Controllable Ion Channel Expression through Inducible Transient Transfection

1. Ligating the Gene of Interest into the Repressible Site of Vector

1. Obtain an inducible vector such as pcDNA5/FRT/TO or pcDNA4/TO.
2. 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.
3. 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.
4. Digest both the vector and insert with restriction enzymes (chosen in step 1.2) at enzymes' 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.
5. Load the digested DNA onto a 1% agarose gel and separate the cleaved segments by electrophoresis⁹.
6. Using UV light, cut with a blade the pieces of gel containing the desired segments of vector and insert to be ligated.
7. 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.
8. 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.
9. For bacterial transformation, add 10 ng of vector alone and vector + gene into 50 μL competent E. coli. Incubate the tube on ice for 30 minutes followed by 45 seconds incubation at 42 °C. Immediately place back on ice for two minutes and transfer the transformed bacteria to 500 μL LB medium. Incubate for one hour at 37 °C under agitation at 220 rpm.
10. For selection of successfully transformed bacteria, plate 100 μL of each reaction (as described in 1.9) on a prepared LB agar plate containing the suitable antibiotic (e.g., 100 μg/mL ampicillin when using pCDNA4/TR).
11. Allow to grow overnight at 37 °C. Plates may be stored at 4 °C for up to two weeks by tightly wrapping them in plastic paraffin film.
12. Lift individual colonies using a 10 μL tip, and allow to grow overnight in 3 mL LB + antibiotic medium at 37 °C under agitation at 220 rpm. Bacteria with DNA of interest may be frozen (-20 °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 °C.
13. Extract DNA through mini-prep and measure the final concentration by spectrophotometer at 260 nm⁵.
14. 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.

2. Culturing Cell Lines Expressing TetR

1. 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.
2. Seed cells in 100 mm tissue culture plate with Dulbecco'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 °C and 5% CO₂. All work with cells should be done in a biological hood under sterile conditions.
3. In order to maintain the expression of Tet repressor gene, aspirate the medium and replace it with Full DMEM supplemented with 5 µg/mL blasticidin. Incubate cells at 37 °C and 5% CO₂.
4. Once cells reach 80 – 90% confluency, aspirate the medium and gently wash the cells twice with DPBS (without calcium and magnesium) warmed to 37 °C.
5. In order to gently lift the cells without causing mechanical damage, incubate the culture in DMEM containing 0.05% trypsin warmed to 37 °C for 1.5 min.
6. 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.
7. Centrifuge the cells at 200 x g for 5 min.
8. 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.
9. Seed 1 – 2 x 10⁶ cells in 100 mm tissue culture dish containing 12 mL Full DMEM with 5 µg/mL blasticidin. Split the cells twice a week as they reach 90% confluency.

3. Transfecting the Plasmid of Interest into Cells

1. 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 °C in a CO₂ incubator.
2. Prepare a transfection mixture with pcDNA4/TO containing the gene of interest, an inert plasmid to bring the total DNA amount to 1 µg (if needed), 3 µL lipid transfection reagent and DMEM to make the final volume 100 µL. 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.
3. Incubate the transfection mixture at RT for 30 min.
4. 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.
5. Incubate the transfected cells O/N at 37 °C and 5% CO₂.
6. Confirm that the cells were successfully transfected by observing the fluorescence signal of EGFP under UV lamp the following morning if performing electrophysiology.

4. Plating Cells on Poly-D-Lysine (PDL) Coverslips/Wells

1. 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.
2. Pipette a solution of 0.1 – 0.2 mg/mL PDL solution onto each coverslip or well of a calcium imaging chamber.
3. Allow to sit for 30 min at 37 °C in a 5% CO₂ incubator.
4. 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.
5. For electrophysiology, fill each well containing a PDL coated coverslip with 500 µL Full DMEM warmed to 37 °C.
6. Perform the same method of splitting the transfected cells as described above. After resuspending the cells following trypsin treatment and blocking, transfer 80 µL 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 °C in a 5% CO₂ incubator.
7. For calcium imaging transfer and spot 20 µL 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 and add 180 µL full DMEM to each well.

5. Inducing Gene Expression

1. Prepare a doxycycline stock solution of 1 mg/mL in DDW according to manufacturer's instructions. Keep stock solution protected from light at 4 °C for up to 3 weeks. For long term storage keep doxycycline stock solution at -20 °C.
2. Prepare a fresh 2 µg/mL (electrophysiology) or 3 µg/mL (calcium imaging) doxycycline solution in Full DMEM and warm it to 37 °C.
3. For electrophysiological recordings, pipette 500 µL of 2 µg/mL doxycycline solution to each well to make a final concentration of 1 µg/mL doxycycline. For calcium imaging, add 100 µL of 3 µg/mL doxycycline solution to each well to make a final concentration of 1 µg/mL doxycycline. Write down the hour of induction.
4. Incubate for the desired amount of time for induction at 37 °C and 5% CO₂.

6. Calibrating Timeline of Protein Expression

1. Calibrating protein expression through Calcium Imaging^5,10
   1. Prepare Ringer'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 °C.
   2. Prepare saturating concentrations of channel agonist in Ringer’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. This also makes the amount of bath solution in each well critical to control.
   3. Prepare a solution of non-ionic F-127 20% W/V in DMSO. Heat the solution to 40 °C until non-ionic F-127 is dissolved. Store the non-ionic F-127 solution at RT and warm it to 40 °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.
   4. Aspirate medium and replace it with Fura-2AM loading solution (Ringer's solution supplemented with 2 – 3 µM Fura-2AM, 0.02 mg/mL non-ionic F-127 and 10 mM D-glucose).
   5. Incubate for 60 min at RT in the dark.
   6. Aspirate Fura-2AM solution and wash cells with Ringer's + 10 mM D-glucose solution to remove extracellular dye. Repeat this step twice.
   7. Leave 200 µL Ringer's + 10 mM D-glucose solution in each well and incubate for 30 min at RT in the dark.
   8. Place chamber on stage above the microscope nose piece and secure it with holder.
   9. Turn on the lamp, camera and microscope and select Fura-2 filter to detect emission at 510 nm wavelength.
   10. Adjust for the desired magnification and focus cells in the selected field.
   11. In the dark, subtract background light and set exposure time.
   12. Set picture sampling at the desired rate and record fluorescence response of Fura-2 loaded cells by exciting them at 340 and 380 nm wavelengths. The ratio of 340/380 signals is an indicator for intracellular Ca²⁺ concentration and thus also for TRPV1 activity.
   13. Add 2 µM capsaicin in 200 µL Ringer's solution by pipetting to create a final saturating concentration of 1 µM capsaicin in order to evaluate the expression level of TRPV1.
   14. Analyze TRPV1 response according to induction time and determine protocol for the protein of interest.
2. Adjusting protein expression in heterologous systems using the whole cell configuration of the patch clamp technique^4,11.
   1. 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.
   2. Prepare saturating concentrations of channel agonist in extracellular solution.
   3. Pull glass electrodes with an inner diameter (I.D) of 1.10 mm and fire-polish to a resistance of 2 – 4 MΩ.
   4. 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.
   5. 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.
   6. Locate an EGFP positive cell, as visualized with the epifluorescent microscope, on the prepared coverslip and move to the center of the visualized field.
   7. 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.
   8. As the pipette touches the cell, apply a small amount of negative pressure in order to create a GΩ seal between the glass pipette and cell membrane.
   9. 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.
   10. Switch the amplifier mode to whole cell, and lower the Bessel filter and output gain to 1 – 5 kHz and 0.5α, respectively.
   11. Record the current response to the saturating concentrations of channel agonist by using voltage ramps or gap free protocol.
   12. Because expression efficiencies change between different proteins, repeat the recordings using different induction length times.
3. Determining ideal induction time for single channel recordings⁴.
   1. 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.
   2. Repeat whole cell procedure using I.D. 0.86 mm glass pipettes fire-polished to a resistance of 10 – 12 MΩ and set holding potential to -40 mV.
   3. Create a seal on the membrane as described previously and rupture the membrane.
   4. Using the micromanipulator, lift the pipette with the membrane patch away from the cell.
   5. Position the perfusion system directly next to the pipette containing the membrane patch.
   6. Lower the Bessel filter and output gain to 2 – 5 kHz and 10α, respectively.
   7. Perfuse saturating concentrations of agonist onto the membrane patch, evaluating if the patch contains one or more channels according to the response.
   8. 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.