Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.
The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 – 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.
Xenopus oocytes are widely used as an expression system (References 2 – 3, and references therein). They are able to properly assemble and incorporate functionally active multisubunit proteins into their plasma membranes. Using this system, it is possible to functionally investigate membrane proteins alone or in combination with other proteins, in order to study the properties of mutated, chimeric, or concatenated proteins, and to screen potential drugs.
Advantages of using oocytes over other heterologous expression systems include the simple handling of the giant cells, the high proportion of cells expressing foreign genetic information, the simple control of the environment of the oocyte by means of bath perfusion, and the control of the membrane potential.
The drawback of this expression system is the seasonal variation observed in many laboratories17-20. The reason for this variation is far from clear. Additionally, the quality of oocytes is often observed to vary strongly. Traditional methods7-9 have included the isolation of ovary lobes, the exposure of ovary lobes to collagenase for some h, the selection of denuded oocytes, and the oocyte microinjection. Here, a number of alternative, fast procedures are reported that have allowed us to work with this expression system for more than 30 years with no seasonal variation and little variation in oocyte quality.
The modified and improved methods described here for the isolation of oocytes, microinjection with cRNA, and removal of follicular cell layers can be used for the expression of any protein of choice in the Xenopus oocyte. The very simple method for fast solution changes of the medium around the oocyte may be applied to the study of any ligand-gated ion channel and of carriers.
Animal experiments have been approved by the local committee of the Canton Bern Kantonstierarzt, Kantonaler Veterinärdienst Bern (BE85/15).
1. Preparation of Xenopus Oocytes
2. Microinjection of mRNA into the Cytoplasm
Note: The microinjection system described here is derived from that reported by Kressmann and Birnstiel22.
3. Stripping of the Follicles (Figure 3)
NOTE: As mentioned above, the oocyte covered by a vitelline layer, follicle cells, and connective tissue, which contains the blood vessels24, is known as a "follicle." All layers except for the vitelline layer, which provides mechanical stability without preventing the access of solutions to the cell surface, must be removed before electrophysiological experiments. The follicle without the surrounding cell layers has previously been termed a "denuded" oocyte25. This step is usually performed on the same day as the electrophysiological experiments.
4. Fast Solution Change around the Oocyte
Xenopus oocytes were mechanically singled out using a platinum loop (Figure 1). The oocytes were microinjected with mRNA coding for the GABAA receptor subunits α4, β2, δ, 0.5:0.5:2.5 fmol/oocyte (Figure 2). After 4 d, follicular cell layers were removed (Figure 3). Oocytes were voltage clamped at -80 mV and exposed to increasing concentrations of γ-aminobutyric acid (GABA) in the presence of 1 µM 3α,21-dihydroxy-5α-pregnan-20-one (THDOC), a potent positive allosteric modulator of the GABAA receptor. Figure 4A shows original current traces recorded in such an experiment. Figure 4B shows elicited current amplitude depending on the GABA concentrations. This allows determination of the sensitivity of this subunit combination toward GABA. The individual curves were fitted and standardized to Imax and subsequently averaged. The equation used was I(c)=Imax/(1+(EC50/c)n), where c is the concentration of GABA, EC50 the concentration of GABA (in the presence of 1 µM 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 α4β2δ GABAA receptor amounted to 0.41 ± 0.12 µM, and n was 0.76 ± 0.04.
Figure 1: Platinum Loop. The platinum wire loop mentioned in protocol step 1.4. Platinum is a metal that can be bent without producing splinters, and it does not stick to biological material. Please click here to view a larger version of this figure.
Figure 2: Homebuilt Microinjection Apparatus. The apparatus is described in protocol step 2. a, grill motor; b, micrometer screw; c, spring between syringe and plunger; d, 10 µL glass syringe; e, thick-walled polytetrafluoroethylene tubing; f, hand drill chuck; g, injection pipette; h, stereomicroscope; and i, motor control. Please click here to view a larger version of this figure.
Figure 3. Scheme Showing the Defolliculation of an Oocyte. Oocytes are first incubated in a collagenase solution in a water bath at 36 °C for 20 min. Oocytes are then washed in a modified Barth medium. The final incubation step is in a hypertonic EGTA solution at RT for 4 min. As a result, the connective tissue and the follicle cells are removed to give way to the denuded oocyte. Please click here to view a larger version of this figure.
Figure 4. Current Traces from a Two-electrode Voltage Clamp Experiment. A, Current traces from a GABA concentration response curve in the presence of 1 µM THDOC obtained from a Xenopus oocyte expressing the α4β2δ GABAA receptor. The bars indicate the time period of GABA/1 µM THDOC perfusion. Increasing concentrations of GABA were applied to the oocytes, and the corresponding current amplitudes were determined. GABA concentrations are indicated above the bars. B, Averaged concentration response curves of the α4β2δ GABAA receptor. Individual curves were first normalized to the fitted maximal current amplitude and were subsequently averaged. The data are shown as mean ± SD, n = 3. Please click here to view a larger version of this figure.
The methods described in this article deviate from those used traditionally7-9. It is standard to expose the lobes of the ovary to a 1 to 2 h collagenase treatment8; isolate undamaged, denuded oocytes; and inject them with mRNA using commercial injection devices. This classical procedure has the following drawbacks: 1) Oocytes are likely to be damaged by the long exposure to high concentrations of collagenase. 2) The unstable denuded oocytes must be stored until the experiment. 3) Denuded oocytes are more likely to suffer during microinjection than follicles. Commercial injection devices use large-diameter (about 20 µm) injection needles, likely to result in a relatively high rate of damage. The advantages of the improved procedure described here are as follows: exposure to collagenase is limited to 20 min, denuded oocytes do not have to be stored, and the small tip diameter of the pipettes (about 12 – 15 µm) used for microinjection does not require prior "defolliculation" of the follicles for the mRNA injection. The single critical step is that the follicle incubation temperature in collagenase solution should be carefully adjusted to 36 °C and should not exceed it.
For solution changes in electrophysiological experiments, very often the solution in the measurement chamber is changed, which takes a substantial amount of time, depending upon size of the chamber and the perfusion rate. Using the perfusion capillary avoided this.
The major drawback of the expression of ion channels in Xenopus oocytes is their giant size. Voltage control faster than about 2 ms is difficult, and very fast (<0.5 s) solution changes require elaborate procedures. Furthermore, strongly hydrophobic substances may almost be irreversibly bound to the egg yolk of the oocyte.
The methods outlined here have allowed us to investigate in a time-saving manner the functional properties of recombinant GABAA receptors, their modulation by synthetic compounds and plant substances, their subunit arrangement, and the location of drug binding sites in receptors of different subunit composition. The methods described here are suited for the expression of any protein of choice in the Xenopus oocyte. The very simple method for fast solution changes of the medium around the oocyte may be applied to the study of any ligand-gated ion channel or carrier.
The authors have nothing to disclose.
This work was supported by the Swiss National Science Foundation grant 315230_156929/1. M.C.M. is a recipient of a fellowship (Beca Chile Postdoctorado from CONICYT, Ministerio de Educacion, Chile).
NaCl | Sigma | 71380 | |
KCl | Sigma | P-9541 | |
NaHCO3 | Sigma | S6014 | |
MgSO4 | Sigma | M-1880 | |
CaCl2 | Sigma | 223560 | |
Ca(NO3)2 | Sigma | C1396 | |
HEPES | Sigma | H3375 | |
Penicilin/streptomycin | Gibco | 15140-148 | 100 μg penicillin/ml and 100 μg streptomycin/ml |
Platinum wire loop | home-made | ||
Micropipette puller | Zeitz-Instruments GmBH | DMZ | |
Hamilton syringe | Hamilton | 80300 | 10 μl, Type 701N |
Thick walled polytetrafluoroethylene tubing | Labmarket GmBH | 1.0 mm OD | |
Paraffin oil | Sigma | 18512 | |
Nylon net, gauge 0.8 mm | ZBF Züricher Beuteltuchfabrik AG | ||
Borosilicate glass tube | Corning | 99445-12 | PYREX |
Collagenase NB Standard Grade | SERVA | 17454 | |
Trypsin inhibitor type I-S | Sigma | T-9003 | |
EGTA | Sigma | E3389 | |
Glass capillary | Jencons (Scientific ) LTD. | H15/10 | 1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited |
Borosilicate glass capillary | Harvard Apparatus Limited | 30-0019 | 1.0 OD X 0.58 ID X 100 Length mm (for microinjection) |
Borosilicate glass capillary | Harvard Apparatus Limited | 30-0044 | 1.2 OD X 0.69 ID X 100 Length mm (for two-electrode voltage clamp) |
γ-Aminobutyric acid (GABA) | Sigma | A2129 | |
3α,21-Dihydroxy-5α-pregnan-20-one (THDOC) | Sigma | P2016 | |
grill motor | Faulhaber | DC micromotor Type 2230 with gear Type 22/2 | |
micrometer screw | Kiener-Wittlin | 10400 | TESA, AR 02.11201 |
Sterile plastic transfer pipettes | Saint-Amand Mfg. | 222-20S |