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.
