The overall goal of this procedure is to express new proteins, such as ion channels in Xenopus oocytes. This method can help answer key questions in any field that requires a functional expression system. The main advantages of the variations of this technique are the speed, reliability, and improved survival of the oocytes.
This method can provide insight into functioning of ion channels. Additionally, it can be applied to the study of any other protein. We first had the idea for this method when we realized that classical methods resulted in a low rate of oocyte survival.
After removing the lobes of the ovaries from female frogs according to the text protocol, place the lobes in sterile Modified Barth's Solution supplemented with penicillin and streptomycin. Cut open the lobes to allow access of the tissue to oxygen. To single out stage five to six follicles, use forceps to hold the ovary and then lower a platinum loop over the follicles and gently withdraw the loop to disrupt the connective tissue with the follicle from the ovary tissue.
Sort out healthy-looking oocytes using the platinum loop. Use a plastic Pasteur pipette with the tip cut to a 1.5 millimeter diameter to transfer selected follicles to a 35 millimeter diameter Petri dish. After preparing cRNA's for the GABA A receptor and micro-injection pipettes according to the text protocol, use paraffin oil and a 10 milliliter syringe with a thin needle to backfill a micro-injection pipette.
Then mount the pipette onto a home-built micro-injection apparatus. Using a pipette with a sterile plastic tip, place a droplet of mRNA solution onto the clean side of a piece of moisture-resistant thermoplastic. Then immerse the tip of the injection pipette into the droplet and turn on the motor to retract the plunger.
mRNA solution should enter the pipette and form a visible interface with the oil. Next, retract the tip of the pipette from the droplet, and apply a positive pressure to the inside of the injection pipette. A droplet of mRNA solution should form at the tip of the injection pipette.
Using a 60 millimeter Petri dish with nylon mesh glued to the bottom and covered with MBS for injection, line up the follicles with their vegetal poles pointing upwards. With the micromanipulator, position the injection pipette over an individual follicle. Insert the injection needle into the center of the vegetal pole and apply positive pressure to the inside of the pipette to inject 50 nanoliters of mRNA at a flow rate of 0.6 microliters per minute.
Wait five to 10 seconds before removing the injection pipette tip from the follicle to avoid messenger RNA escape. Transfer injected follicles to a new Petri dish filled with two milliliters of MBS. Place the dish into a wine cooler set at 18 degrees Celsius.
Incubate the injected follicles for one to seven days before recording, depending on the identity of the newly-expressed protein. To strip follicles, transfer ten injected follicles to a borosilicate glass tube containing 0.5 milliliters of MBS, one milligram per milliliter of collagenase, and 0.1 milligram per milliliter of trypsin inhibitor. Immerse the tube in a 36-degree Celsius water bath and incubate the follicles for 20 minutes.
The temperature here is critical because one degree more may kill the oocytes. Rinse the follicles by transferring them to a second tube containing one milliliter of MBS at room temperature, and leave them in the tube for about ten seconds. Then transfer the follicles to a third tube containing 0.5 milliliters of doubly-concentrated MBS containing four millimolars EGTA, and incubate the samples at room temperature for four minutes, occasionally shaking the tubes.
The incubation time of four minutes is critical to ensure follicular layers detach from the oocyte. Rinse the follicles by transferring them to another tube containing one milliliter of MBS at room temperature, and leave them in the tube for about 10 seconds. Transfer the oocytes to a 35 millimeter Petri dish containing two milliliters of MBS.
Then, under a stereo microscope, use a platinum loop to separate the outer envelopes from the follicles by simply pushing the naked oocyte away. Store the denuded oocytes until use. In this experiment, mechanically singled-out Xenopus oocytes were micro-injected with mRNA coating for GABA A receptor sub-units.
Then follicular cell layers were removed. The oocytes were voltage-clamped at negative-80 millivolts and exposed to increasing concentrations of GABA and the presence of THDOC, a potent positive allosteric modulator of the GABA A receptor. This panel shows elicited current amplitudes depending on the GABA concentrations, indicating the sensitivity of this sub-unit combination toward GABA.
The equation used was I(c)Imax/n)where c is the concentration of GABA, the EC50 is the concentration of GABA in the presence of one micromolar THDOC, eliciting a half-maximal current amplitude. Imax is the maximal current amplitude. I is the current amplitude, and n is the Hill coefficient.
The EC50 of the alpha four beta two delta GABA A receptor amounted to 0.41 0.12 micromolar and n was 0.76 0.04 Once mastered, folliculal cell layers can be removed in half an hour if performed properly. After it's development, this technique paved the way for researchers to improve protein expression procedure in Xenopus oocytes. After watching this video, you should have a good understanding of how to isolate Xenopus oocytes surrounded by follicular cell layers their micro-injection with mRNA and subsequent removal of follicular cell layers.
