentration by spectrophotometer at 260 nm5.</li>
	<li>Confirm successful insertion of gene of interest by sequencing the purified construct4. Alternatively, diagnostic digestion of the construct by restriction enzymes using electrophoresis to separate the cut segments and evaluate their presence and length could also be utilized to this end4.</li>
</ol>
2. Culturing Cell Lines Expressing TetR
<ol>
	<li>Obtain a cell culture line harboring a Tet repressor plasmid incorporated into their genomic DNA. Herein the protocol will be written using Human embryonic kidney 293T cells (HEK-293T) harboring pcDNA6/TR plasmid as an example.</li>
	<li>Seed cells in 100 mm tissue culture plate with Dulbecco&#39;s Modified Eagles Medium (DMEM) supplemented with 10% FBS, 1% Penicillin-Streptomycin, 2 mM l-Glutamine, and 25 mM HEPES, pH 7.3 (herein: Full DMEM) and incubate O/N at 37 &#176;C and 5% CO2. All work with cells should be done in a biological hood under sterile conditions.</li>
	<li>In order to maintain the expression of Tet repressor gene, aspirate the medium and replace it with Full DMEM supplemented with 5 &#181;g/mL blasticidin. Incubate cells at 37 &#176;C and 5% CO2.</li>
	<li>Once cells reach 80 - 90% confluency, aspirate the medium and gently wash the cells twice with DPBS (without calcium and magnesium) warmed to 37 &#176;C.</li>
	<li>In order to gently lift the cells without causing mechanical damage, incubate the culture in DMEM containing 0.05% trypsin warmed to 37 &#176;C for 1.5 min.</li>
	<li>Block the trypsin action with an equal volume of Full DMEM. Gently pipette up and down in order to lift the cells and transfer them to a sterile tube.</li>
	<li>Centrifuge the cells at 200 x g for 5 min.</li>
	<li>Aspirate the supernatant and replace it with 1 mL Full DMEM. Pipette the solution with the cells up and down until no cell clumps can be seen. Count and calculate the number of cells using a hemocytometer.</li>
	<li>Seed 1 - 2 x 106 cells in 100 mm tissue culture dish containing 12 mL Full DMEM with 5 &#181;g/mL blasticidin. Split the cells twice a week as they reach 90% confluency.</li>
</ol>
3. Transfecting the Plasmid of Interest into Cells
<ol>
	<li>Using the same method of splitting cells as described above, transfer cells to wells in a 12-well plate prepared with 0.9 mL Full DMEM, sufficient to make 50% confluent (~200,000 cells) and incubate overnight at 37 &#176;C in a CO2 incubator.</li>
	<li>Prepare a transfection mixture with pcDNA4/TO containing the gene of interest, an inert plasmid to bring the total DNA amount to 1 &#181;g (if needed), 3 &#181;L lipid transfection reagent and DMEM to make the final volume 100 &#181;L. The optimal amount of plasmid DNA to be used varies widely as different proteins express in different efficiency. For electrophysiological experiments, include EGFP in mammalian expression plasmid in the transfection cocktail in order to visualize cells that successfully undergo transfection4.</li>
	<li>Incubate the transfection mixture at RT for 30 min.</li>
	<li>Transfect cells by pipetting the transfection mixture drop-wise onto the plated cells in the 12-well plate and rock the plate vigorously to ensure a homogenous dispersion of DNA and transfection reagent. Cells should be ~80% confluent at the time of transfection.</li>
	<li>Incubate the transfected cells O/N at 37 &#176;C and 5% CO2.</li>
	<li>Confirm that the cells were successfully transfected by observing the fluorescence signal of EGFP under UV lamp the following morning if performing electrophysiology.</li>
</ol>
4. Plating Cells on Poly-D-Lysine (PDL) Coverslips/Wells
<ol>
	<li>Sterilize 12 mm coverslips by dousing with 70% isopropyl ethanol solution. Dry and place a single coverslip in each well of a 24-well plate for electrophysiological recording.</li>
	<li>Pipette a solution of 0.1 - 0.2 mg/mL PDL solution onto each coverslip or well of a calcium imaging chamber.</li>
	<li>Allow to sit for 30 min at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Wash with cell culture grade double distilled water (DDW) three times, aspirating between each wash. Following the final wash, dry thoroughly and allow to sit at RT.</li>
	<li>For electrophysiology, fill each well containing a PDL coated coverslip with 500 &#181;L Full DMEM warmed to 37 &#176;C.</li>
	<li>Perform the same method of splitting the transfected cells as described above. After resuspending the cells following trypsin treatment and blocking, transfer 80 &#181;L of cells (or approximately 30,000 cells) to the center of each PDL coated coverslip for electrophysiology recording. Allow cells to settle for at least 1.5 hours or incubate overnight at 37 &#176;C in a 5% CO2&#160;incubator.</li>
	<li>For calcium imaging transfer and spot 20 &#181;L of cells (approximately 20,000 cells) to the center of each PDL coated calcium imaging well. Allow cells to settle for at least 30 min&#160;and add 180 &#181;L full DMEM to each well.</li>
</ol>
5. Inducing Gene Expression
<ol>
	<li>Prepare a doxycycline stock solution of 1 mg/mL in DDW according to manufacturer&#39;s instructions. Keep stock solution protected from light at 4 &#176;C for up to 3 weeks. For long term storage keep doxycycline stock solution at -20 &#176;C.</li>
	<li>Prepare a fresh 2 &#181;g/mL (electrophysiology) or 3 &#181;g/mL (calcium imaging) doxycycline solution in Full DMEM and warm it to 37 &#176;C.</li>
	<li>For electrophysiological recordings, pipette 500 &#181;L of 2 &#181;g/mL doxycycline solution to each well to make a final concentration of 1 &#181;g/mL doxycycline. For calcium imaging, add 100 &#181;L of 3 &#181;g/mL doxycycline solution to each well to make a final concentration of 1 &#181;g/mL doxycycline. Write down the hour of induction.</li>
	<li>Incubate for the desired amount of time for induction at 37 &#176;C and 5% CO2.</li>
</ol>
6. Calibrating Timeline of Protein Expression
<ol>
	<li>Calibrating protein expression through Calcium Imaging5,10
	<ol>
		<li>Prepare Ringer&#39;s solution (140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 2 mM MgSO4, 20 mM HEPES, pH adjusted to 7.4 with NaOH) and warm it to 37 &#176;C.</li>
		<li>Prepare saturating concentrations of channel agonist in Ringer&#8217;s solution. Because the amount of agonist is diluted when added to the calcium imaging chambers containing the cells and extracellular solution, double the amount of the desired final concentration of agonist.&#160;This also makes the amount of bath solution in each well critical to control.</li>
		<li>Prepare a solution of non-ionic F-127 20% W/V in DMSO. Heat the solution to 40 &#176;C until non-ionic F-127 is dissolved. Store the non-ionic F-127 solution at RT and warm it to 40 &#176;C before use. Non-ionic F-127 is used to help disperse acetoxymethyl (AM) esters of fluorescent ion indicators such as Fura-2 in aqueous solutions.</li>
		<li>Aspirate medium and replace it with Fura-2AM loading solution (Ringer&#39;s solution supplemented with 2 - 3 &#181;M Fura-2AM, 0.02 mg/mL non-ionic F-127 and 10 mM D-glucose).</li>
		<li>Incubate for 60 min at RT in the dark.</li>
		<li>Aspirate Fura-2AM solution and wash cells with Ringer&#39;s + 10 mM D-glucose solution to remove extracellular dye. Repeat this step twice.</li>
		<li>Leave 200 &#181;L Ringer&#39;s + 10 mM D-glucose solution in each well and incubate for 30 min at RT in the dark.</li>
		<li>Place chamber on stage above the microscope nose piece and secure it with holder.</li>
		<li>Turn on the lamp, camera and microscope and select Fura-2 filter to detect emission at 510 nm wavelength.</li>
		<li>Adjust for the desired magnification and focus cells in the selected field.</li>
		<li>In the dark, subtract background light and set exposure time.</li>
		<li>Set picture sampling at the desired rate and record fluorescence response of Fura-2 loaded cells by exciting them at 340&#160;and 380 nm wavelengths. The ratio of 340/380 signals is an indicator for intracellular Ca2+ concentration and thus also for TRPV1 activity.</li>
		<li>Add 2 &#181;M capsaicin in 200 &#181;L Ringer&#39;s solution by pipetting to create a final saturating concentration of 1 &#181;M capsaicin in order to evaluate the expression level of TRPV1.</li>
		<li>Analyze TRPV1 response according to induction time and determine protocol for the protein of interest.</li>
	</ol>
	</li>
	<li>Adjusting protein expression in heterologous systems using the whole cell configuration of the patch clamp technique4,11.
	<ol>
		<li>Prepare the bath solution of (mM) 140 NaCl, 2.3 KCl, 2 MgSO4, 5 HEPES, and 5 2-(N-morpholino) ethanesulfonic acid (MES), adjusted to pH 7.4 with NaOH.</li>
		<li>Prepare saturating concentrations of channel agonist in extracellular solution.</li>
		<li>Pull glass electrodes with an inner diameter (I.D) of 1.10 mm and fire-polish to a resistance of 2 - 4 M&#937;.</li>
		<li>Prepare a pipette solution of (mM) 130 KCl, 4 NaCl, 2 MgSO4, 0.5 CaCl2, 1 EGTA and 10 HEPES adjusted to pH 7.2 with KOH. Filter the pipette solution prior to use. Before each experiment, a single, fire-polished glass pipette is back filled by submerging the tip in the pipette solution for at least a minute, and then filled using melted and stretched tip with a syringe filled with the same pipette solution.</li>
		<li>Gently lift a single PDL coated coverslip with the plated cells from the well to a 35 mm plastic petri dish filled with approximately 2 mL bath solution at RT. Observe under an epi-fluorescent microscope.</li>
		<li>Locate an EGFP positive cell, as visualized with the epifluorescent microscope, on the prepared coverslip and move to the center of the visualized field.</li>
		<li>Attach the filled pipette to the pipette holder of the micromanipulator, and inject a small amount of positive pressure into the system to avoid contamination of the intracellular pipette solution. Lower it to just above the GFP - positive cell, checking for proper resistance.</li>
		<li>As the pipette touches the cell, apply a small amount of negative pressure in order to create a G&#937; seal between the glass pipette and cell membrane.</li>
		<li>With a short, sharp pulse of suction, break the cell membrane, allowing the pipette to come into contact with the intracellular contents of the cell.</li>
		<li>Switch the amplifier mode to whole cell, and lower the Bessel filter and output gain to 1 - 5 kHz and 0.5&#945;, respectively.</li>
		<li>Record the current response to the saturating concentrations of channel agonist by using voltage ramps or gap free protocol.</li>
		<li>Because expression efficiencies change between different proteins, repeat the recordings using different induction length times.</li>
	</ol>
	</li>
	<li>Determining ideal induction time for single channel recordings4.
	<ol>
		<li>Prepare a solution of (mM) 150 Na-gluconate, 15 NaCl, 5 EGTA, and 10 HEPES, adjusted to pH 7.4 with NaOH, as a pipette solution. For the bath solution, use the solution from step 6.2.1.</li>
		<li>Repeat whole cell procedure using I.D. 0.86 mm glass pipettes fire-polished to a resistance of 10 - 12 M&#937; and set holding potential to -40 mV.</li>
		<li>Create a seal on the membrane as described previously and rupture the membrane.</li>
		<li>Using the micromanipulator, lift the pipette with the membrane patch away from the cell.</li>
		<li>Position the perfusion system directly next to the pipette containing the membrane patch.</li>
		<li>Lower the Bessel filter and output gain to 2 - 5 kHz and 10&#945;, respectively.</li>
		<li>Perfuse saturating concentrations of agonist onto the membrane patch, evaluating if the patch contains one or more channels according to the response.</li>
		<li>Analyze the number of single channel patches successfully recorded against the induction time applied to cells. Adjust the induction time according to the desired results.</li>
	</ol>
	</li>
</ol>

<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. 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The authors have nothing to disclose.

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.

<table><tbody><tr><td>pcDNA&amp;trade;4/TO Mammalian Expression Vector</td><td>ThermoScientific Fisher</td><td>V102020</td><td></td></tr><tr><td>pcDNA&amp;trade;5/TO Mammalian Expression Vector</td><td>ThermoScientific Fisher</td><td>V103320</td><td></td></tr><tr><td>PureLink Quick PCR Purification Kit</td><td>Invitrogen</td><td>K310001</td><td></td></tr><tr><td>Swift&amp;trade;&amp;nbsp;MaxPro Thermal Cycler</td><td>Esco&amp;nbsp;</td><td>n.a</td><td></td></tr><tr><td>Restriction Enzymes</td><td>ThermoScientific Fisher</td><td>ER0501</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>PureLink Quick Gel Extraction Kit</td><td>Invitrogen</td><td>K210012</td><td></td></tr><tr><td>NanoDrop 2000c</td><td>ThermoScientific Fisher</td><td>ND-2000C</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>ThermoScientific Fisher</td><td>EL0011</td><td></td></tr><tr><td>One Shot&amp;reg; TOP10 Chemically Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>ThermoScientific Fisher</td><td>C404006&amp;nbsp;</td><td></td></tr><tr><td>Ampicillin (Sodium), USP Grade</td><td>Gold Bio</td><td>A-301-5</td><td></td></tr><tr><td>Tryptone for microbiology</td><td>Merck</td><td>6.19305E+13</td><td></td></tr><tr><td>Yeast Extract</td><td>BD worldwide</td><td>212750</td><td></td></tr><tr><td>SIF6000R Incubated Shaker</td><td>LAB COMPANION</td><td>45H118</td><td></td></tr><tr><td>NucleoSpin&amp;reg;plasmid</td><td>Macherey Nagel</td><td>740588.25</td><td></td></tr><tr><td>MS 300V Power Supply</td><td>Major Science</td><td>MP-300V</td><td></td></tr><tr><td>Owl&amp;trade; EasyCast&amp;trade; B1A Mini Gel Electrophoresis System</td><td>ThermoScientific Fisher</td><td>B1A</td><td></td></tr><tr><td>T-REx&amp;trade;-293 cell line</td><td>Invitrogen</td><td>R710-07</td><td></td></tr><tr><td>DMEM (1x), liquid (high glucose)</td><td>Gibco</td><td>41965-039</td><td></td></tr><tr><td>HindIII-HF&amp;reg;</td><td>NEB</td><td>R3104S</td><td></td></tr><tr><td>ApaI</td><td>NEB</td><td>R0114S</td><td></td></tr><tr><td>CutSmart&amp;reg; Buffer</td><td>NEB</td><td>B7204S</td><td></td></tr><tr><td>pcDNA&amp;trade;6/TR&amp;nbsp; Mammalian Expression Vector</td><td>ThermoScientific Fisher</td><td>V102520</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), qualified, E.U.-approved, South America origin</td><td>Gibco</td><td>10270106</td><td></td></tr><tr><td>HEPES Buffer Solution (1 M)</td><td>Biological Industries</td><td>03-025-1B</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Biological Industries</td><td>03-031-1B</td><td></td></tr><tr><td>L-Alanyl-L-Glutamine (Stable Glutamine) (200 mM)</td><td>Biological Industries</td><td>03-022-1B</td><td></td></tr><tr><td>Heracell&amp;trade; 150i CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>ThermoScientific Fisher</td><td>51026406</td><td></td></tr><tr><td>MSC-Advantage&amp;trade; Class II Biological Safety Cabinet</td><td>ThermoScientific Fisher</td><td>51025411</td><td></td></tr><tr><td>Blasticidine S hydrochloride</td><td>Sigma-Aldrich</td><td>15205-25MG</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-buffered Saline (DPBS) Modified, without calcium chloride and magnesium chloride</td><td>Sigma-Aldrich</td><td>D8537-500ML</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>Double Neubauer Ruled Metallized Hemacytometer</td><td>Hausser Scientific</td><td>31000</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985070</td><td></td></tr><tr><td>TransIT&amp;reg;-LT1 Transfection Reagent</td><td>Mirus</td><td>MIR 2300</td><td></td></tr><tr><td>glass coverslips, #1 thickness, 12 mm diameter round</td><td>Knittel Glass</td><td>GG-12</td><td></td></tr><tr><td>BioCoat&amp;trade; Poly-D-Lysine (PDL)</td><td>Corning</td><td>354210</td><td></td></tr><tr><td>Water, Cell Culture Grade</td><td>Biological Industries</td><td>03-055-1A</td><td></td></tr><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891-1G</td><td></td></tr><tr><td>Fura-2, AM ester</td><td>Biotium</td><td>BTM-50034</td><td></td></tr><tr><td>Pluronic&amp;reg; F-127</td><td>Sigma-Aldrich</td><td>P2443-250G</td><td></td></tr><tr><td>&amp;micro;-Slide 8 Well</td><td>ibidi</td><td>80826</td><td></td></tr><tr><td>(E)-Capsaicin</td><td>Tocris</td><td>462</td><td></td></tr><tr><td>Olympus IX70 Fluorescence Microscope</td><td>Olympus</td><td>n.a</td><td></td></tr><tr><td>Lambda DG-4 Wavelength Switcher</td><td>Sutter Instruments</td><td>n.a</td><td></td></tr><tr><td>EXi Blue Fluorescence Microscopy Camera</td><td>QImaging</td><td>n.a</td><td></td></tr><tr><td>MetaFluor Fluorescence Ratio Imaging Software</td><td>Molecular Devices</td><td>n.a</td><td></td></tr><tr><td>Thin Walled Borosilicate Tubing</td><td>Sutter Instruments</td><td>B150-110-7.5HP</td><td></td></tr><tr><td>Standard Walled Borosilicate Tubing</td><td>Sutter Instruments</td><td>B150-86-7.5HP</td><td></td></tr><tr><td>Dimethyl sulfoxide anhydrous</td><td>Sigma-Aldrich</td><td>276855</td><td></td></tr><tr><td>P1000 micropipette puller</td><td>Sutter Instruments</td><td>P-1000</td><td></td></tr><tr><td>MF-900 Microforge</td><td>NARISHIGE</td><td>n.a</td><td></td></tr><tr><td>ValveBank perfusion sysytem</td><td>AutoMate Scientific</td><td></td><td></td></tr><tr><td>Digidata&amp;reg; 1440A Low-noise Data Acquisition System</td><td>Molecular Devices</td><td>n.a</td><td></td></tr><tr><td>Axopatch 200B Amplifier</td><td>Molecular Devices</td><td>n.a</td><td></td></tr><tr><td>pCLAMP 10.6 Software</td><td>Molecular Devices</td><td>n.a</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instruments</td><td>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 ...

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Research

JoVE Journal

Genetics

9.3K Views.  The Hebrew University of Jerusalem (HUJI).  Het bestuderen van ionkanalen via een heteroloog tot expressie brengen systeem is uitgegroeid tot een kern techniek in het biomedisch onderzoek. In dit manuscript presenteren we een tijd efficiënte methode om strak ionkanaal expressie te komen door het uitvoeren transiënte transfectie onder controle van een induceerbare promoter. 

Video: Controllable Ion Channel Expression through Inducible Transient Transfection

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based approaches. For example, although chimeric and Cre-LoxP targeting strategies have been widely used, the intrinsic limitations of these strategies (i.e., leaky promoter activity, mosaic Cre expression, etc.) have significantly restricted their application. Moreover, a complete deletion of many endogenous genes is embryonically lethal, making it impossible to study gene function in postnatal life. To address these challenges, we have made significant changes to an early genetic engineering protocol and combined a short (transgenic) version of the Rb1 gene with a lysosomal protease procathepsin B (CB), to generate a DN mouse model of Rb1 (CBRb). Due to the presence of a lysosomal protease, the entire CB-RB1 fusion protein and its interacting complex are routed for proteasome-mediated degradation. Moreover, the presence of a tetracycline inducer (rtTA) element in the transgenic construct enables an inducible and reversible regulation of the RB1 protein. The presence of a ubiquitous ROSA-CAG promoter in the CBRb mouse model makes it a useful tool to carry out transient and reversible Rb1 gene ablation and provide researchers a resource for understanding its activity in virtually any cell type where RB1 is expressed.

Inducible and Reversible Dominant-negative DN Protein Inhibition

Here we present a protocol to develop a dominant-negative inducible system, in which any protein can be conditionally inactivated by reversibly overexpressing a dominant-negative mutant version of it.

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based ...

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

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Immunology and Infection

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

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

An Introduction to Transfection

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for transfected RNA is the cytoplasm, DNA must be transported to the nucleus for effective transfection. There, the DNA can be transiently expressed for a short period of time, or become incorporated into the genomic DNA, where the change is passed on from cell to cell as it divides.
This video describes the basics behind chemical mediated transfections and introduces some of the most commonly-used reagents, including charged lipids, polymers, and calcium phosphate. Each step is described from the preparation of cells for transfection through analysis of transfection efficiency. Additionally, the applications section of this video-article describes the use of electroporation and a biolistic transfection as alternative methods for introducing nucleic acid into mammalian cells. It also describes an advanced use of transfection where co-transfection of interfering RNA and DNA are introduced as a way to down-regulate a naturally occurring protein while at the same time producing a mutant variant of it within the same cell.

Transfection: Inserting Genetic Materials into Mammalian Cells

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell ...

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Basic Methods in Cellular and Molecular Biology

Inducible Tet-Off Regulatable System: An In Vitro Method to Modulate Gene Expression in Cultured Cells Using Tetracycline-Off System

Source: Migneault, F. et al, <a href="https://www.jove.com/v/60654">Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells.</a> J. Vis. Exp. (2020).
This video describes the method of regulating gene expression in cultured cells by transfection with a plasmid containing a gene of interest and a regulatory plasmid. The expression of this plasmid can be controlled by tetracycline or its synthetic analogs, making this system a useful model to study regulatable gene expression.

Source: Migneault, F. et al, Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in ...

Controllable Ion Channel Expression through Inducible Transient Transfection

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