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 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.
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 rate8.
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, 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.
1. Ligating the Gene of Interest into the Repressible Site of Vector
2. Culturing Cell Lines Expressing TetR
3. Transfecting the Plasmid of Interest into Cells
4. Plating Cells on Poly-D-Lysine (PDL) Coverslips/Wells
5. Inducing Gene Expression
6. Calibrating Timeline of Protein Expression
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. Adding doxycycline to the medium prevents TR-TetO interaction, thus allowing the expression of the transfected protein. To control the expression of rat TRPV1 (rTRPV1) we first inserted this channel's gene into a pcDNA4/TO vector using the above-described protocol. Next, T-REx-293 cells were transfected with the rTRPV1- pcDNA4/TO plasmid according to the steps in point 3 of the protocol. Following transfection, rTRPV1 expression was induced by incubating the cells in the presence of doxycycline (1 μg/mL) for 1 – 4 h. Importantly, different doxycycline concentrations can be used to achieve desired expression rate. We used electrophysiological and calcium imaging methods to evaluate rTRPV1 expression level at each time point by applying its agonist, capsaicin. As shown in Figure 1, using live-cell calcium imaging, intracellular calcium level after capsaicin application is increased gradually with longer induction times. Activation of uninduced cells may be attributable to a basal leak in the TR activity, overexpression of the plasmid, or residual tetracycline in the cells media components. Next, we recorded currents from cells using the whole-cell configuration of the patch clamp technique applying voltage ramps between −80 mV to +80 mV. Figure 2 shows that higher current amplitudes are obtained with longer induction times. The saturation in current amplitude at 4 h induction is consistent with the saturation seen in calcium response (Figure 1). Finally, we analyzed rTRPV1 expression levels in the outside-out configuration of the patch-clamp technique. One of the major difficulties in studying ion channel structure-function is reaching expression levels suitable for single-channel analysis. Based on the published unitary rTRPV1 conductance4, the recorded current amplitude was used to determine the number of channels in the excised patch. As shown in Figure 3, the number of channels in the patch increases proportionately to the doxycycline incubation time. After one hour of induction, we were not able to detect any channel activity (0 out of 8). However, in both two- and three-hour time points we recorded single channel activity in similar success rates. Of note, while in two hours most patches did not respond, in three hours most patches showed single-to-multi -channel activity. The chances of recording multiple channels after three hours of induction is high, thus the optimal induction time to record current from a single channel is between two to three hours under the presented conditions. Together these results demonstrate that expression of ion channels can be tightly controlled and regulated after transient transfection using this protocol.
Figure 1. Response of TRPV1 Activation Increases according to Induction Time, as Visualized through Calcium Imaging. (A) Pseudo-colored images of T-REx-293 cells transiently expressing rTRPV1, before ('Basal') and after capsaicin (2 μM) application. White bars represent 30 μm. Scale bar indicates levels of intracellular calcium. (B) Changes with time of intracellular calcium levels in transfected T-REx-293 cells treated as shown in A. Each graph represents an average of 50 capsaicin sensitive cells. Note the stepwise increases in capsaicin responses in relation to induction time. Please click here to view a larger version of this figure.
Figure 2. TRPV1 Current Increases as a Result of Increasing Induction Time in Whole Cell Patch Clamp Recordings. (A) Whole-cell recordings from T-REx-293 transiently transfected with rTRPV1 at a holding potential of -40 mV. Cells were induced with doxycycline (1 μg/mL) for the indicated time and then exposed to capsaicin ('Cap'; 1 μM; red bar). Shown is a representative trace of 6 – 11 independent recordings. (B) Mean/scatter-dot plot representing the whole-cell amplitude evoked as shown in A. Statistical significance between groups was determined with ANOVA with multiple comparisons, where *** represents p ≤0.001 and ns- not statistically significant (n = 6 – 11 cells). Please click here to view a larger version of this figure.
Figure 3. Success Rate of Single Channel Patch in TRPV1 Recordings is Dependent on Induction Time. (A) Outside-out recordings from T-REx-293 transiently transfected with rTRPV1 at a holding potential of +50 mV. Cells were induced with doxycycline (1 μg/mL) for the indicated time and were exposed to capsaicin ('Cap'; 1 μM; red bar). Shown is a representative trace of 6 – 9 independent recordings. (B) Mean/scatter-dot plot representing the number of channels in the excised patch as shown in A. Statistical significance between groups was determined with ANOVA with multiple comparisons, where *** represents p ≤0.001 and ns- not statistically significant (n = 6 – 9 cells). Please click here to view a larger version of this figure.
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 electrophysiology2. Alternatively, stable cell lines under the tetracycline repressible expression system have been developed to offer a way to control the expression level of the protein of interest12. This approach provides consistent expression of all cells in a culture with the introduction of a tetracycline derived supplement, such as doxycycline. While this allows for consistent and precise control of protein levels, the time and effort that go in to establishing a stable cell line makes it inconvenient and disadvantageous when studying a wide variety of proteins8.
The protocol presented here offers a middle-of-the-road solution to the current transfection protocols offered. By transiently transfecting a gene under a controllable promoter into cells possessing a Tet repressor, we can achieve controllable expression while avoiding lengthy and complicated procedures. Here, we used the non-selective cation channel TRPV1 with calcium imaging and electrophysiology analysis. We found that the ideal induction time for the controlled expression of this specific protein for single channel analysis was between 2 – 3 h incubation with 1 μg/mL of doxycycline (Figures 1 – 3). On a broader scale, this technique can also be applied to any protein of interest. This protocol also offers a solution to controlling expression when a large range of different proteins or mutations in protein sequences are of interest and creating a stable cell line for each variant is impractical. Importantly, while the results here represent the optimal time and concentrations applicable for TRPV1 expression, each gene has its individual and specific translation and trafficking pace, thus the calibration and expression times of each protein will differ greatly13.
While this technique offers a convenient solution to controlled expression, it is not without limitations. Only commercially available cell lines harboring the Tet system can be used with this system, which presents a problem if these particular cell lines are unsuitable for the type of analysis that is to be performed. Another pitfall compared to stably transfected lines is that not all cells undergo transfection, and thus not all cells express the protein of interest. Overall, we offer a convenient way to control protein expression in a heterologous system that can be applied to a wide array of biomedical research.
The authors have nothing to disclose.
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.
pcDNA™4/TO Mammalian Expression Vector | ThermoScientific Fisher | V102020 | |
pcDNA™5/TO Mammalian Expression Vector | ThermoScientific Fisher | V103320 | |
PureLink Quick PCR Purification Kit | Invitrogen | K310001 | |
Swift™ MaxPro Thermal Cycler | Esco | n.a | |
Restriction Enzymes | ThermoScientific Fisher | ER0501 | |
Agarose | Lonza | 50004 | |
PureLink Quick Gel Extraction Kit | Invitrogen | K210012 | |
NanoDrop 2000c | ThermoScientific Fisher | ND-2000C | |
T4 DNA Ligase | ThermoScientific Fisher | EL0011 | |
One Shot® TOP10 Chemically Competent E. coli | ThermoScientific Fisher | C404006 | |
Ampicillin (Sodium), USP Grade | Gold Bio | A-301-5 | |
Tryptone for microbiology | Merck | 6.19305E+13 | |
Yeast Extract | BD worldwide | 212750 | |
SIF6000R Incubated Shaker | LAB COMPANION | 45H118 | |
NucleoSpin®plasmid |
Macherey Nagel | 740588.25 | |
MS 300V Power Supply | Major Science | MP-300V | |
Owl™ EasyCast™ B1A Mini Gel Electrophoresis System | ThermoScientific Fisher | B1A | |
T-REx™-293 cell line | Invitrogen | R710-07 | |
DMEM (1X), liquid (high glucose) | Gibco | 41965-039 | |
HindIII-HF® | NEB | R3104S | |
ApaI | NEB | R0114S | |
CutSmart® Buffer | NEB | B7204S | |
pcDNA™6/TR Mammalian Expression Vector | ThermoScientific Fisher | V102520 | |
Fetal Bovine Serum, qualified, E.U.-approved, South America origin | Gibco | 10270106 | |
HEPES Buffer Solution (1 M) | Biological Industries | 03-025-1B | |
Penicillin-Streptomycin Solution | Biological Industries | 03-031-1B | |
L-Alanyl-L-Glutamine (Stable Glutamine) (200 mM) | Biological Industries | 03-022-1B | |
Heracell™ 150i CO2 Incubator | ThermoScientific Fisher | 51026406 | |
MSC-Advantage™ Class II Biological Safety Cabinet | ThermoScientific Fisher | 51025411 | |
Blasticidine S hydrochloride | Sigma-Aldrich | 15205-25MG | |
Dulbecco’s Phosphate Buffered Saline Modified, without calcium chloride and magnesium chloride | Sigma-Aldrich | D8537-500ML | |
Trypsin-EDTA (0.05%), phenol red | Gibco | 25300054 | |
DOUBLE NEUBAUER RULED METALLIZED HEMACYTOMETER | Hausser Scientific | 31000 | |
Opti-MEM I Reduced Serum Medium | Gibco | 31985070 | |
TransIT®-LT1 Transfection Reagent | Mirus | MIR 2300 | |
glass coverslips, #1 thickness, 12mm diameter round | Knittel Glass | GG-12 | |
BioCoat™ Poly-D-Lysine | Corning | 354210 | |
Water, Cell Culture Grade | Biological Industries | 03-055-1A | |
Doxycycline hyclate | Sigma-Aldrich | D9891-1G | |
Fura-2, AM ester | Biotium | BTM-50034 | |
Pluronic® F-127 | Sigma-Aldrich | P2443-250G | |
µ-Slide 8 Well | ibidi | 80826 | |
(E)-Capsaicin | Tocris | 462 | |
Olympus IX70 Fluorescence Microscope | Olympus | n.a | |
Lambda DG-4 Wavelength Switcher | Sutter Instruments | n.a | |
EXi Blue Fluorescence Microscopy Camera | QImaging | n.a | |
MetaFluor Fluorescence Ratio Imaging Software | Molecular Devices | n.a | |
Thin Walled Borosilicate Tubing | Sutter Instruments | B150-110-7.5HP | |
Standard Walled Borosilicate Tubing | Sutter Instruments | B150-86-7.5HP | |
Dimethyl sulfoxide anhydrous | Sigma-Aldrich | 276855 | |
P1000 micropipette puller | Sutter Instruments | P-1000 | |
MF-900 Microforge | NARISHIGE | n.a | |
ValveBank perfusion sysytem | AutoMate Scientific | ||
Digidata® 1440A Low-noise Data Acquisition System | Molecular Devices | n.a | |
Axopatch 200B Amplifier | Molecular Devices | n.a | |
pCLAMP 10.6 Software | Molecular Devices | n.a | |
micromanipulator | Sutter Instruments | MP-225 |